Molecular Biological and Immunological Techniques and Applications for Food Chemists

MOLECULAR BIOLOGICAL AND IMMUNOLOGICAL TECHNIQUES AND APPLICATIONS FOR FOOD CHEMISTS

Bert Popping Eurofins Scientific Group Yorkshire, England

Carmen Diaz-Amigo U.S. Food and Drug Administration Maryland, USA

Katrin Hoenicke Eurofins WEJ Contaminants GmbH Hamburg, Germany

MOLECULAR BIOLOGICAL AND IMMUNOLOGICAL TECHNIQUES AND APPLICATIONS FOR FOOD CHEMISTS

MOLECULAR BIOLOGICAL AND IMMUNOLOGICAL TECHNIQUES AND APPLICATIONS FOR FOOD CHEMISTS

Bert Popping Eurofins Scientific Group Yorkshire, England

Carmen Diaz-Amigo U.S. Food and Drug Administration Maryland, USA

Katrin Hoenicke Eurofins WEJ Contaminants GmbH Hamburg, Germany

Copyright  2010 by John Wiley &Sons, Inc. All rights reserved. Published by John Wiley & Sons, Inc., Hoboken, New Jersey Published simultaneously in Canada No part of this publication may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, recording, scanning, or otherwise, except as permitted under Section 107 or 108 of the 1976 United States Copyright Act, without either the prior written permission of the Publisher, or authorization through payment of the appropriate per-copy fee to the Copyright Clearance Center, Inc., 222 Rosewood Drive, Danvers, MA 01923, 978-750-8400, fax 978-750-4470, or on the web at www.copyright.com. Requests to the Publisher for permission should be addressed to the Permissions Department, John Wiley & Sons, Inc., 111 River Street, Hoboken, NJ 07030, 201-748-6011, fax 201-748-6008, or online at http: //www. wiley.com/go/permission. Limit of Liability/Disclaimer of Warranty: While the publisher and author have used their best efforts in preparing this book, they make no representations or warranties with respect to the accuracy or completeness of the contents of this book and specifically disclaim any implied warranties of merchantability or fitness for a particular purpose. No warranty may be created or extended by sales representatives or written sales materials. The advice and strategies contained herein may not be suitable for your situation. You should consult with a professional where appropriate. Neither the publisher nor author shall be liable for any loss of profit or any other commercial damages, including but not limited to special, incidental, consequential, or other damages. For general information on our other products and services or for technical support, please contact our Customer Care Department within the United States at 877-762-2974, outside the United States at 317-572-3993 or fax 317- 572-4002. Wiley also publishes its books in a variety of electronic formats. Some content that appears in print may not be available in electronic formats. For more information about Wiley products, visit our web site at www.wiley.com. Library of Congress Cataloging-in-Publicatton Data: Popping, Bert. Molecular biological and immunological techniques and applications for food chemists / Bert Popping, Carmen Diaz-Amigo, Katrin Hoenicke. p. cm. Includes index. ISBN 978-0-470-06809-0 (cloth) 1. Food–Analysis. 2. Molecular biology. 3. Immunoassay. I. Diaz-Amigo, Carmen. II. Hoenicke, Katrin. III. Title. TX545.P67 2009 6640 . 07–dc22 2009009723 Printed in the United States of America 10 9 8 7 6 5 4 3 2 1

To Sue Hefle Sue Hefle was an internationally recognized food scientist with major contributions in the area of Food Allergy and Food Allergens where she was considered a pioneer. She was the recipient of numerous national and international awards and because of her expertise she was member of numerous advisory panels, task forces, and working groups. During her career at the University of Nebraska she worked very closely with the food industry, governments, and consumer organizations. Unfortunately, we lost her in 2006 at the age of 46 after a long fight against cancer. She fought the disease with the same energy and positive attitude she showed during her professional career. To recognize her hard work and significant contributions in the field of Food Science and in particular Food Allergens—something she did with passion—we are dedicating this book to Sue, our friend and colleague.

CONTENTS

CONTRIBUTORS PREFACE

PART Ia

xi xiii

MOLECULAR BIOLOGICAL METHODS: TECHNIQUES EXPLAINED

1. Molecular Biology Laboratory Layout

3

Rainer Schubbert

2. Polymerase Chain Reaction

41

Hermann Broll

3. Quantitative Real-Time PCR

59

Hermann Broll

4. Polymerase Chain Reaction–Restriction Fragment Length Polymorphism Analysis

85

Klaus Pietsch and Hans-Ulrich Waiblinger

5. Single-Stranded Conformation Polymorphism Analysis

105

Hartmut Rehbein

6. Sequencing

119

Rainer Schubbert

PART Ib

MOLECULAR BIOLOGICAL METHODS: APPLICATIONS

7. Meat

135

Ines Laube

8. Genetically Modified Organisms

157

Bert Popping

vii

viii

CONTENTS

9. Detection of Food Allergens

175

Carmen Diaz-Amigo and Bert Popping

10. Offal

199

Neil Harris

11. Aquatic Food

209

Hartmut Rehbein

PART IIa

IMMUNOLOGICAL METHODS: TECHNIQUES EXPLAINED

12. Antibody-Based Detection Methods: From Theory to Practice

223

Carmen Diaz-Amigo

PART IIb

IMMUNOLOGICAL METHODS: APPLICATIONS

13. Animal Specification in Speciation

249

Bruce W. Ritter and Laura Allred

14. International Regulatory Environment for Food Allergen Labeling

267

Samuel Benrejeb Godefroy and Bert Popping

15. Japanese Regulations and Buckwheat Allergen Detection

293

Hiroshi Akiyama, Shinobu Sakai, Reiko Adachi, and Reiko Teshima

16. Egg Allergen Detection

311

Masahiro Shoji

17. Soy Allergen Detection

335

Marcello Gatti and Cristina Ferretti

18. Milk Allergen Detection

349

Sabine Baumgartner

19. Gluten Detection

359

Ulrike Immer and Sigrid Haas-Lauterbach

20. Nut Allergen Detection

377

Richard Fielder, Warren Higgs, and Katie Barden

21. Fish Allergen Detection

407

Christiane Kruse Fæste

22. Lupin Allergen Detection Christiane Kruse Fæste

423

CONTENTS

23. Mustard Allergen Detection

ix

445

Anne E. Ryan and Michael S. Ryan

24. Celery Allergen Detection

451

Charlotta Engdahl Axelsson

INDEX

459

CONTRIBUTORS

Reiko Adachi, Division of Novel Foods and Immunochemistry, National Institute of Health Sciences, Tokyo, Japan Hiroshi Akiyama, Division of Novel Foods and Immunochemistry, National Institute of Health Sciences, Tokyo, Japan Laura Allred, ELISA Technologies, Inc., Gainesville, Florida Charlotta Engdahl Axelsson, Eurofins Food & Agro Sweden AB, Ldink€oping, Sweden Katie Barden, Tepnel Research Products and Services, Deeside Industrial Park, Flintshire, UK Sabine Baumgartner, Center for Analytical Chemistry, University of Natural Resources and Applied Life Sciences, Tulln, Austria Hermann Broll, Bundesinstitut f€ ur Risikobewertung, Berlin, Germany Carmen Diaz-Amigo, Center for Food Safety and Applied Nutrition, U.S. Food and Drug Administration, Maryland, USA Christiane Kruse Fæste, National Veterinary Institute, Oslo, Norway Cristina Ferretti, Microbiotech Department, Neotron S.p.a., Modena, Italy Richard Fielder, Tepnel Research Products and Services, Deeside Industrial Park, Flintshire, UK Marcello Gatti, Microbiotech Department, Neotron S.p.a., Modena, Italy Samuel Benrejeb Godefroy, Food Directorate, Health Products and Food Branch, Health Canada, Ottawa, Ontario, Canada Sigrid Haas-Lauterbach, R-Biopharm AG, Darmstadt, Germany Neil Harris, LGC Limited, Teddington, Middlesex, UK Warren Higgs, Tepnel Research Products and Services, Deeside Industrial Park, Flintshire, UK Katrin Hoenick, Eurofins WEJ Contaminants GmbH, Hamburg, Germany Ulrike Immer, R-Biopharm AG, Darmstadt, Germany xi

xii

CONTRIBUTORS

Ines Laube, Institut f€ ur Lebensmitteltechnologie und Lebensmittelchemie, Technische Universit€at Berlin, Berlin, Germany Klaus Pietsch, Chemisches und Veterin€aruntersuchungsamt Freiburg, Freiburg, Germany Bert Popping, Eurofins Scientific Group, Pocklington, Yorkshire, UK Hartmut Rehbein, Max Rubner-Institut, Hamburg, Germany Bruce W. Ritter, ELISA Technologies, Inc., Gainesville, Florida Anne E. Ryan, ELISA Systems Pty. Ltd., Brisbane, Queensland, Australia Michael S. Ryan, ELISA Systems Pty. Ltd., Brisbane, Queensland, Australia Shinobu Sakai, Division of Novel Foods and Immunochemistry, National Institute of Health Sciences, Tokyo, Japan Rainer Schubbert, Eurofins Medigenomix GmbH, Ebersberg, Germany Masahiro Shoji, Morinaga Institute of Biological Science, Inc., Yokohama, Japan Reiko Teshima, Division of Novel Foods and Immunochemistry, National Institute of Health Sciences, Tokyo, Japan Hans-Ulrich Waiblinger, Chemisches und Veterin€aruntersuchungsamt Freiburg, Freiburg, Germany

PREFACE

From a historical point of view, the analysis of food is performed predominantly using typical chemical or physicochemical methods. These are, for example, wet chemical methods for proximity analysis or chromatographic methods for the analysis of pesticides and veterinary drug residues. In addition, food chemists use such physics-based methods as viscosity measurement and atomic absorption spectroscopy for the analysis of heavy metals. Food chemists in general tend to have good knowledge in some areas of biological analysis: in particular, regarding methods for the detection and enumeration of microorganisms and enzymatic methods for the analysis of single sugars or nitrite/ nitrate. In the past, use of these methods was sufficient for compliance with regulations and for quality assurance of food. However, in recent years several new issues have arisen in the field of food analysis which cannot be solved simply by applying chemical methods. Companies and regulators alike now have higher demands for safer food and product quality. This is reflected in more stringent customer product specifications as well as new regulations issued across the world. An example is an allergen-labeling regulation introduced in the United States on January 1, 2006. Similar regulations with an expanded scope have come into force in Europe and, some years ago, in Japan. Another example is the introduction of regulations for the labeling and traceability of genetically modified organisms. Although at present these are not regulated in the United States, regulations exist in Europe, Japan and other Asian countries, and in other parts of the world. Other regulations cover the protection of consumers from deception by mislabeling or adulteration of products. Examples are the adulteration of mandarine/tangerine juice, mislabeling of premium products such as Angus beef, and the protection of ethnic minorities from eating products forbidden by their religion. These analytical challenges can be solved easily using methods based on molecular biological or immunological principles: polymerase chain reaction (PCR), real-time PCR, and restriction fragment length polymorphism, or in the immunological field, enzymelinked immunosorbent assay and dot and Western blot, to name but a few. For the typical food chemist this tends to be a generally new field, since molecular biological and immunological methods are based on other principles. But especially over the past few years, borderlines between biological and physicochemical techniques have moved and fields previously separate have amalgamated somewhat as techniques have been combined to make it possible to provide answers faster and more xiii

xiv

PREFACE

cost-efficiently. One example is the invention of biochips, which, packed with antibodies, are being used to determine amounts of veterinary drug residues such as chloramphenicol, which until recently could only be determined by gas chromatographic or high-performance liquid chromatographical methods. Such methods serve not only to answer questions faster but also complement each other by confirming results through a completely independent method. As biological methods gain more importance in food analysis, it is prudent for the food chemist to become familiar with the techniques and to know the advantages and disadvantages, the fields of useful application, and the pitfalls of biological methods. For scientists with a basic chemical education, the contributors provide, in a simple and understandable but still comprehensive manner, descriptions of the most important methods used in routine molecular biology and immunology and give selected examples of important applications of these techniques in food analysis. The book is aimed at students and professional food chemists as well as quality assurance managers and can serve as guidance in understanding the techniques as well as implementing them in a laboratory to expand and complete a service portfolio. BERT POPPING Yorkshire, England CARMEN DIAZ-AMIGO Maryland, USA KATRIN HOENICKE Hamburg, Germany

Note: Several of the figures that appear in the book may be viewed in color at ftp://ftp.wiley.com/public/sci_tech_med/molecular_biological.

PART Ia

MOLECULAR BIOLOGICAL METHODS: TECHNIQUES EXPLAINED

CHAPTER 1

Molecular Biology Laboratory Layout RAINER SCHUBBERT Eurofins Medigenomix GmbH, Ebersberg, Germany

1.1

INTRODUCTION

In this chapter methods for the analysis of biological samples using molecular biological methods are described. The main focus will be on topical methods used in routine laboratories. However, the developmental rate of analytical methods and instruments is high in this field, and every year new applications are established in routine laboratories. Perhaps in a few years some types of routine DNA analysis will be performed with transportable instruments directly in food production facilities or food stores. The applications described herein are examples that represent the wide field of analyses performed by molecular biological methods in daily analysis work. Generally, the success of the analysis depends on correct sampling and storage, the DNA content of the sample, the correct DNA extraction method, and the corresponding analysis method. All methods described here are based on polymerase chain reaction (PCR), which is described later. For some of the analyses described, the methods are defined by legislation, for some analyses commercially available kits can be used, and for other analyses in-house methods must be developed and validated directly in the laboratory. The protocols for DNA extraction depend on the method used and are available from the manufacturer of the respective kit. Also, PCR reaction mixes and cycling conditions are specific for each assay and therefore are not described here. Generally, guidelines for forensic labs describe a very high standard and are therefore recommended (ILAC, 2002).

Molecular Biological and Immunological Techniques and Applications for Food Chemists Edited by Bert Popping, Carmen Diaz-Amigo, and Katrin Hoenicke Copyright  2010 John Wiley & Sons, Inc.

3

4

MOLECULAR BIOLOGY LABORATORY LAYOUT

1.2

LABORATORY

The laboratory design depends on the type of analysis performed. Detailed recommendations for a laboratory design are available, for example, at www.ilac.org/ publicationslist.html, www.dach-gmbh.de/, and www.eurachem.ul.pt/. In this chapter, only principles are explained. Generally, a molecular biological laboratory should be separated into three departments (pre-PCR, thermocycler, post-PCR), as shown in Figure 1.1. 1.2.1

Pre-PCR Department

At least three different rooms are necessary: . .

Room 1: Sample registration. In this room the biological samples are registered (e.g., barcoded) and subsamples are taken if necessary. Room 2: DNA extraction. In this room the DNA is extracted. All working steps should be performed with filter pipette tips. Coats and gloves must be worn to protect both the lab personal and the samples. Air conditioning is recommended. For work with samples with small amounts of DNA, a specific portion of the room

FIGURE 1.1 A molecular biological laboratory should be separated into three departments: pre-PCR (rooms 1 to 3), thermocycler (room 4), and post-PCR (room 5). A computer for sample tracking should be present in every room. In rooms where work with liquids is expected, a basin or separate waste bin is recommended. Equipment such as laminar flow or thermocycler are positioned in the schematic. Freezers or refridgerators should be planned depending on the number of samples expected in every room.

METHODS

.

5

should be separated off. If samples with infectious content are expected, laminar flow should be present. Room 3: PCR setup. In this room the PCR reaction is pipetted using filter tips. If possible, all PCR reagents are pipetted on one bench and the genomic DNA is added on a second bench to avoid contamination of the PCR reagents with the DNA. Air conditioning is recommended.

After each working step the benches have to be cleaned with suitable reagents. It is absolutely necessary that no PCR products be treated in one of these rooms. If contamination has occurred, all surfaces, instruments, and coats must be cleaned, and all chemicals and working solutions must be exchanged. 1.2.2

Thermocycler Department

PCR cycling is performed in the thermocycler department (room 4). No plastic material or solutions should be transferred from this department to the pre-PCR department. Air conditioning is recommended. 1.2.3

Post-PCR Department

In the post-PCR department (room 5) PCR products are handled using agarose gel electrophoresis, capillary electrophoresis, and other procedures. For this work a separate set of pipettes, plastic material, gloves, and coats are necessary. Air conditioning is recommended and is required if the analysis is performed using genetic analyzers with a laser and CCD (charge-coupled device) camera as the detection system. As mentioned above, instruments, coats, single-use plastic, and all other materials must not be transferred from this department into the pre-PCR department. If it should be necessary to reamplify PCR products, the PCR master mix has to be prepared in the pre-PCR department, transferred to the post-PCR department, and the PCR product added here.

1.3 1.3.1

METHODS Collection of Samples and Storage of Sample Material

One major aspect of the success of the analysis is correct sampling of the biological material and storage of the samples. Any mistake at this point would deeply influence succeeding steps of the analysis and could lead to a complete failure or to incorrect analysis results. Even if DNA extraction from clotted blood, decomposed meat, or swabs overgrown with fungi might be successful, it should only be used in forensic casework or in cases where no other biological material is available. Swabs for DNA extraction should be air-dried after sampling. Swabs stored in any gel or liquid (swabs for cultivation of bacteria) must not be used. The best storage conditions for biological materials are listed in Table 1.1. For all biological material, freez–thaw cycles should

6

MOLECULAR BIOLOGY LABORATORY LAYOUT

TABLE 1.1

Sampling and Storage Conditions for Biological Material

Biological Material

Sampling and Storage Conditions

Liquid blood for DNA extraction

Preserved with EDTA (first choice) or heparin (second choice); short-term storage and transport at þ 4 C, long-term storage at 20 C. Avoid thawing and freezing cycles; transport at 20 C; thawing in a water bath at 37 C directly before DNA extraction. Preserved with specific buffers [e.g., RNAlater solution (content of Qiagen RNA extraction kits)], storage at 80 C; transport on dry ice. Blood up to 200 mL dropped on filter paper [e.g., FTA, FTA-elute (Whatman)]; long-term storage under dry conditions at room temperature. Short-term storage (up to 24 h) at þ 4 C, long-term storage and transport longer than 24 h at 20 C. Strictly avoid thawing of frozen material. 10 to 15 min drying at room temperature after sampling; storage and transport under dry conditions at room temperature. Short-term storage at þ 4 C, long-term storage 20 C. Avoid thawing and refreezing. Storage and transport frozen in liquid nitrogen. If transported at þ 4 C or 20 C, do not store again in liquid nitrogen. Dry and protect from light at room temperature.

Liquid blood for RNA extraction Blood spots (only for DNA extraction) Fresh meat, fish meat, meat from seafood Swabs from surfaces, buccal swabs Bones, teeth, connective tissues Sperm samples (conserved for artificial insemination) Dried sperm spots

be avoided. If a frozen sample has thawed and has been refrozen, the laboratory must be informed in order to choose the most suitable DNA extraction method. Example 1: If a frozen sperm sample normally used for artificial insemination was thawed and later refrozen in liquid nitrogen, the heads of the sperm cells may have been destroyed. In routine protocols for DNA extraction from sperm cells a special step is included to pretreat the heads. After this step the used buffer is not reused in later extraction steps because for a native sperm sample the DNA is in the pellet and not in the buffer. In contrast, for thawed and refrozen samples, most of the DNA can be in this buffer and therefore DNA extraction from the pellet would fail. Example 2: Experiments have shown that fish meat frozen directly after capture contains sufficient amounts of DNA for analysis in about 100 mg of sample. But after thawing and refreezing, 1 to 2 g of fish is needed to obtain sufficient amounts of DNA; analysis with only 100 mg could fail. However, for meat from mammals, the influence is not that strong. During the sampling process, wearing of gloves, cleaning of instruments, and the use of single-use material is strictly enforced to avoid contamination. Especially for samples

METHODS

7

with a low DNA content (e.g., decomposed samples, degraded samples, bones, teeth), the risk of contamination is high and can lead to incorrect or unreliable results. 1.3.2

DNA Extraction

In recent years, several methods for the isolation of DNA from biological material have been developed, and kits are commercially available. The method used depends on the consistency of the biological material, the ratio expected for the amount of DNA per amount of biological material, the potential presence of PCR inhibitors in the biological material, and the instruments or pipetting machines present in the laboratory. Treatment of the sample with proteinase K and an EDTA buffer, followed by extraction with phenol and chloroform and ethanol precipitation of the DNA, leads to very pure DNA but has all the disadvantages inherent in handling organic substances. Therefore, most of the kits available work without phenol and chloroform. The principle of these kits is treatment of the sample with a low-salt lysis buffer which contains proteinase K and the addition of a binding buffer containing a chaotropic salt. In the presence of the correct concentration of the salt (e.g., guanidinium thiocyanate) the DNA binds to silica which is fixed on membranes (column-based DNA extraction kits) or coated on magnetic beads. Proteins, salts, and other components from the biological material do not bind to silica. After different washing steps, the DNA is eluted into water or TE buffer. Other kits are based, for example, on the characteristics of DNA at various levels of pH (Charge Switch, Invitrogen). Kits for low throughput, where all steps are processed manually, to kits for high throughput, where most or all steps are processed on pipetting machines, are available from most suppliers (see Table 1.2). 1.3.3

Measurement of DNA Concentration

For a successful analysis it is necessary to determine the DNA concentration. The presence of high concentrations of DNA can influence downstream applications, which can lead to a total or partial inhibition of PCR, especially for commercially available multiplex PCR kits. For DNA concentrations greater than 10 ng/mL, the measurement of DNA concentration by a determination of OD (optical density) 260/ 280 nm with a photometer will lead to reliable results. With this method all DNA that is present in the solution is measured. This is sufficient with DNA from fresh blood or meat samples, for example. If an analysis were performed to prove the identity of a degraded tissue sample, it would be necessary to determine separately the amount of DNA from the tissue and from bacteria and fungi grown on this tissue. These methods are described in Section 1.3.8. 1.3.4

Variants in the Sequences of Genomic DNA

The DNA of higher organisms is separated into DNA located in the nucleus (genomic DNA) and DNA located in the mitochondria (mtDNA). The genomic DNA is separated on the chromosomes. At every somatic cell two copies of the autosomal

8

MN

Supplier/ Biological Material

TABLE 1.2

Blood on Filter Paper

Animal Tissue or Meat Bones and Teeth

NucleoSpin NucleoSpin NucleoSpin NucleoSpin DNA Blood Tissue Tissue Trace (740951.10/.50/ (740952.10/.50/ (740952.10/.50/ (740942.4/.25) .250) .250) .250) NucleoSpin NucleoSpin 8 NucleoSpin 8 þ NucleoSpin Blood L Trace Tissue Trace Bone (740954.20) (740722/.1) (740740/.5) Buffer Set (740943.25) NucleoSpin NucleoSpin 96 NucleoSpin 96 Blood XL Trace Tissue (740950.10/.50) (740726.2/.4) (740741.2/.4/.24) NucleoSpin 8 NucleoMag 96 NucleoMag 96 Blood Trace (744600.1/ Tissue (740664/.5) .4/.24) (744300.1/.4/.24) NucleoSpin 96 NucleoSpin Food Blood (740665.1/ (740945.10/.50/ .4/.24) .250) NucleoMag 96 NucleoSpin 8 Food Blood (740975/. 5) (744500.1/.4/.24) NucleoSpin 96 Food (740976.2/. 4/.24)

Liquid Blood

Kits for DNA Extraction a

Swabs

Homepage

NucleoSpin NucleoSpin 96 Plant XL Trace (740540.6) (740726.2/.4) NucleoSpin 8 NucleoMag 96 Plant Trace (740662/.5) (744600.1/.4/.24) NucleoSpin 96 Plant (740661.2/ .4/.24) NucleoMag 96 Plant (74400.1/ .4/.24)

NucleoSpin NucleoSpin www.mnPlant II Tissue net.com (740770.10/.50/ (740952.10/.50/ .250) .250) NucleoSpin NucleoSpin 8 Plant L Trace (740539.20) (740722/. 1)

Plant Material

9

QIAamp DNA Blood Mini Kits QIAamp DNA Micro Kit Generation Capture Card Kit

—

DNA IQ System DNA IQ System Wizard Genomic ReadyAmp DNA Purification Genomic DNA Kit Purification System

Gentra Puregene Blood Kits FlexiGene DNA Kits Generation Capture Kits

EZ1 DNA Blood Kits (200 or 350 m L) QIAamp DNA Blood BioRobot MDx Kit

QIAamp DNA Blood Kits (Mini, Midi, Maxi) QIAamp 96 DNA Blood Kits

XK02-04 Genisol ABgene/ Maxi-Prep Kit Thermo (single preps) Fisher Scientific

Promega

Qiagen

XK02-04 Genisol Maxi-Prep Kit (single preps)

Wizard SV Genomic DNA Purification System

DNeasy Blood and Tissue Kit

XK02-04 Genisol Maxi-Prep Kit (single preps)

—

—

QIAamp 96 DNA Swab BioRobot Kit

QIAamp DNA Micro Kit

Wizard Genomic DNA IQ System DNA Purification Kit

DNeasy Plant Maxi Kit

DNeasy Blood and Tissue Kit

QIAamp DNA Mini Kits

DNA IQ System

DNeasy Plant Mini Kit

QIAamp DNA Micro Kit

(continued)

www. abgene. com

www. promega. com

www.qiagen. com

10

For 1, 8, and 96 samples as noted.

a

GeneCatcher gDNA Blood Kits (1) DNAzol BD Reagent (1)

ChargeSwitch gDNA Serum Kits (1)

ChargeSwitch Forensic DNA Purification Kit (1)

ChargeSwitch gDNA Mini or Micro Tissue Kit (1) PureLink Genomic DNA Purification Kit (1) DNAzol Reagent (1)

DNAzol Reagents (1) (bone marrow)

PureLink Plant DNA Purification Kit (1) Plant DNAzol Reagent (1)

ChargeSwitch gDNA Plant Kit (1)

Nucleon Phytopure

—

—

—

First-DNA all tissue 10/50/ 100/500

First-DNA all tissue 10/50/ 100/500

First-DNA all tissue 10/50/ 100/500

First-DNA all tissue 10/50/ 100/500

First-DNA all tissue 10/50/ 100/500 —

Plant Material

Bones and Teeth

Animal Tissue or Meat

Blood on Filter Paper

Liquid Blood

Invitrogen ChargeSwitch gDNA Blood Kits (96)

Tepnel Life Sciences

Genial

Supplier/ Biological Material

TABLE 1.2 (Continued)

ChargeSwitch Forensic DNA Purification Kit (1)

First-DNA all tissue 10/50/ 100/500

Swabs

www. invitrogen. com

www. genial.de

Homepage

METHODS

11

chromosomes are present (diploid chromosome set). Therefore, from all genetic information located on these chromosomes, two copies (alleles) are present in every cell. From the gonosomal chromosomes two copies of one variant or one copy of each of the two variants is present, depending on the gender of the individual (X and Y chromosomes in mammals, W and Z chromosome in birds). In the germ cells only one copy of the autosomal chromosomes and one gonosomal chromosome are present. The genomic DNA is separated into introns and exons as shown in Figure 1.2. An exon is any region of the DNA within a gene that is transcribed to the final messenger RNA (mRNA) molecule, which is translated into proteins, for example (Gilbert, 1978). Therefore, mutations in these regions can have a strong influence on the organism where they occur. Examples are variants in BRCA genes, which lead to a higher risk of developing breast cancer in humans; mutations at the PKD 1 gene, which leads to polycystic kidney disease in cats; or variations at the PrP gene in sheep or goat, which leads to higher or lower risk to develop scrapie after exposure to the infectious agent. Mutation in the exon regions can be insertions (new bases are added to the DNA), deletions (single bases up to longer parts of DNA are missing), point mutations [singlenucleotide polymorphisms (SNPs)], duplications, or translocations. Therefore, most of the DNA sequences of exonic regions are highly conserved in an animal or plant species. Some can be used for animal species determination. One example is SNPs at the mitochondrial cytochrome b gene, which can be detected, for example, by RFLP (restriction fragment length polymorphism) followed by agarose gel electrophoresis. Depending on the DNA sequence, specific restriction enzymes cut the PCR products. From the number and length of the fragments it is possible to conclude the DNA sequence at the restriction sites (see Section 1.3.9). In contrast to these conserved exonic sequences, at intronic sequences (sections that are spliced out after transcription but before the RNA is used) mutations have in most cases no influence on the individual in which they occurred and therefore are passed on to the next generation. Furthermore, insertions, deletions, SNPs, duplications, and translocations exist in the intronic regions. In addition, regions with repeated DNA motifs are present, known as STRs (short tandem repeats) or VNTRs (variable number tandem repeats). From the length of the repeated motif they are separated into

Intron

Exon

Intron

Exon

Intron

DNA

RNA

Protein

FIGURE 1.2 Genomic DNA is separated into introns (white) and exons (black). Sequences from exons were transcribed to RNA and translated to proteins.

12

MOLECULAR BIOLOGY LABORATORY LAYOUT

Intron

Exon

Intron

Exon

Intron

DNA

Short Tandem Repeat

Sample A: 11 Repeats

ACGTCAGATAGTTGCAT CG CG CG CG CG CG CG CG CG CG CG TTAAAGCCGATAG TGCAGTCTATCAACGTA GC GC GC GC GC GC GC GC GC GC GC AATTTCGGCTATC

Sample B: 9 Repeats

ACGTCAGATAGTTGCAT CG CG CG CG CG CG CG CG CG TTAAAGCCGATAG TGCAGTCTATCAACGTA GC GC GC GC GC GC GC GC GC AATTTCGGCTATC

Sample C: 8 Repeats

ACGTCAGATAGTTGCAT CG CG CG CG CG CG CG CG TTAAAGCCGATAG TGCAGTCTATCAACGTA GC GC GC GC GC GC GC GC AATTTCGGCTATC

Sample D: 5 Repeats

ACGTCAGATAGTTGCAT CG CG CG CG CG TTAAAGCCGATAG TGCAGTCTATCAACGTA GC GC GC GC GC AATTTCGGCTATC

FIGURE 1.3 Microsatellites and other variable regions are located in introns. In this scheme, four different sequences of a microsatellite with the dinucleotide motif CG with 11, 9, 8, and 5 repeats are shown.

microsatellites and minisatellites. At microsatellites the repeat motif contains 1 to 5 base pairs (bp) (Figure 1.3), at minisatellites more than 15 bp. Even if the repeat motif is specific for any microsatellite (e.g., main motif AGAAn for the human marker D18S51), incomplete repeats also exist. Alleles with incomplete repeats are described as microvariants. At every microsatellite, different numbers of repeats can be found (e.g., at the very polymorphic human STR SE33/ACTBP2, there are about 100 different alleles with 4 to 50 complete and incomplete repeats (Schubbert, 2002). For example, at most markers used in routine analysis for human identification, the difference between the shortest and longest alleles is 20 to 30 bp. It is necessary to distinguish between the nearest possible fragments, which can be 1 bp at several markers. In principle, PCR products can be separated by highly concentrated agarose gels, combined agarose–polyacrylamide (PAA) gels, PAA gels, or capillary electrophoresis with liquid polymer, as described in Section 1.3.7. 1.3.5

PCR

Since the first publication of the PCR method (Mullis, 1990), thousands of applications for DNA analysis have been developed. The principle of PCR is shown in Figure 1.4. 1. Melting step. Double-stranded DNA is denatured (single-stranded) in a first temperature step at 94 to 95 C for 15 to 30 s. 2. Annealing step. The reaction mixture is cooled down to 48 to 60 C for 15 to 30 s. At this lower temperature, primers (short DNA molecules with 15 to 40 bp specific for the DNA fragment, which should be amplified) bind

METHODS

13

1 2 Primer Taq - Polymerase

3 4 5

FIGURE 1.4 In PCR double-stranded DNA become denaturated (1) to single strands (2). Sequence-specific primers bind to single-stranded DNA (3) and Taq polymerase starts the duplication of DNA from these primers (4). The next cycle starts with these duplicated fragments (5).

to the single-stranded DNA. The optimal temperature at this step depends on the melting temperature of the primers. 3. Elongation step. At 72 C, Taq polymerase starts elongation of the DNA strand in the 50 ! 30 direction, starting from the primer, for 30 s up to some minutes, depending on the length of the fragment amplified. These three steps (cycles) are repeated 30 to 40 times, depending on the amount of DNA measured at the beginning of the reaction. The complete analysis runs automatically in combined heating–cooling instruments known as thermocyclers. These are available from a variety of suppliers for the analysis of one up to 2  384 samples in parallel. In the past year, several thermocyclers with very high heating and cooling rates have been developed to reduce the PCR time (Table 1.3). PCR for microsatellite analysis or other multiplex analysis can be performed with labeled primers as shown in Figure 1.5. Depending on the analysis system, different dyes are used which can be detected with ultraviolet (UV) or infrared light. Combinations of dyes used in routine analysis are listed in Table 1.4. If only a few samples should be analyzed with a higher number of markers, it may be more economical to elongate a specific primer with a universal DNA sequence tail. PCR will than be performed with a mixture of the specific primers and a labeled primer that binds to the universal tail in a singleplex reaction (Qin et al., 2006). If one anticipates that a specific marker set will be used in routine analysis in the future, it could be useful to redesign the primers. With optimized primer sets it is possible to perform multiplex PCR reactions with 10 to 15 markers. In this case one specific primer from every marker should be labeled.

14

MOLECULAR BIOLOGY LABORATORY LAYOUT

TABLE 1.3

Thermocycler Suppliers

Manufacturer ABI Eppendorf

Stratagene (Robocycler)

1.3.6

Number of Samples Processed in Parallel 96/384/2  96/2  384 Various devices/ configurations available: . 0.5-mL reaction volume: 16 or 77 samples . 0.2-mL reaction volume: 25 or 96 (tubes or plate) samples . 384-well format 96

Homepage www.appliedbiosystems.com www.eppendorf.com

http://www.stratagene.com/ products/displayproduct. aspx?pid¼260

Agarose Gel Electrophoresis

DNA fragments can be separated by agarose gel electrophoresis and stained with dyes such as ethidium bromide or PicoGreen. These dyes interact with double-stranded DNA and emit fluorescent light after stimulation with UV light. As ethidium bromide is

FIGURE 1.5 Dye-labeled DNA fragments are produced by PCR with dye-labeled primers (a). The size of two different alleles (b) with 7 or 5 repeats (4 bp) differs from that of 8 bp. By coseparation of a size standard (c, black lines) and an allelic ladder (c, gray lines), the size can be determined and the alleles determined correctly.

METHODS

TABLE 1.4

Dye Sets Used in Routine Analysis on ABI Genetic Analyzers

Filter Set/ Channel

Blue

Green

Yellow

FAM FAM FAM FAM

HEX JOE JOE VIC

NED NED TMR JOE

D F G5

15

Orange (Used with Five-Dye Sets)

Red (Used as Internal Size Standard)

— — — PET

ROX ROX RXN LIZ

carcinogenic and toxic, nitrile gloves should be worn to protect the hands when handling dyes, stained gels, or contaminated buffers. Depending on the workflow in the laboratory, the dye is already mixed with the melted agarose and is present in the gel during electrophoresis, or the gel is stained after electrophoresis. Also, ready-to-use agarose gels are available with or without dyes from some manufacturers. If prestained gels are used, special attention has to be paid because the buffers and chambers will be contaminated with the dye. In this case, strict rules should be established in the laboratory and chambers, pipettes, and instruments contaminated with the dye must be controlled. Contaminated objects should always be handled with gloves. Depending on the size of the DNA, the concentration of agarose, and its quality, fragments with differences of at least 4 bp can be separated. Agarose Gel Electrophoresis of Genomic DNA The concentration of DNA can be determined by OD measurement. However, this technique gives no information about possible degradation of the DNA, which it is necessary to know for some applications. For these reasons, agarose gel electrophoresis of genomic DNA can be performed. A size marker that covers the sizes expected (up to 40 kb) has to be co-separated on the same gel. In Figure 1.6, examples of different grades of degradation of genomic DNA are demonstrated. If agarose gel electrophoresis shows that almost all DNA is degraded to fragments shorter than 400 bp, for example, it will be very difficult to amplify a PCR fragment of about 450 or 1000 bp length, which is used routinely for the analysis of mtDNA in humans or animals. With the information from this agarose gel, the strategy has to be changed and the amplification of two or three smaller fragments would lead to a successful analysis. Agarose Gel Electrophoresis of PCR Products Agarose gel electrophoresis of PCR products can be performed as quality control before further analyses. For RFLP analysis or sequencing of the PCR product, it is necessary to determine whether a PCR product is present and how much PCR product is present. Therefore, a size marker that again covers the expected fragment sizes (at PCR products, normally about 100 to 1200 bp) with known concentration has to be co-separated (see Figure 1.12). After detection of PCR products and estimation of the concentration, the following analyses will be more successful because optimal amounts of DNA can be applied to downstream reactions. For proof of the presence of fungi, bacteria, or viruses, PCR followed by agarose gel electrophoresis is sometimes sufficient for diagnosis. This is the case if the PCR product is specific

16

MOLECULAR BIOLOGY LABORATORY LAYOUT

FIGURE 1.6 Agarose gel picture. The quantity and quality of isolated genomic DNA can be determined in comparison with defined size standards (lanes 1 and 8) From dried fish (lane 2), degraded muscle (lane 5), and heart tissue (lane 7) only weak amounts of mostly degraded DNA can be isolated. From freshly frozen fish (lane 3) or prawns (lane 4) and bone marrow (lane 6), high-molecular DNA can be isolated.

for the organism, all controls show the results expected, and subtyping is not necessary (e.g., detection of Chlamydia). 1.3.7

PAA Gel Electrophoresis and Capillary Electrophoresis

Today, high-throughput microsatellite analysis is performed with PAA gels or capillary electrophoresis (CE) with automated instruments (Table 1.5). When using older instruments, a swab gel has to be prepared and the samples must be loaded manually. In the first step two panes of glass are treated with NaOH, washed, fixed together, and a PAA solution is placed between the panes. After polymerization, the panes are mounted on the instrument and a pre-run is performed to stabilize the electrophoresis conditions. Finally, the samples, mixed with running buffer, are loaded manually to the instrument using an eight-channel pipette. After every run the glass panes have to be cleaned and the buffer has to be exchanged. The advantage of this type of instrument is better resolution for specific types of samples and robustness if only a few runs are performed per week. With the current generation of capillary electrophoresis instruments, PCR products are mixed with formamide and put into the instrument. Filling the capillaries with viscous polymer, loading the samples and the size standard to the capillary, and starting

METHODS

TABLE 1.5

17

Instruments for PAA or Capillary Electrophoresis

Numbers of Samples Processed in Number Swab Gel Parallel of Dyes or CE Homepage Manufacturer Instrument ABI Amersham LiCor

3130 XL MegaBACE 4300

96 96

5 4

48

2

CE CE

www.appliedbiosystems.com www.4.amershambiosciences. com Swab gel www.licor.com

and performing electrophoresis are carried out by the instrument automatically. In one capillary analyzer, the ABI 3130 (Figures 1.7 and 1.8), 30 runs of 16 samples with a read length of 600 bp can be run within 24 h. Routine work for this instrument is reduced to refilling the buffers and viscous polymers or capillary arrays and routine cleanup of the instrument (buffer chambers, injection pumps), which should be carried out once a week. One disadvantage of this type of instrument is the aging of the polymer and array if mounted on the instrument, which can be critical if only a few runs are performed per week. For PAA electrophoresis or CE with automated fragment detection and size calling, the PCR products must be labeled with dyes (Table 1.4). During the establishment of a new assay, for best results several dilutions of PCR products should be tested after PCR. For electrophoresis the PCR products have to be mixed with a loading dye according to the concentrations given by the manufacturer and an internal size standard that is co-separated in every line or capillary. During electrophoresis a laser stimulates

FIGURE 1.7

Genetic analyzer ABI 3130 with control computer.

18

MOLECULAR BIOLOGY LABORATORY LAYOUT

FIGURE 1.8 Detailed view of ABI 3130. Liquid polymer stored in a bottle (a) became transported to a capillary array (b) by a pump (c). Samples prepared for electrophoresis became stored in a tray (d). By electrophoresis, PCR products migrate to the detection window (e) and become measured by a CCD camera.

the dyes to emit fluorescent light, which is measured by a CCD camera. This camera scans the detection window 5000 to 10,000 times per run. Thereby, the data collection software of the instrument collects the data of four or five different dye channels. By comparing the raw data from the red channel (on ABI instruments, the internal size standard is recorded in the red channel) with data from the other channel, the analysis software (e.g., GeneScan or Genemapper for instruments from Applied Biosystems) calculates the fragment size of the PCR products (Figure 1.9). For a reproducible allele calling, categories can be defined by the software (for instruments from Applied Biosystems, e.g., Genotyper or GeneMapper) for every marker at additional analysis steps which can be used for further analyses. Depending on the instrument and size standard used, the same allele/PCR fragment can be defined with different lengths (Figure 1.10). Therefore, it is necessary to standardize the results for intra- and interlaboratory data exchange. For some commercially available microsatellite multiplex PCR kits, allelic ladders are available (Figure 1.11). These ladders should be analyzed within every run in a separate line or capillary to assure the quality of the analysis. For individual marker sets it is recommended that an allelic ladder be developed or at least that one or two control samples with a known genotype be analyzed in every batch of samples. For some marker sets, ring trials were organized [e.g., by ISAG (International Society for Animal Genetics) for horse, cattle, sheep, goat, dogs, and cats; and by ISFG (International Society for Forensic Genetic) and DGRM (German Society for Legal Medicine) for humans].

METHODS

19

FIGURE 1.9 Data from GeneScan analysis. Size of PCR products (blue, green, and black peaks) is measured by comparison with an internal size standard co-separated in the same capillary. (a) Size standard ROX500 from ABI; (b) PCR fragments and size standard. (See insert for color representation.)

FIGURE 1.10 Comparison of data from Genotyper analysis. Identical PCR was co-separated with size standard ILS600, Promega (a, b) and with size standard ROX500 (ABI) (c, d) in parallel on an ABI 3100. Using ROX500, fragment lengths seems to be 2 to 3 bp longer than when using ILS.

20

MOLECULAR BIOLOGY LABORATORY LAYOUT

FIGURE 1.11 Comparison of data from Genotyper analysis. Comparing PCR products with fragments of allelic ladders, intra- and interlaboratory data exchange is possible. Upper panel: allelic ladder of markers D3S1358, TH01, and D18S51 used for human identification shown; at the lower panel: DNA profiles from four different DNA samples.

1.3.8

Real-Time PCR

For some applications it is necessary to determine the amount of specific DNA or RNA in a biological sample. At the beginning of every PCR reaction only a few amplified fragments are present. In the following logarithmic phase the number of PCR products is doubled in every cycle under optimal conditions. Depending on the amount of DNA at the beginning of the reaction, after various cycles the reaction reaches the plateau phase. This is influenced by several factors. During PCR the amount of free nucleotides available, which are necessary to synthesize a new DNA strand, and the amount of free primers decrease. At a later time, high numbers of PCR fragments are present. These fragments also reanneal and are not available to bind primers. Finally, the efficiency of the enzyme weakens from cycle to cycle. Applying PCR with, for example, 35 cycles and a separation on agarose gel, no difference can be detected, whether 10, 25, 50, or 100 ng of genomic DNA is analyzed in the PCR reaction (Figure 1.12). In contrast, using real-time PCR it is possible to distinguish between the various amounts of DNA (Figure 1.13). Real-time PCR can be carried out with a pair of unlabeled primers and the presence of SybrGreen, which emits fluorescent light after stimulation when double-stranded DNA is present. However, SybrGreen also binds to the high-molecular DNA that is added to the PCR reaction, to primer–dimmers, and to unspecific PCR products. As a consequence, only one PCR product can be detected per reaction. Therefore, during the development phase of a new assay, the PCR products should be controlled by an agarose gel electrophoresis after real-time PCR. The optimal amount of high-molecular DNA added to the PCR also has to be determined. The performance of a melting curve after the PCR reaction can assure that the PCR products expected are amplified.

METHODS

21

FIGURE 1.12 Agarose gel picture. In comparism with defined size standard (lane 1) the quantity and fragment size length of PCR products can be determined. Different amounts of DNA (60 ng, 30 ng, 15 ng, 7.5 ng, 3.75 ng, 1.8 ng, and 900 pg; lanes 2 to 8) were analyzed by real-time PCR with primers specific for mitochondrial DNA. In contrast to online measurement during real-time PCR by agarose gel electrophoresis, quantification of the amount of genomic DNA put into this PCR is not possible.

FIGURE 1.13 Different amounts of DNA (60 ng, 30 ng, 15 ng, 7.5 ng, 3.75 ng, 1.8 ng, and 900 pg) were analyzed by real-time PCR with primers specific for mitochondrial DNA. Depending on the amount of DNA put into PCR, the amplification curves cross the threshold (horizontal line) with one cycle difference (upright gray lines).

22

MOLECULAR BIOLOGY LABORATORY LAYOUT

Real-time PCR is more specific when primer and labeled probes are used. By a combination of specific primers and probes with a 10 C higher melting temperature, it is possible to detect, for example, 1-bp mutations (SNPs) by real-time PCR. Different types of probes are developed. At dual-labeled probes at the 50 end a reporter dye is labeled, which emits fluorescent light after stimulation. At the 30 end a quencher dye is labeled. If reporter and quencher are localized nearby, the fluorescent light of the reporter dye is quenched. The Taq polymerase used for PCR also has exonuclease activity. Therefore, during the elongation phase of the PCR the probe is not melted from the DNA strand and is destroyed by the Taq polymerase. The reporter and quencher dyes are separated and the light emitted from the reporter can be measured by the detection system of the thermocycler. At the beginning of the reaction the instrument measures the background signal from the reporter dye. During every cycle the instrument measures the signal intensity and determines the cycle at which the signal is significant higher than the background signal of the samples at the start of the analysis. This cycle is called a cycle of threshold (Ct). High Ct values mean that less DNA was present at the beginning of the PCR. Real-time PCR can also be used for the quantitative analysis of DNA or RNA. In expression analysis, the Ct values of a constant-expressed gene (a housekeeper gene) and a variable-expressed gene (a gene of interest) are compared. Expression analysis is not often used in the analysis of meat or food products. Currently, different instruments are available for real-time PCR. Depending on the manufacturer, the stimulating light is emitted from a laser or from a tungsten bulb in combination with a filter. The fluorescent light emitted from the reporter dye is measured through a prism or filter system. The number of parallel dyes detected varies from three to five. Instruments currently available are listed in Table 1.6. 1.3.9

RFLP Analysis

For RFLP (restriction fragment length polymorphism) analysis, the qualities of restriction endonuleases are used. These cut genomic DNA or PCR products at or near specific sequences. At a single basepair mutation, two different sequences are present. The corresponding enzyme cleaves only one of the two possible DNA strands. The fragments can be detected by agarose gel analysis. 1.4

APPLICATIONS

1.4.1

PCR and Detection of PCR Fragments

Gender Determination of Animals Cattle

The gender determination of cattle is necessary to answer two questions:

1. During embryogenesis of twins, anastomotic blood vessels can be developed between the placentas, which can lead to problems with dioecious twins. Through these blood vessels, stem cells and hormones can be exchanged. If the female twin

23

Roche Stratagene (Mx3005P)

96/384 96

96

ABI

Eppendorf

48/96/384

Manufacturer 5

Number of Dyes

Two options available: up to two or four different dyes detectable 4 5

Instruments for Real-Time PCR

Number of Samples Processed in Parallel

TABLE 1.6

Halogen lamp; photomultiplier

LED/Halogen/ Laser 96 LEDs for excitation; channel photomultiplier for detection

Stimulating Light/Detection System

yes yes

yes

yes

Performance of Melting Curve Possible?

www.roche-applied-science.com www.stratagene.com/qpcr

www.eppendorf.com

www.appliedbiosystems.com

Homepage

24

MOLECULAR BIOLOGY LABORATORY LAYOUT

receives the anti-Mueller hormone from the male twin, sexual organ development will be inhibited. This can range from missing organs in the newborn calf to functional problems even when the organs are present. In almost all cases such female twins will not become pregnant. Development of the sexual organs of the male twin is not influenced. A farmer thus has two choices after the birth of dioecious twins: He or she can feed the female calf and slaughter it as he or she would a male calf, or can determine by PCR whether blood cells that carry a Y chromosome are present in the circulating blood of the female calf. If these cells are present, it is very likely that the anti-Mueller hormone was transferred to the female twin. The analysis has to be performed with EDTA or heparin blood. Hairs with roots would lead to an incorrect result because the cells transferred are present only in the blood. The cells transferred are underrepresented when using muscle biopsies and buccal swabs and therefore may not be detected. 2. If female calves or cattle are slaughtered, female meat might be declared to be male meat, perhaps unintentionally. Because male meat receives higher prizes and higher prices are paid for exports, female meat might intentionally and deceitfully be declared to be male meat. According to European Commission (EC) Regulation 2002/765/EC of 3/5/2002, the analysis of gender determination has to be performed by PCR with primers specific for DNA fragments which are located on both the X and Y chromosomes: .

.

Forward and reverse amelogenin (Ennis and Gallagher, 1994); the length of a PCR fragment specific for the X chromosome is 280 bp, one specific for the Y chromosome is 218 bp. ZFX and ZFY forward and ZFX/ZFY reverse (Zinovieva et al., 1995); the length of a PCR fragment specific for the X chromosome is 132 bp, one specific for the Y chromosome is 282 bp.

The use of two primer pairs reduces the risk of an incorrect result. Since mutations of DNA sequences are spread over the complete genome, a mutation can also be present at any primer binding site. If this occurs at the binding site of genes located on the Y chromosome but not on the X chromosome, only the fragment specific for the X chromosome is amplified. In this case, male meat can be determined incorrectly to be female meat. The use of two independent DNA fragments for the analysis reduces the risk of a wrong result dramatically. PCR products can be detected by agarose gel electrophoresis or capillary electrophoresis (with one labeled primer per pair). In Figure 1.14, genotypes of a male and a female sample detected by CE are shown. Birds Gender determination can be necessary for bird species without external gender differences. As surgery (laparatomy) with anesthesia for gender determination can lead to the death of the birds, DNA analysis from blood or feathers is a noninvasive alternative. Normally, gender determination by DNA analysis is not performed for

APPLICATIONS

25

FIGURE 1.14 Gender determination of beef meat. Samples of male (a) or female (b) origin were analyzed by PCR with primers specific for bovine amelogenin locus. With samples of male origin, two PCR products can be detected; with samples of female origin, one PCR product can be detected.

poultry such as geese but, rather, for parrots, parakeets, and some birds of prey. For gender determination of a wide variety of birds, except the ratites (ostrich, rhea), which are sometimes grown for meat production, universal primers were used (Griffiths et al., 1998). For gender determination of cattle, PCR products can be detected by agarose gel electrophoresis or CE. In Figure 1.15 genotypes of a male and a female sample detected by CE are shown. Analysis of Special Ingredients in Food Products In modern food production different groups of additives are used, which sometimes cannot be detected or distinguished by methods other than PCR. Examples for those

FIGURE 1.15 Gender determination of birds. Samples of male parrot (a) or female parrot (b) origin were analyzed by PCR. With samples of male origin, one PCR product can be detected; with samples of female origin, two PCR products can be detected.

26

MOLECULAR BIOLOGY LABORATORY LAYOUT

additives are hydrocolloids [e.g., xanthan (E415), guar gum (E412), or locust bean gum (E410)], which are added to products such as yogurt, ketchup, or instant soup for technical reasons. However, in some cases these additives may not be declared. Xanthan, which is allowed to be added to organic food, is produced by the fermentation of wood by Xanthomonas campestris strains. Xanthan can therefore be detected with primers specific for the DNA of these bacterial strains. Cellulose and pectin are produced from the shells of apples and citrus fruits. Theoretically, these substances can be detected with primers specific, for example, for apple chloroplast genes. However, experiments have shown that for most samples the DNA was too degraded during the production process for a successful analysis. The detection of guar gum (E412) or locust bean gum (E410) is possible with primers specific for the DNA of Cyamopsis (guar bean) and Ceratonia (carob), as described elsewhere (Urdiain et al., 2004). For the analysis of food products the DNA extraction should be performed with specially developed kits. With these types of kits it is possible, for example, to extract DNA from chocolate, cheese and other milk products, or marmalade without coextraction of such PCR inhibitors as salts, fatty acids, or humid acids. Examples of kits are the food kits from MN and Genial (see Table 1.2). For some highly processed food products the DNA content might be very low. For these applications the Funnel Food Kit from MN allows lysis of the sample material in volumes up to 10 mL and the elution of the DNA into volumes less than 100 mL. PCR products can be detected with agarose gels. Bacterial Species and Antibiotic Resistance Determination As described in Chapter 6, determination of bacterial species may be necessary for pathogen detection in meat and food products: identification of infectious pathways of persons working in food production or other sensitive departments, or for identification of bacterial strains used in food production. With sequencing of PCR products of the bacterial 16S RNA gene, identification is possible if only one strain is present. For mixed samples, interpretation of sequencing data can be difficult or impossible. For this analysis at least 6 h is necessary after DNA extraction. In contrast, PCR with primers specific for single strains followed by agarose gel electrophoresis gives results in 3 to 4 h following DNA extraction. Using real-time PCR, results can be available as quickly as 2 h after DNA extraction. For both applications, kits are available from different manufacturers. For several bacterial strains, sequences of plasmids causing antibiotic resistances are known. Therefore, PCR detection of specific sequences can reduce the analysis time and accelerate the therapy. Example: Testing of Enterococcus against vancomycin and teicoplanin resistance with other methods (e.g., VITEK, bioMerieux) can lead to unclear or variable results. By PCR four different plasmids that encode for high-level resistance against vancomycin and teicoplanin (plasmid VanA), high-level resistance against vancomycin and variable resistance against teicoplanin (plasmid VanB), or lowlevel resistance against vancomycin, and practically no resistance against teicoplanin (plasmids VanC 1, VanC 2/3) can be detected specifically (Dutka-Malen et al., 1995; Ballard et al., 2005).

APPLICATIONS

TABLE 1.7 Manufacturer

Commercially Available Kits for Microsatellite Analysis Animal Species

Number of Markers

Allelic Ladder Available?

Homepage

Applied Biosystems

Cattle Horse Dog

11 16 10

no no no

www.appliedbiosystems. com/

Finnzymes

Cattle

—

no

www.finnzymes.fi

Biotype

Pork

—

yes

www.biotype.de

1.4.2

27

Microsatellites and Variable Number of Tandem Repeats

DNA Profiling By microsatellite analysis DNA fingerprints can be made from almost all mammals. Primers are published for a wide variety of mammals, birds, and fishes. For some animal species used in agriculture, commercial PCR kits are available (Table 1.7); for other species, sets of markers are available some of which were tested in ring trials (sheep, goat, pig). In contrast to the analysis of human DNA, currently no allelic ladders are commercially available for these marker sets except for porcine microsatellite analysis with a kit from Biotype. For proof of the identity of animals grown for food production or of the origin of meat, DNA profiling can be used based on several concepts: 1. DNA profiles from all animals used for breeding are collected within a database. From every offspring a DNA profile is generated, compared with the profile of the parents, and put into the database. For every marker analyzed, the offspring must have one common allele with the biological father and one with the biological mother (Figure 1.16). When, for example, the animal is sold or transported and there is any doubt about the identity, a new profile can be generated from the blood, tissue, or hairs. After the animal has been slaughtered, a sample is taken from the meat and the DNA profile is compared with the existing profile. 2. From all sires used for breeding the DNA profile is collected in a database. With a specific type of eartag, a small piece of tissue is collected during the collecting process. This tissue sample is stored until the animal loses the eartag. Before the animal gets a new eartag, the DNA is extracted from the tissue collected earlier and compared with the current DNA profile and/or with the profile of the putative father. After the animal has been slaughtered, the DNA profile from the meat can be compared with the profile of the tissue sample collected earlier. 3. From all newborn animals, tissue is collected as described above. DNA profiling is performed only if there is any doubt about the identity of the animal. The sires used for breeding are not tested in this concept. With all these concepts, controls can be exercised randomly. Depending on the statistical concept, the costs for the analysis can be reduced, but there remains a

28

MOLECULAR BIOLOGY LABORATORY LAYOUT

FIGURE 1.16 Paternity testing. DNA profiles from offspring (a), dam (b), and two possible fathers (c, d) with two markers specific for canine DNA (PEZ10 and FH2361) are shown. At PEZ 10, offspring and dam share allele 283. Therefore, allele 299 present in the offspring has to be present in the biological father (present only at sample c). At FH2361, offspring and dam share allele 338. Therefore, allele 342 present in the offspring has to be present in the biological father (present at both samples c and d). From marker PEZ10, sample d could not be from the biological father of sample a.

probability of up to 99% that a wrong declaration can be detected. DNA for the analysis can be extracted from EDTA blood, heparine blood, blood spots on filter paper, tissue/ meat, bones, or teeth. It is also possible to extract DNA from hairs with roots, but hairs can be contaminated with DNA from the saliva of other animals or with feces. With most farm animals, DNA extracted from buccal swabs contains less genomic DNA from the animal. In ruminants a swab contains a large amount of saliva and bacteria from the rumen but a small amount of cells from the mucosa. In addition, PCR inhibitors from food may be present. The quantity and the quality of DNA extracted from meat should be determined by OD measurement and/or agarose gel control. Applying PCR with multiplex kits, the amount of DNA suggested by the manufacturer should be added to the PCR to provide balanced DNA profiles (Figure 1.17). Population Genetic or Animal Breed Determination For some reason it may be necessary to determine whether an individual animal is part of a specific population or breed. The first step in answering this question is to define the population or breed and identify all animals that are typical for the respective group. DNA profiles can then be generated and compared. Example 1: Information was requested from a breeding organization, the Aberdeen Angus Society, as to which sires were used most often for artificial insemination and natural insemination. Sperm samples were collected from these sires and also from

APPLICATIONS

29

FIGURE 1.17 DNA profile with 11 markers specific for bovine DNA. From left to right: (a) TGLA 227, BM2113, TGLA53, ETH10, SPS115; (b) TGLA126, TGLA122, INRA23; (c) ETH3, ETH225, BM1824.

other breeds present in the same region that could deliver meat which could wrongly be declared to be Aberdeen Angus (AA) meat. The DNA was extracted and analyzed with about 120 markers. The allele frequencies of these 120 markers from the AA samples and non-AA samples were compared. Markers were identified that show typical alleles of the AA samples and other alleles of the non-AA samples. With these markers, blind tests were performed with meat samples of known origin. All of these samples were typed correctly. Example 2: At a control on a farm, several animals without eartags were found. Normally, all such animals have to be slaughtered. According to documents of the farmer, the biological mothers of some of the animals were still on the farm, some were sold or slaughtered, but closely related animals were still present. Blood samples were collected from all animals on the farm and DNA profiles were generated. Parallel samples from other animals of the same breed were collected and analyzed. By comparison of the DNA profiles of the offspring with those of the putative parents, some of the animals could be identified. Profiles from the other animals were compared with profiles of the closely related animals of the putative mothers and the unrelated animals. The likelihood that these animals could be offspring of the animals as documented by the farmer was calculated. Example 3: A fish retailer declared smoked salmon to be wild from salmon captured in a specific river. The food analyst has doubts about this declaration; he thinks that the meat is from farmed salmon. In this case reference samples must be collected from salmon captured in the river and from salmon raised on all farms. After microsatellite analysis, allele frequencies have to be determined and the likelihood has to be calculated whether the salmon can be from the river population or from one of the farm populations. Determination of Basmati Rice Basmati rice is a long-grain rice that grows in the Himalaya region of India and Pakistan. Actually, 17 Basmati varieties are recognized (Table 1.8). Since Basmati rice

30

MOLECULAR BIOLOGY LABORATORY LAYOUT

TABLE 1.8

Approved Basmati Rice Varieties

Variety from India

Variety from India or Pakistan

Variety from Pakistan

Basmati 217 Ranbir Basmati 370 Basmati 386 Taraori Dehradun Pusa Kasturi Mahi Suganda Haryana Punjab

Basmati 370

Basmati 370 Super Kernel Basmati 198 Basmati 385

is more expensive than other long-grain rice and upon import into the European Union (EU) lower tax rates have to be paid, controls are necessary as to whether a sample contains only Basmati rice or a mixture with other rice varieties. A DNA microsatellite method was developed by the Food Standards Agency (FSA) in London to check retail sales of Basmati rice in the UK market (FSA, 2004). In the UK, Basmati is defined by a code of practice (COP) agreed to among the rice industry, retailers, and the enforcement authorities. The COP lists the varieties that can be described as Basmati (Table 1.8) and outlines a specification for the rice in which a realistic level of unavoidable contamination with non-Basmati rice varieties is set. Contamination can happen during harvesting of the rice, transport to local traders, and export into the EU. The contamination allowed following COP is a maximum of 7%. Because the detailed analysis protocol may change in the near future and because it can be retrieved from the FSA homepage on the web, only the principle of analysis is explained. About 100 g of rice is milled with a coffee grinder. From this powder DNA should be extracted in triplicate. From each of the three DNA samples, PCR is performed with at least 10 microsatellite markers (actually, the list of markers RM1, RM16, RM44, RM55, RM171, RM201, RM202, RM223, RM229, RM241). The genotypes detected are compared with known genotypes of the approved varieties. In mixtures, contamination with nonapproved varieties is documented. In 2006 a ring trial of the quantitative determination of non-Basmati rice varieties in a mixture with Basmati rice varieties was organized by the FSA. The results from 9 of the 11 participating laboratories differed by less than 0.6% from the weighted mixtures; two laboratories had bigger differences. This test demonstrated that laboratories with experience in microsatellite analysis can deliver reliable results in analyses of Basmati rice or mixtures of Basmati- and non-Basmati varieties. In Figure 1.18 profiles from Basmati rice and a mixture of Basmati and non-Basmati varieties are shown.

APPLICATIONS

31

FIGURE 1.18 DNA profiles with two markers specific for rice DNA (left marker, RM171; right marker, RM55: (a, b) two different mixtures of rice varieties Pusa and Dehradun; (c) pure sample of rice variety Pusa; (d) pure sample of rice variety Dehradun.

Similar analyses can also be made on other rice varieties, such as Jasmine rice. Authentic samples of typical Jasmine rice and of other rice varieties that can be used to minic Jasmine rice have to be collected and analyzed with a broad range of markers. The next step is performed similar to that described earlier for the establishment of a breed-specific analysis for Aberdeen Angus cattle. Basmati and Jasmine rice are both famous for their specific flavor, caused by a mutation at the putative betaine aldehyde dehydrogenase 2 (BAD2) gene, which can also be determined by DNA analysis (Bradbury et al., 2005a, b). As the predisposition “flavor” is recessive, only rice grains that are homozygous for the mutation develop the flavor (Figure 1.19) Identification of Bacterial Strains by VNTR Analysis For some bacterial strains, sequencing of 16S rRNA or real-time PCR with specific primers cannot provide all the information needed. Especially if infectious pathways have to be followed, subtyping with VNTR is the method of choice. For several bacterial species, VNTR (and STR) analysis methods are described that can be used. In Figure 1.20 DNA profiles received by VNTR analysis of Francisella strains are shown. The method can also be used to determine whether reference strains are pure or contain a mixture of two or more substrains (Bystr€ om et al., 2005).

32

MOLECULAR BIOLOGY LABORATORY LAYOUT

FIGURE 1.19 DNA profiles with PCR product coding for fragrance. Rice sample contains (a) fragrant and nonfragrant grains; (b) only fragrant grains; (c) only nonfragrant grains.

1.4.3

Real-Time PCR

Determination of DNA Concentrations As described above, for several applications it is necessary to determine the DNA concentration before PCR. For DNA concentrations higher than 10 ng/mL, measurement of the DNA concentration by a determination of OD 260/280 with a photometer will lead to reliable results. These amounts of DNA can be expected after DNA

FIGURE 1.20 DNA profiles with two markers specific for Francisella DNA (left marker, FtM08; right marker, FtM21). Strain (a) can be distiguished from strains (b) and (d) by marker FtM08 and from strain (c) by marker FtM21. Strain (b) can be distiguished from strains (a), (c), and (d) by marker FtM08 and from strains (c) and (d) by marker FtM21. Strain (c) can be distiguished from strains (b) and (d) by marker FtM08 and from strains (a), (b), and (d) by marker FtM21. Strain (d) can be distiguished from strains (a), (b), and (d) by markers FtM08 and FtM21.

APPLICATIONS

TABLE 1.9

33

DNA Yield of Biological Material

Biological Material EDTA or heparin blood Muscle tissue, liver tissue Plant material Rice grains Processed food products

Average DNA Concentration of Optimally Stored or Fresh-Drawn Samples 5 ng/mL blood 40–70 mg/100 mg tissue 1–30 mg/100 mg tissue 15–300 ng/ g grains 0.1–5 mg/100 mg product

extraction from blood, fresh tissue samples, and buccal swabs with high numbers of attached cells. In Table 1.9 ranges of DNA concentrations are listed. By OD measurement, all DNA present in a sample is detected. This should be no problem for fresh-drawn or optimally stored blood or tissue samples because in this case it is expected that only DNA from the donor of the sample is present. For older or nonoptimally collected or stored buccal swabs or decomposed samples, it can happen that a part of the measured DNA comes from bacteria or fungi. In addition, for those samples the DNA yield expected will be lower than that listed in Table 1.9. Other biological material, such as teeth, bones, or connective tissue, or boiled, grilled, or smoked meat, contains less DNA. In this case a determination of DNA concentration by real-time PCR is necessary. Using a real-time instrument it is possible to determine the total amount of DNA in a sample or the amount of DNA from a specific species. Total DNA Kits are available from some suppliers to determine the concentration of DNA or RNA by staining with PicoGreen (e.g., Quant-iT PicoGreen dsDNA Assay Kit, Invitrogen). The kit also contains a control DNA of known concentration. Normally, analysis with this kit should be performed with a fluorometer, but it is also possible to perform the analysis on some real-time instruments. For this, the DNA solution is mixed with a very low concentration of PicoGreen and a melting curve is analyzed (Figure 1.21). By comparing the signal intensities of controls and samples at a specific temperature, the DNA concentration can be determined. DNA from Vertebrates For the differentiation of DNA from bacteria and the DNA from vertebrates, analysis can utilize primers specific for genomic or mitochondrial DNA. Real-time PCR with primers specific for conserved sequences of the mitochondrial cytochrome b gene detects DNA from all mammals and most fishes. For the detection of chondrichtyes (ray, shark) and prawns, other primers have to be chosen. The analysis can be performed with unlabeled primers. Amplicons can be detected by SybrGreen. For most instruments, control samples with known concentrations can be used as markers, and using these the DNA concentration of samples is calculated automatically. DNA from Specific Species For the analysis of genomic human DNA, kits from two suppliers (i.e., Applied Biosystems, Promega) are currently available. With these

34

MOLECULAR BIOLOGY LABORATORY LAYOUT

FIGURE 1.21 Different amounts of DNA were analyzed using a melting curve from a realtime PCR instrument (ABI 7900). A mixture of genomic DNA and PicoGreen melted. During cooling the DNA rehybridizes and PicoGreen intercalates with the DNA. Measured fluorescence is relative to the amount of DNA.

kits the detection of concentration of total human DNA or male human DNA is possible. Dilutions of the control DNA contained in the kit allow calculation of the concentration down to 6 pg/mL, which corresponds to one genome equivalent. Results of analysis with a Quantifiler kit from Applied Biosystems are shown in Figure 1.22. Several publications describe assays for genomic animal DNA. Analysis of short interspersed nuclear elements (SINEs) allows specific detection of DNA from many species, including cattle, horse, pig, deer, and dog. SINEs are repeated, unblocked, and dispersed throughout the genome sequences. They represent retroposons (included in the genome transcripts of intracellular RNA) and constitute more than 20% of the genome of humans and other mammals. Unique sequences could be identified for every species and used for the development of a species-specific PCR assay (Walker et al., 2003). For this application the laboratory has to prepare its own control samples with the DNA extracted from reference samples. Specific detection of DNA can also be carried out by real-time PCR with primers specific for microsatellites. Examples of microsatellites that are specific to one species are listed in Table 1.10. In principle, all DNA sequences that are known as species specific can be used for the development of an assay for the detection of DNA from specific animals, plants, or bacteria. In control experiments it must be shown that no other DNA fragments are amplified using the primers chosen. Generally, assays with primers and a specific

APPLICATIONS

35

FIGURE 1.22 Quantification of human DNA with a quantifiler kit. Serial dilutions (1:3) of human DNA leads to corresponding differences in Ct values (b) which should be located on a straight line (a) with a regression coefficient as high as 0.99.

labeled probe will be more specific and show fewer background signals than will assays with unlabeled primers and detection with SybrGreen. Assays specific for mtDNA are more sensitive than assays for genomic DNA. They can be used if analysis of mtDNA is necessary (e.g., for sequencing of human HV I and II regions) (see Chapter 6). For all assays specific for genomic DNA, the limit of detection should be 6 pg DNA per reaction for assays specific for single-copy genes (one copy per haploid genome). If the self-developed assay is less sensitive, the PCR conditions may not be optimized or the reference DNA will have a lower concentration than expected or measured. If the assay is more sensitive, it may be because the reference DNA has a higher concentration than expected or the assay detects a multicopy gene (more than one copy per haploid genome). Degradation of the DNA In most real-time PCR assays, fragments of 60 to 100 bp are amplified. For degraded DNA these short fragments can be overrepresented. This TABLE 1.10 Species-Specific Microsatellites Species Horse Cattle Pig Dog Cat

Microsatellite Marker

Fragment Size (bp)

AHT 4 TGLA 53 SW 0155 PEZ 1 F85

140–160 150–180 150–160 96–120 183–301

36

MOLECULAR BIOLOGY LABORATORY LAYOUT

can lead to failed microsatellite or other PCR analysis even when the optimal amount of DNA was given to the PCR following the results of real-time PCR analysis. To overcome this problem, species-specific PCR assays can be developed with longer fragments. The size of these fragments should correspond that of analysis by real-time PCR. Comparison of Ct values for shorter (e.g., 100 bp) and longer (e.g., 300 bp) fragments provides a hint of degradation of the DNA in the sample. Animal Species Determination (Known Species With or Without Quantification) Animal species determination in processed material or food products can be performed by PCR amplification of mitochondrial DNA followed by sequencing analysis of the product (see Chapter 6) or by RFLP. RFLP analysis can be successful only if the DNA detected is from a species with a known RFLP pattern. In addition, if DNA from only one animal is detected by sequencing analysis or RFLP, there is actually no guarantee that DNA from other species is also present at very low amounts. Low contaminations can also be detected by real-time PCR. For sequencing analysis or RFLP, PCR primers are used that amplify DNA from almost all mammals. In contrast, for real-time PCR, primers specific for each species have to be used. Example 1: DNA Analysis of Carcass Meal After DNA extraction, PCR with universal primers should be performed to exclude PCR inhibition. In addition, PCR with primers specific for species that are allowed to be processed as carcass meal (e.g., pig, chicken) and with primers specific for species that are not allowed to be processed (e.g., cattle, sheep) is carried out. Example 2: Analysis of Raw Gelatin Gelatin can be produced from both bovine and porcine material. In Europe, gelatin from pig is preferred; in other countries, gelatin from pigs cannot be used, for ethical or religious reasons. To determine whether gelatin is produced from pigs or cattle, a primer pair should be used that amplifies DNA from both species with the same efficiency. The primers have to be located in a conserved region of the mitochondrial genome, with identical sequences for pig and cattle. In addition, two probes must be designed, one specific for bovine DNA labeled with one dye (e.g., FAM) and one specific for porcine DNA labeled with another dye (e.g., VIC). This assay design has the advantage that controls are included. Since DNA extracted from gelatin has to be amplified with the primers, a negative result means that an insufficient amount of DNA was extracted from the gelatin. For pure gelatin samples one signal (FAM or VIC) is detected; for mixed samples both signals have to be analyzed. If both signals are detected, the result of the analysis should be interpreted carefully because how strong the DNA was degraded during the production process of the gelatin cannot be calculated. When analyzing cow milk products for gelatin, bovine DNA is detected in every case and it is not possible to determine whether the bovine DNA extracted is from the gelatin or from the cells of the milk. Therefore, the detection of porcine DNA cannot exclude the admixture of bovine gelatin. For milk products from sheep or goat milk, bovine DNA can be analyzed from intentional or unintentional contamination with cow’s milk or from the addition of bovine gelatin.

APPLICATIONS

37

Example 3: Analysis of Chondroitine Sulfate Chondroitine sulfate can be produced from raw material of cattle, pigs, chicken, or sharks. With universal primers for cytochrome b used for species determination, the amplification of DNA from cattle and pigs is more efficient than the amplification of DNA from chicken. DNA from shark is not amplified using these primers. Therefore, universal primers for the detection of DNA from all shark species have to be designed. Four independent realtime PCR reactions have to be carried out for the analysis. As described for the analysis of gelatin, it cannot be guaranteed that DNA will be degraded during the production of chondroitine sulfate from the various raw materials. Therefore, the quantitative results of mixed samples have to be interpreted very carefully. Amplified PCR products can be used for sequencing (e.g., to determine shark species, if necessary). ^ e A sample of duck liver p^ate was Example 4: Analysis of Duck Liver Pat declared as a mixture of 20% duck meat and liver and 80% porcine meat, liver, and fat. For PCR with universal primers for cytochrome b, amplification from porcine DNA is more efficient than that from duck DNA. Therefore, in a first sequencing analysis, no DNA from duck can be detected. However, real-time PCR with primers specific for DNA from pig and duck can confirm the ingredients declared. DNA/RNA Contamination on Surfaces and DNAse/RNAse Contamination For some applications or for quality assurance, it can be necessary to control whether instruments, surfaces, reaction tubes, or used buffers are contaminated with DNA, RNA, DNAse, or RNAse. For the detection of DNAse or RNAse, known amounts of DNA or RNA are incubated with the suspected buffers or in the tubes at 37 to 40 C for 30 to 60 min. After this, real-time PCR is performed with the untreated DNA or RNA as control. If the analysis of incubated DNA or RNA shows a higher Ct value than the control, and the internal process control gives no hint of inhibition, DNAse or RNAse was present. For the detection of DNA or RNA contaminations, humid swab samples (in TE or phosphate-buffered saline buffer) have to be collected from surfaces or instruments and the nucleic acids extracted into the TE buffer. The buffer can also be incubated in the suspected reaction tube or pipette tip. Real-time PCR is performed with universal primers specific for cytochrome b, human DNA, or any other DNA/RNA that is identified as a source of contamination in the laboratory. Detection of SNPs and InDel SNPs and InDel (Insertion/deletion) are distributed equally over the total genome of animals, plants, fungi, and bacteria. They have very low mutation rates and therefore can be used for animal or plant species determination, identification of fungi or bacterial strains, proof of identity, and authenticity testing. Detection of SNPs can be carried out on both genomic and mitochondrial DNA. For high-throughput screening of SNPs, techniques such as MALDI-TOF-MS (matrix-assisted laser desorption–Ionization time-of-flight mass spectroscopy) are used. Examples of projects are population studies/gene diagnostics, clinical studies, analysis of mitochondrial DNA or Y- chromosomal SNPs, and DNA forensics. The

38

MOLECULAR BIOLOGY LABORATORY LAYOUT

advantages of this technique are possible multiplex analyses, competitive costs per analysis (a few cents per SNP for multiplex analysis), high throughput (production of 10,000 data points per hour), and the detection of SNP and InDel in one assay or reaction. The disadvantages are that only the detection of known mutations is possible, the fixed costs are high, and a complex infrastructure is necessary for analysis. In laboratories with lower throughput, SNP detection for analysis of food or food products can be performed by real-time PCR or sequencing analysis. Detection of Genetically Modified Organisms Due to legislative guidelines (e.g., EU Guideline 90/220), it is necessary to carry out qualitative and quantitative detection of genetically modified organisms (GMOs). If GMOs are imported into the EU, the product has to be declared as a GMO and information as to how the GMO can be identified must be available. However, it has been shown in the past that this does not always happen (e.g., import of nondeclared GMO rice into the EU from the United States in 2006). Since GMO and non-GMO products are stored, transported, and processed in the same facilities, contamination of non-GMO products by GMO products can occur unintentionally. Therefore, quantitative analysis is performed to determine if the GMO content is higher than a defined threshold. For the quantitative analysis of several GMOs, reference material with different GMO/non-GMO ratios is commercially available. By an analysis of a housekeeper gene specific for the plant species, the total amount of DNA present in the single reactions could be determined. PCR is performed in parallel specifically for the sequences of the transgenic region that have been published (e.g., a promoter of cauliflower mosaic virus). From the ratio of the Ct values of the two genes of the reference samples with a known content of GMO and the samples to be analyzed, the GMO content of the samples analyzed can be determined. De novo detection of GMO is more difficult. If there is any doubt that a sample contains transgenic DNA sequences, an analysis has to be performed specifically for all known sequences used for the development of the GMO. This analysis should be done in specialized laboratories. After the new transgenic sequence is detected and published, the analysis can also be performed in other laboratories.

REFERENCES Ballard SA, Grabsch EA, Johnson PDR, Grayson ML (2005). Comparison of three PCR primer sets for identification of vanB gene carriage in feces and correlation with carriage of vancomycin-resistant enterococci: interference by vanB-containing anaerobic bacilli. Antimicrob. Agents Chemother., Jan., 49(1):77–81. Bradbury L, Robert H, Jin Q, Reinke RF, Waters DLE (2005a). A perfect marker for fragrance genotyping in rice. Mol. Breed., Nov., 16(4):279–283. Bradbury LMT, Fitzgerald TL, Henry RJ, Jin Q, Waters DLE (2005). The gene for fragrance in rice. Plant Biotechnol. J., 3(3):363–370. Bystr€om M, B€ocher S, Magnusson A, Prag J, Johansson A (2005). Tularemia in Denmark: identification of a Francisella tularensis subsp. holarctica strain by real-time PCR and

REFERENCES

39

high-resolution typing by multiple-locus variable-number tandem repeat analysis. J. Clin. Microbiol., Oct., 43(10):5355–5358. Dutka-Malen S, Evers S, Courvalin P (1995). Detection of glycopeptide resistance genotypes and identification to the species level of clinically relevant enterococci by PCR. J. Clin. Microbiol., 33:24–27. Gallagher ES (1994). A PCR-based sex-determination assay in cattle based on the bovine amelogenin locus. Anim. Genet., Dec. 25(6):425–427. FSA (Food Standards Agency) (2004). Survey on Basmati Rice. FSIS 47/04. Food safety and Inspection service, U.S. Department of Agriculture, Washington, DC. Gilbert W (1978). Why genes in pieces? Nature, Feb. 9, 271(5645):501. Griffiths R, Double MC, Orr K, Dawson RJG (1998). A DNA test to sex most birds. Mole. Ecol., 7(8):1071–1075. ILAC (International Laboratory Accreditation Cooperation) (2002). Guidelines for Forensic Science Laboratories. ILAC-G19:2002. ILAC, silver water, Australia. Mullis KB (1990). Target amplification for DNA analysis by the polymerase chain reaction. Ann. Biol. Clin. (Paris), 48(8):579–582. Qin Y, Liu X, Zhang H, Zhang G, Guo X (2006). Segregation and linkage analysis of 75 novel microsatellite DNA markers in pair crosses of Japanese abalone (Haliotis discus hannai) using the 50 -tailed primer method. Mar. Biotechnol. (NY), Sept.–Oct. 8(5):453–466. Schubbert R (2002). Detection of microvariants and large alleles in microsatellite systems SE33/ ACTBP 2, FGA/FIBRA and D21S11. Presented at the 14th International Symposium on Human Identification. Urdiain M, Domenech-Sanchez A, Albertı S, Benedı VJ, Rosselló JA (2004). Identification of two additives, locust bean gum (E-410) and guar gum (E-412), in food products by DNAbased methods. Food Addit. Contam., 21(7):619–625. Walker JA, Hughes DA, Anders BA, Shewale J, Sinha SK, Batzer MA (2003). Quantitative intra-short interspersed element PCR for species-specific DNA identification. Anal. Biochem., May 15, 316(2):259–269. Zinovieva N, Palma G, M€uller M, Brem G (1995). A rapid sex determination test for bovine blastomeres using allele-specific PCR primers and capillary PCR. Theriogenology, 43:365.

CHAPTER 2

Polymerase Chain Reaction HERMANN BROLL € r Risikobewertung, Berlin, Germany Bundesinstitut fu

2.1

INTRODUCTION

In 1983, while driving on a moonlit California road, Kary Mullis from the Cetus Corporation came up with a simple and very elegant concept: the basic idea for the polymerase chain reaction (PCR), which solved a core problem in genetics: how to make copies of a strand of DNA in which you are interested. In 1993, Kary Mullis received the Nobel Prize in Chemistry, emphasizing the great importance of this very simple idea. At the beginning the method was slow, expensive, and imprecise. PCR turns the job over to the biomolecules that nature uses for copying DNA: two primers, which comprise the beginning and end of the DNA stretch to be copied; an enzyme called polymerase, which walks along part of the DNA, reading its code, and assembling a copy; and plenty of the DNA building nucleotides that the polymerase needs to make the copy. PCR is a technique that amplifies a specific DNA template to produce identical DNA fragments in vitro. To get enough material for subsequent analysis, traditional methods of cloning a DNA sequence into a vector and replicating it in a living cell usually require days or even weeks of work. In contrast, amplification of DNA sequences by PCR requires only minutes up to a few hours. Whereas most biochemical analyses, including nucleic acid detection with radioisotopes or fluorescence labels, require the input of significant amounts of biological material, the PCR process requires very little starting material. Thus, PCR can achieve more sensitive detection and higher levels of amplification of specific sequences in less time than can other techniques used so far. These features make the technique extremely useful not only in basic research but also in applied science, including genetic authenticity testing, forensics, industrial quality control, and in vitro diagnostics. Simple PCR has become ordinary in many molecular biology labs, where it is used to amplify DNA fragments and detect DNA or, after reverse Molecular Biological and Immunological Techniques and Applications for Food Chemists Edited by Bert Popping, Carmen Diaz-Amigo, and Katrin Hoenicke Copyright  2010 John Wiley & Sons, Inc.

41

42

POLYMERASE CHAIN REACTION

transcription, RNA sequences in a cell or environment. Nonetheless, PCR has evolved far beyond easy amplification and detection, and many further developments of the original PCR method have been described. In this chapter we provide an overview of the PCR method, applications, and optimization. The reference section will provide a valuable guide to researchers who require more comprehensive information. 2.2

ORIGINAL PCR

The PCR process was developed originally to amplify short segments of a longer DNA molecule (Saiki et al., 1985). A typical amplification reaction includes the target DNA, a thermostable DNA polymerase, two oligonucleotide molecules used as primers, deoxynucleotide triphosphates (dNTPs), a reaction buffer, and magnesium. Once combined, the reaction is placed in a thermal cycler, an instrument that subjects the reaction to a series of temperatures for varying amounts of time. This series of temperature and time adjustments is referred to as one cycle of amplification. Theoretically, each PCR cycle doubles the amount of targeted sequence in the reaction. Today, the term amplicon is used widely, although it has been defined in conjunction with plasmids containing the origin of replication and the cleavage/packaging site of herpesvirus genomes since 1984. Theoretically, 10 cycles multiply the amount of products amplified by a factor of about 1000 in a matter of hours; 20 cycles, by a factor of more than 1 million (Figure 2.1 and Table 2.1). PCR is carried out in cycles, each of which can be separated into three phases, including steps for template denaturation, primer annealing, and primer extension

target cycle 1

cycle 2

cycle 3

cycle 4

35 to 45 cycles

Exponential amplification: 21 = 2 copies

22 = 4 copies

23 = 8 copies

24 = 16 copies

235 = 34 billion copies

FIGURE 2.1 Theoretical amplification efficiency in PCR. From a theoretical point of view, the amount of target DNA is duplicated in each individual cycle, resulting in 34 billion identical copies of the same DNA after 35 cycles.

ORIGINAL PCR

TABLE 2.1

43

Amplification Efficiency in PCR a

Theory Average amplification factor per cycle Amplification after: 10 cycles 20 cycles 30 cycles 40 cycles

In Practice 2

1.65

1.60

1.55

1.50

1.45

103 106 109 101

150 2.2  104 3.3  106 5  108

110 1.2  104 1.3  106 1.5  108

80 6.4  103 5  105 4.1  107

58 3.3  103 1.9  105 1.1  107

41 1.7  103 7  104 2.8  106

100%

82.5%

80%

77.5%

75%

72.5%

a

Depending on amplification factors in PCR, different amounts of PCR products are amplified after a certain number of cycles. Because the factor is influenced by a number of different factors not known in advance, PCR is in general a qualitative procedure. However, in practice, an amplification factor of about 75 to 80% is reachable.

(Figures 2.2 and 2.3). The initial step denatures the target DNA by heating it to 94 C or even higher for a few seconds up to 2 min. In the denaturation process, the two closely intertwined DNA strands separate from one another, producing the necessary singlestranded DNA (ssDNA) template for replication by the thermostable DNA polymerase. In the next step of a cycle, the temperature is reduced to approximately 40 to 60 C. At this temperature the oligonucleotide primers can form stable associations (anneal) with the denatured target DNA and serve as an initiation for the DNA polymerase to

FIGURE 2.2 Principle of PCR. As indicated PCR is running in cycles. Each cycle is separated in three individual steps (1, denaturation; 2, annealing; 3, polymerization), resulting in doubling the amount of target DNA. A typical PCR temperature–time profile is given in Table 2.2, and a standard PCR setup in Table 2.3.

44

POLYMERASE CHAIN REACTION 1,60E+06 1,40E+06

fluorescence

1,20E+06 1,00E+06 8,00E+05 6,00E+05

exponential phase

4,00E+05 2,00E+05 0,00E+00 0

5

-2,00E+05

10

15

20

cycles

25

30

35

40

FIGURE 2.3 Process of amplification. Two samples containing different amounts of target DNA were analyzed by PCR. In conventional PCR the amount of products amplified from samples indicated an almost similar amount at the endpoint. However, the amplification curve of the sample containing more target molecules left the background earlier, indicating a higher amount of target DNA introduced into the PCR.

synthesize a new DNA strand. This step lasts approximately 15 to 60 s. Finally, the synthesis of new DNA begins as the reaction temperature is raised to the optimum for the DNA polymerase. For most thermostable DNA polymerases, this temperature is in the range 70 to 74 C. The extension step lasts approximately 1 to 2 min. The next cycle begins again with a return to 94 C for denaturation. The original procedure used the DNA polymerase I Klenow fragment from Escherichia coli, but it had the significant drawback that between each cycle the DNA had to be denaturated and new enzyme added. Today the enzyme of choice is the Taq DNA polymerase isolated from the bacterium Thermus aquaticus YT1, which grows in the hot springs of Yellowstone National Park. The enzyme works optimally at 72 C and can withstand heating to over 90 C for short periods of time. Therefore, no more enzyme addition is necessary during 20 cycles of PCR (Saiki et al., 1988). Each step of the cycle should be optimized for each template–primer combination. In certain cases in which the temperature during the annealing and extension steps are similar, the two steps can be combined into a single step in which both primer annealing and extension take place. After 20 to 45 cycles, the products amplified may then be analyzed for size, quantity, sequence, and so on, or used in further experimental procedures. A typical PCR temperature–time profile is given in Table 2.2 and a standard PCR setup in Table 2.3. 2.3

NESTED PCR

A typical problem that occurs in PCR analysis is the very low presence of template DNA isolated from the various samples in the reaction. Although in theory only one

NESTED PCR

TABLE 2.2

45

Example Temperature–Time Program Temperature ( C)

Step Initial denaturation a Denaturation Annealing Extension Final extension Hold

95 95 42–65 b 72 72 4

Time (min) 2  0.5–1 0.5–1 1 min/kb b, c 7 Indefinite

Number of Cycles 1 25–45 1 1

a The thermal cycling protocol has an initial denaturation step where samples are heated at 95 C for 2 min to ensure that the target DNA is denatured completely. This step is also necessary for the activation of today’s most used hot-start Taq DNA polymerase, which is inactive until an initial heating step at 95 C. b Annealing temperature needs to be optimized for each primer set based on the primer melting temperature. c The extension time should be at least 1 min per kilobase pair of target DNA. Typically, target DNA smaller than 1 kb uses a 1-min extension; for target DNA >1 kb, 2 or even more minutes are necessary.

target molecule should be sufficient to generate a positive signal in PCR, in reality it is often not the case. Above 40 cycles, an increase in the number of cycles will not give a satisfying result because of a tendency to generate more background amplification products. A way to overcome this problem and generate a positive signal even from a very low level of target DNA is known as nested PCR. The principle is quite simple and

TABLE 2.3

Standard PCR Reagents a

Reagent

Volume

Final Concentration

Sterile water 10  PCR buffer b MgCl2 dNTP solution, 10 mmol/L Primer 1 c Primer 2 c Taq DNA polymerase, 5 IU/mL Template DNA Final volume

Variable 5 mL Variable 1 mL Variable Variable 0.4 mL Variable 50 mL

1 1.0–2.5 mmol/L 0.2 mmol/L 0.1–1.0 mmol/L 0.1–1.0 mmol/L 2 IU 0.1–1.0 mg

a

A standard PCR is running at a volume of 50 mL. However, if the procedure is optimized, the total volume can be decreased even to 10 mL. b Taq buffer (10) 600 mM Tris-HCl (pH 9.5, room temperature), 150 mM (NH4)2SO4, and 20 mM MgCl2. c The primer sequences should have the following characteristics wherever practicable: .

Length of each primer ¼ 18 to 30 nucleotides.

.

Optimal annealing temperature 60 C (established experimentally; i.e., estimated melting temperature 65 C).

.

GC/AT ratio ¼ 50 : 50 if possible, or as close to this ratio as possible. Avoid concentration of G’s and C’s in short segments of primers (internal high stability).

.

No 30 -end complementarity, to avoid primer–dimer formation.

.

No internal secondary structure.

.

No possible dimer formation with other primers used in multiplex PCR.

.

46

POLYMERASE CHAIN REACTION

based solely on two independent subsequently performed PCR routines. As a target for the second PCR, the amplified products generated in the first PCR are used. The primer pair used in the second PCR needs to be complementary and anneals within the DNA sequence amplified first. By doing this, the background DNA amplified is reduced drastically, due to the selection of the primer pair used in the second PCR, resulting in more specific amplified product afterward. In addition, the sensitivity is increased.

2.4

MULTIPLEX PCR

Multiplex PCR is a variant of PCR that enables simultaneous amplification of several targets of interest in one reaction by using more than one pair of primers. A primary reason to run multiplex PCR is to save money and time when analyzing a lot of samples in parallel. Since its description in 1988 by Chamberlain et al. this method has been employed in many areas of DNA testing, including analyses of deletions, mutations, and polymorphisms, or quantitative assays and reverse transcription PCR. Typically, it is used for genotyping applications where simultaneous analysis of multiple markers is required, the detection of pathogens or the parallel identification of genetically modified organisms (GMOs), or for microsatellite analyses. Multiplex assays can be tedious and time consuming to establish, requiring lengthy optimization procedures. However, attempts to combine more than three to five primer pairs in just one single reaction often fails, because the limit of detection for one or the other target is decreased significantly. In particular, if the presence of different targets varies drastically, multiplexing becomes very complicated. It seems to be very tricky in practice to establish multiplex PCR and is therefore not recommended.

2.5

PCR CONTROLS

In theory, PCR is able to generate a positive signal if only one or a very few target molecules are present in the reaction. Therefore, it is a very powerful tool in various approaches to identify trace amounts of agents such as viruses, bacteria, or even GMOs. This unique advantage, on one hand, is also a disadvantage on the other, due to the possibility of contamination, resulting in false-positive signals. To avoid such cases or at least to identify such contaminations, appropriate positive (analyte present) and negative (analyte absent) controls should be included at each step of the analysis. Some controls should follow the samples to be analyzed in the successive steps of an analysis. Additional controls should be included at regular intervals and always if one of the other controls does not yield the results expected and when contamination is suspected. An exhaustive list of various controls applied in PCR follows. 1. Positive DNA target control: reference DNA or DNA extracted from a certified reference material or known positive sample representative of the sequence or organism under study. The control is intended to demonstrate what the result of analyses of test samples containing the target sequence will be.

ANALYSIS OF PCR PRODUCTS

47

2. Negative DNA target control: reference DNA or DNA extracted from a certified reference material or known negative sample not containing the sequence under study. The control is intended to demonstrate what the result of analyses of test samples not containing the target sequence under study will be. 3. PCR inhibition control: control containing a known amount of positive template DNA added in the same amount of analyte DNA as the reaction (that is to be controlled) (this could be the original target or a spike, e.g., a slightly modified target such as a competitor plasmid). This control allows determination of the presence of soluble PCR inhibitors, particularly necessary in the case of negative amplification. 4. Amplification reagent control: control containing all the reagents except test sample template DNA extracted. Instead of the template DNA, a corresponding volume of nucleic acid–free water is added to the reaction. 5. Extraction blank control: control performing all steps of the extraction procedure except addition of the test portion (e.g., by substitution of water for the test portion). It is used to demonstrate the absence of contaminating nucleic acid during extraction. If many PCR analyses are performed on DNA extracted in separate series, all the appropriate extraction blank controls are included. It can also be used instead of the amplification reagent control. 6. Positive extraction control: control sample meant to demonstrate that the nucleic acid extraction procedure has been performed in a way that will allow for extraction of the target nucleic acid (i.e., by using a sample material known to contain the target nucleic acid).

2.6

ANALYSIS OF PCR PRODUCTS

Independent of the starting copy number of target molecules and number of cycles performed in PCR, the products amplified are not visible in the reaction vessel! It is necessary to run a post-PCR gel electrophoresis to identify the amplified product predicted and to assess the sample under investigation. In the case of nucleic acids the preferred matrix is purified agarose (a component of agar, which is a red seaweed extract), which forms a solid but porous matrix that looks and feels like clear gelatine dessert. For DNA, the direction of migration, from negative to positive electrodes, is due to the natural negative charge carried on their sugar–phosphate backbone. Doublestranded DNA fragments naturally behave as long rods, so their migration through the gel is relative to their size. Shorter molecules move faster and migrate farther than longer ones. Increasing the concentration of agarose in the gel reduces the migration speed and enables separation of smaller DNA molecules. The higher the voltage, the faster the DNA migrates. However, voltage is limited by the fact that it heats and ultimately causes the gel to melt. High voltages also decrease the resolution (above about 5 to 8 V/cm) (Sambrook and Russell, 2001). The most common dye used for agarose gel electrophoresis is ethidium bromide (EtBr). It fluoresces under ultraviolet (UV) light when intercalated into DNA. By running DNA through an EtBr-treated gel and visualizing it with UV light, distinct bands of DNA become visible (Figure 2.4).

48

POLYMERASE CHAIN REACTION

FIGURE 2.4 Agarose gel electrophoresis of PCR products. Along with the PCR products, reference DNA fragments are separated to estimate the size of fragments amplified by PCR.

A range of issues important to the analysis of PCR products are discussed below. Denaturation Denaturation is a very fast step in the process, which starts at a temperature above 70 C. All reaction components are very sensitive with respect to increasing temperatures: The DNA polymerase as well as nuleotides begin to denaturate; template DNA and primer will depurinate by high temperatures. Therefore, temperature as well as time should be selected carefully and reduced to a minimum. In practice, a few seconds (ca. 5 s) are enough to separate the double-stranded DNA molecules. However, at the beginning of the PCR process, an initial denaturation step of approximately 2 min should be used to generate only single-stranded template DNA. Annealing The temperature in the annealing phase is most heavily dependent on primer length and the sequence used. A variety of computer programs are available to calculate the theoretical melting point (Tm) and secondary structure of the primers chosen. All possible calculations are only theoretical considerations. The most convenient and simplest way to calculate Tm for a given primer is based on the GC content and is called a 2 þ 4 rule: Tm ¼ 4ðnumber of G and CÞ þ 2ðnumber of A and TÞ This approximation is valid only for short primers (up to 20 bases). A more sophisticated calculation is the nearest-neighbor approach (Breslauer et al., 1986),

ANALYSIS OF PCR PRODUCTS

49

also taking into account the primer sequence and the fact that nucleotides influence their neighbors. The melting temperature alone is defined as the temperature at which 50% of the primers are no longer annealed to the target; it does not determine reliably the temperature at which the primer is hybridized to the target sequence. Therefore, it is advisable to determine the most appropriate annealing temperature empirically at a temperature 5 to 10 C lower with respect to the one calculated theoretically. Elongation The polymerization time depends on the length of the amplified PCR product predicted. If too-short a temperature is chosen, DNA polymerase cannot complete the polymerization; if it is chosen for too long a period of time, false, nonwanted DNA could be synthesized. Typically, 0.5 to 1 min per 1 kb of amplified product length is calculated if Taq DNA polymerase is used. For proofreading polymerases, a prolonged time should be considered. PCR Efficiency In a simplified way it is considered that during each individual PCR cycle the amount of DNA is duplicated. Based on this assumption the introduction of only one starting template molecule in PCR will result in 1 billion identical copies of the template after 30 cycles and a trillion copies after 40 cycles. If a single molecule 1 kb in length is used as a template, it would endup in 1 mg of amplified products, which is a significant amount of DNA! Unfortunately, this does not reflect what is really going on in the reaction. In reality, the amplification factor is between 1.6 and 1.7 on average; sometimes it is a bit better, sometimes a bit worse (Figure 2.4). The efficiency is not the same even over the entire PCR process. At the beginning the efficiency is lower, probably due to the low probability that template, primer, and Taq DNA polymerase need more time to find each other to establish a complex initiating amplification. During the accumulation of amplified products the efficiency here increased close to 100%. After a certain number of cycles the efficiency decreased again, due to residuals such as the pyrophosphates accumulating as the result of the hydrolyzation of dNTPs. Moreover, the DNA polymerase will be affected by the temperature shift and the products amplified will rehybridize, avoiding the annealing of primers to their complementary sequence. Template Quality The quality of the DNA introduced into PCR is of critical importance. Highly purified template DNA and the absence of inhibitors are the best guarantee of enormous quantities of amplified products. Thus putting a lot of effort into this step of the analysis is highly recommended. Today, a variety of extraction protocols are available from different suppliers, including all components required to get sufficient amounts of DNA from various samples. In particular, for bacterial suspensions and for mammalian cell cultures, ready-to-use kits are on the market. For food samples there are also kits developed to extract amplifiable DNA from the sample. Moreover, the integrity of the

50

POLYMERASE CHAIN REACTION

template DNA influences the outcome of the PCR and hence the analytical results obtained. The applicability of a specific method may therefore depend on whether the sample or material to be analyzed is processed or refined or neither, and on the degree of degradation of the DNA. Template Quantity The amount of template DNA necessary for successful amplification depends on the complexity of the DNA sample. For example, for 4 kb of viral genomic DNA containing a 1-kb target sequence, 25% of the input DNA is the target of interest. Conversely, a 1-kb target sequence in the soybean genome (1.1  109 bp) represents approximately 0.00001% of the DNA input. Thus, approximately 1,000,000-fold more soybean genomic DNA is required to maintain the same number of target copies per reaction. Common mistakes include inputting too much DNA, too much PCR product, or too little genomic DNA as the template. Reactions with too little DNA template will result in low yields; reactions with too much DNA template can be afflicted by nonspecific amplification. As a general role, approximately 104 copies of the target DNA should be used to obtain a signal in 25 to 30 cycles, but the final DNA concentration of the reaction should be kept at or below 10 ng/mL. When reamplifying a PCR product, the concentration of the specific PCR product is often not known. Therefore, the previous amplification reaction should be diluted at least 1:10 to 1:10,000 before reamplification. . . . . .

1 mg 1 mg 1 mg 1 mg 1 mg

of of of of of

1 kb dsDNA ¼ 9.12  1011 molecules pUC19 Vector DNA ¼ 2.85  1011 molecules lambda DNA ¼ 1.9  1010 molecules E. coli genomic DNA ¼ 2  108 molecules soybean genomic DNA ¼ 8.48  105 molecules

Taking into account the calculations above and that in theory only one target molecule is sufficient to be amplified in PCR, it is obvious that 1 mg of soybean DNA does contain more than enough DNA. From a practical point of view it is usually recommended that the sample DNA be diluted rather than concentrating the DNA amount in PCR in order to get a positive signal. Failure of amplification in PCR is probably due to the presence and accumulation of inhibitors after extraction of DNA from samples rather than the low presence of target molecules. Buffer Almost all reaction buffers contain as a buffering agent Tris-based buffer and salt, commonly KCl. The buffer regulates the pH of the reaction, which finally affects the DNA polymerase activity and fidelity. The highest activity of the Taq DNA polymerase is observed above pH 8. Modest concentrations of KCl increase DNA polymerase activity by 50 to 60% over activities in the absence of KCl; 50 mM KCl is considered optimal (Gelfand, 1989).

ANALYSIS OF PCR PRODUCTS

51

MgCl2 Concentration Magnesium is an essential cofactor for thermostable DNA polymerases, and the magnesium concentration is a crucial factor in the success of amplification. Template DNA concentration, chelating agents present in the sample (e.g., EDTA or citrate), dNTP concentration, and the presence of proteins could all affect the amount of free magnesium in PCR. In the absence of adequate free magnesium ions, Taq DNA polymerase is totally inactive. An excess of free magnesium reduces the enzyme fidelity and possible increases the level of nonspecific amplification. Therefore, the optimal magnesium concentration should be determined empirically for each reaction. To do so, a series of reactions containing 1.0 to 4.0 mM Mg2þ in increments of 0.5 to 1.0 mM should be performed and the results visualized in order to determine which magnesium concentration produced the highest yield of product and the minimum amount of nonspecific product. The effect of magnesium concentration and the optimal concentration range can vary with the particular Taq DNA polymerase used. Many Taq DNA polymerases are supplied with a magnesium-free reaction buffer along with a tube of 25 mM MgCl2, so the most appropriate Mg2þ concentration can be adjusted for each reaction. Magnesium chloride solutions can form concentration gradients as a result of multiple freeze–thaw cycles, and vortex mixing is required to obtain a uniform solution. This step eliminates the cause of many failed experiments. It is sometimes preferred to use reaction buffers that already contain MgCl2 at a final concentration of 1.5 mM. However, it should be noted that Hu et al. (1992) reported the performance variability of reaction buffer solutions containing magnesium. The free magnesium changes of 0.6 mM observed in their experiments affected amplification yields dramatically in an allele-specific manner. The authors found that heating the buffer at 90 C for 10 min restored the homogeneity of the solution. They postulated that magnesium chloride precipitates as a result of multiple freeze–thaw cycles (Hu et al., 1992). Primer Design PCR primers typically range from 18 to 25 nucleotides long and are designed to flank the target DNA region of interest. Primers should have 40 to 60% GC content, and care should be taken to avoid sequences that might produce intermolecular or intramolecular secondary structure. To avoid the development of primer–dimers, the 30 ends of the primers should not be complementary. Primer–dimers unnecessarily sequester primers away from the reaction and result in an unwanted polymerase reaction that is in competition with the intended PCR product. Three G or C nucleotides in a row near the 30 end of the primer should be avoided, as this may result in nonspecific primer annealing, increasing the synthesis of undesirable products. Intramolecular regions of secondary structure can interfere with primer annealing to the template and should also be avoided. Ideally, the Tm value of the primer pair, at which 50% of them are annealed to the complementary target DNA, should be within 5 C, allowing both primers to anneal at approximately the same temperature. Finally, the annealing temperature of PCR is dependent on the primer with the lowest Tm value. However, higher annealing temperatures improve the stringency of primer annealing, resulting in more specific product.

52

POLYMERASE CHAIN REACTION

Several software packages are available to aid in primer design, and it is strongly recommended that they be taken into consideration. PCR Enhancers and Additives The addition of enhancing components can influence the PCR and therefore increase the yield of the intended product or decrease the production of undesired products. Many PCR enhancers are described in the literature that can act through a number of different mechanisms. These reagents do not enhance all PCRs; the beneficial effects are very often template and primer specific, and it will be necessary to determine the effect empirically. Some of the most common enhancing reagents are discussed in more detail below. Among them, dimethyl sulfoxide (DMSO) (up to 10% v/v) and formamide (up to 5% v/v) can be helpful when amplifying GC-rich templates and templates that form strong secondary structures, which can cause DNA polymerases to hold up in DNA synthesis. GC-rich templates can be problematic due to inefficient separation of the two strands of DNA or the tendency for the complementary, GC-rich primers to form intermolecular secondary structures, which will compete with primer annealing to the template. DMSO and formamide are thought to reduce the amount of energy required to separate the strands of DNA templates by interfering with the formation of hydrogen bonds between the two strands of DNA (Geiduschek and Herskovits, 1961). Concentrations of DMSO greater than 10% and formamide greater than 5% can inhibit Taq DNA polymerase as well as other DNA polymerases. In some cases, general stabilizing agents such as bovine serum albumin (BSA) (0.1 mg/mL), gelatine (0.1 to 1.0%) and nonionic detergents (0 to 0.5%) can overcome failures to amplify a region of DNA. These additives can increase Taq DNA polymerase stability and reduce the loss of reagents through adsorption to the tube walls. Nonionic detergents such as PEG 6000, Tween-20, and Triton X-100 have the added benefit of overcoming the inhibitory effects of trace amounts of strong ionic detergents, such as 0.01% sodium dodecyl sulfate (SDS). Ammonium ions can make amplification more tolerant of nonoptimal conditions. Therefore, some reactions include 10 to 20 mM (NH4)2SO4. Additional PCR enhancers mentioned in the literature are glycerol (5 to 20%), polyethylene glycol (5 to 15%), and tetramethylammonium chloride (60 mM). Primer Concentration The primer concentration can also influence the yield of PCR products. A concentration of 0.2 mM of each primer is usually chosen. If the yield of PCR products is low, a higher concentration can be adjusted, but it should exceed a concentration of 2 mM, because the yield will decrease again. The purity of synthesized primers should be considered if it is intended to run critical applications. Then HPLC-purifed primers often improve the result of PCR. Number of Cycles Starting with a certain amount of product (ca. 0.3 to 1 pmol) during the PCR process, a “plateau effect” decreases the efficiency significantly. This is due to the accumulation

ANALYSIS OF PCR PRODUCTS

53

of pyrophosphates, the reduced amount of primers/oligonucleotides present in the reactions, and the amount of intact Taq DNA polymerase decreased during the several times the reaction has been heated up to 95 C. In reality, the majority of amplified products are no more specific, resulting in a higher background. This can be observed during electrophoresis as a smear background along with the PCR product amplified. Therefore, the number of cycles should be selected where the plateau effect begins. It should also be noted that the number of cycles is not the appropriate way to distinguish between positive and negative samples. Cases where only after a large number of cycles (more than 40) can a positive signal be obtained indicate either contamination or nonproper selection of primers. The procedure should then be newly designed and tested before use in routine analysis. Thermostable DNA Polymerases Prior to the use of thermostable DNA polymerases in PCR, scientists had to supplement the reaction laboriously with new enzyme (such as Klenow or T4 DNA polymerase) after each denaturation cycle at 95 C. The identification of thermostable DNA polymerases revolutionized PCR because of their ability to withstand high denaturation temperatures. The use of thermostable DNA polymerases also allowed higher annealing temperatures, which improved the stringency of primer annealing. Although the temperature optima of thermostable DNA polymerases are around 70 C, they are not inactive at other temperatures. For example, Taq DNA polymerase is a processive enzyme with an extension rate above 3600 nucleotides/min at 70 C, so an elongation step of 1 min for 1 kb to be amplified is sufficient to produce full-length PCR products. At 55 C the Taq DNA polymerase can synthesize approximately 2800 nucleotides/min, and even at 37 C approximately 1400 nucleotides/min are polymerized to get a full-length DNA strand. The thermostable DNA polymerases can be divided into two groups: those with a 30 ! 50 exonuclease (proofreading) activity, such as Pfu DNA polymerase, and those without the proofreading function, such as Taq DNA polymerase. Both groups have some important differences. Proofreading DNA polymerases are significantly more accurate than nonproofreading polymerases, due to the 30 ! 50 exonuclease activity, which can remove a false-incorporated nucleotide from an extended chain of singlestranded DNA. When the amplified product is to be cloned in a subsequent procedure, Pfu DNA polymerase (derived from Pyrococcus furiosus) or Pwo DNA polymerase (derived from P. woesei) is a better choice, due to its high fidelity. Pfu DNA polymerase has one of the lowest error rates of all known thermostable DNA polymerases used for amplification, due to the highly active 30 ! 50 exonuclease activity (Cline et al., 1996). For cloning and expressing DNA after PCR, Pfu DNA polymerase is the enzyme of choice. Pfu DNA polymerase can be used alone for the amplification of DNA fragments up to 5 kb by increasing the extension time to 2 min/ kb. However, the proofreading activity can shorten PCR primers, leading to decreased yield and increased nonspecific amplification. This exonucleolytic attack can effectively be avoided by initiating the reaction using hot-start PCR or by introducing a single phosphorothioate bond at the 30 termini of the primers (Byrappa et al., 1995).

54

POLYMERASE CHAIN REACTION

However, for routine analysis, where simple detection of an amplified product is the aim, Taq DNA polymerase is the most commonly used enzyme because yields tend to be higher with a nonproofreading DNA polymerase. Amplification with nonproofreading DNA polymerases results in the template independent addition of a single nucleotide to the 30 end of the PCR product. The single-nucleotide overhang can simplify the cloning of PCR products (A overhang) (Zhou et al., 1995). In contrast, blunt-ended PCR products that are the result of use of a proofreading DNA polymerase need to be cloned into a blunt-ended vector system (Clark, 1988; Hu, 1993). Proofreading DNA polymerases are also used in combination with nonproofreading DNA polymerases, or amino-terminally truncated versions of Taq DNA polymerase, to amplify longer fragments of DNA with greater accuracy than for nonproofreading DNA polymerase alone. The classical Taq DNA polymerase is isolated from T. aquaticus and catalyzes the primer-dependent incorporation of nucleotides into duplex DNA in the 50 ! 30 direction in the presence of Mg2 þ . The enzyme does not possess 30 ! 50 exonuclease activity but has a 50 ! 30 exonuclease activity. The Taq DNA polymerase is isolated from T. aquaticus and suitable for most PCR amplifications that do not require a specific high-fidelity enzyme, such as the detection of specific DNA or RNA sequences. However, the error rate of Taq DNA polymerase is approximately 1  105 errors/base and also depends on the reaction conditions. The fidelity is slightly higher at lower pH, lower magnesium concentration, and relatively low dNTP concentration (Eckert and Kunkel, 1990, 1991). Taq DNA polymerase is normally used to amplify PCR products of 5 kb or less. PCR products in the range of 5 to 10 kb can be amplified with Taq DNA polymerase but often require more optimization than do smaller PCR products. For products larger than approximately 10 kb, it is recommended that an enzyme or enzyme mix and reaction conditions be chosen that are designed specifically for long amplification products. Because Taq DNA polymerase is a nonproofreading polymerase, PCR products generated with Taq DNA polymerase will contain a single-nucleotide 30 overhang, usually a 30 A overhang. The characteristics of Tth DNA polymerase (derived from Thermus thermophilus) is similar to those of the Taq DNA polymerase and catalyze the polymerization of nucleotides into duplex DNA in the 50 ! 30 direction in the presence of Mg2 þ ions. The enzyme is also capable of catalyzing the synthesis of DNA using an RNA template in the presence of MnCl2 (Myers and Gelfand, 1991). Therefore, no further reverse transcriptase (RT) is necessary to generate a DNA template for subsequent PCR analysis. For primer extension and RT-PCR and cDNA synthesis using RNA templates with complex secondary structure, the high reaction temperature of Tth DNA polymerase may be an advantage over that of more commonly used reverse transcriptases such as the AMV and M-MLV transcriptases. Tth DNA polymerase shows a 50 ! 30 exonuclease activity but lacks detectable 30 ! 50 exonuclease activity. The error rate of Tth DNA polymerase has been determined to be approximately 7.7  105 error/base. Tth DNA polymerase has been reported to be more resistant to inhibition by blood components than are other thermostable polymerases (Ehrlich et al., 1991). Characteristics of the DNA polymerases described above are summarized in Table 2.4.

HOT-START PCR

TABLE 2.4

Characteristics of Commonly Used Thermostable DNA Polymerases

50 ! 30 DNA polymerase activity Processivity 50 ! 30 exonuclease activity 30 ! 50 exonuclease activity (proofreading) Producing A overhang Reverse transcriptase activity Approximate error rate

2.7

55

Taq

Pfu, Pwo

Tth

þ High þ  þ  105

þ High þ  þ þ 105

þ High þ þ   106

HOT-START PCR

Today, hot-start PCR is most common technique used to reduce nonspecific amplification due to the assembly of amplification reactions at room temperature or on ice. At room temperature, PCR primers can anneal to template sequences that are not totally complementary. Since thermostable DNA polymerases have activity at these low temperatures, the polymerase can extend misannealed primers. This newly synthesized DNA is 100% complementary to the DNA template, allowing primer extension and the polymerization of undesired amplification products. However, if the reaction is heated to temperatures above 60 C before polymerization begins, the stringency of primer annealing is increased, and subsequent synthesis of undesired PCR products is avoided or reduced. Hot-start PCR can also reduce the amount of primer–dimer formation by increasing the stringency of primer annealing. At lower temperatures, the primers can anneal to each other via regions of complementarity, and the DNA polymerase can extend the annealed primers to produce primer–dimer, which can often be observed as a diffuse band of approximately 50 to 100 bp on an ethidium bromide–stained gel. The formation of nonspecific amplification products and primer–dimer can compete for reagent availability with the amplification of the product desired. Therefore, hot-start PCR can improve the yield of the specific PCR products. To carry out hot-start PCR, the reactions are assembled on ice or at room temperature, but one critical component is omitted until the reaction has been heated to 60 to 65 C, at which point the missing reagent is added. This omission prevents the polymerase from extending primers until the critical component is added at the higher temperature, where primer annealing is more stringent. However, this method is tedious and increases the risk of contamination. A second, much less labor-intensive approach involves the reversible inactivation or physical separation of one or more critical components in the reaction. For example, the magnesium or DNA polymerase can be sequestered in a wax bead, which melts during the reaction as it is heated to 94 C, releasing the component only at higher temperatures. Alternatively, the DNA polymerase can be kept in an inactive state by binding to an oligonucleotide, also known as an aptamer (Dang and Jayasena, 1996; Lin and Jayasena, 1997) or an

56

POLYMERASE CHAIN REACTION

antibody (Scalice et al., 1994). The bond is irreversibly disrupted at the denaturation temperature, releasing the functional DNA polymerase.

2.8

PCR EQUIPMENT

In early PCR experiments, scientists had to rely on a series of water baths to maintain the various temperatures required in order to keep the amplification process ongoing. Cycling involved manual transfer of samples from one water bath to another at specified times. In 1988, Perkin-Elmer introduced the thermal block cycler, a revolutionary device that increased and decreased the temperature of samples automatically and repetitively during the PCR process. This allowed the PCR analysis to be automated. Subsequent refinements of this device extended the flexibility and accuracy of PCR. Today, several suppliers, including Applied Biosystems, Roche Diagnostics, Stratagenes, and Eppendorf, offer thermal cyclers. It is important to validate a certain PCR system before it is used on devices from different suppliers, because ramp rates (time needed for heating and cooling) differ. In the past a drop of mineral oil was used to prevent condensation and evaporation of the liquid. Modern thermal cyclers use a heating lid that reduces the need for the addition of mineral oil.

2.9

LABORATORY ORGANIZATION

Compliance with applicable requirements with respect to safety regulations and manufacturers’ safety recommendations should be assured. Accidental DNA contamination is known to originate from dust and spreading aerosols. As a consequence, the organization of the work area in the laboratory is based logically on the systematic containment of the methodological steps involved in production of the results and a forward-flow principle for sample handling. A minimum of three separately designated contained/dedicated working areas with their own apparatus are required: 1. A working area for extraction of the nucleic acid from the test material 2. A working area dedicated to the setup of PCR/amplification reactions 3. A working area dedicated to subsequent processing, including analysis and characterization of the amplified DNA segments If dust particle–producing grinding techniques are used, they must be carried out in a separate working area. Physical separation through the use of different rooms is the most effective and preferable way of ensuring separate working areas, but other physical or biochemical methods may be used as a protection against contamination provided that their effectiveness is comparable. Laboratory staff should wear different sets of lab coats in each dedicated working area and should wear disposable gloves. Where possible, powdered gloves should be avoided for pre-PCR operations, since the powder can inhibit PCR. Gloves and lab coats should be changed at appropriate

REFERENCES

57

frequencies. All PCR procedures should be carried out under substantially noncontamination conditions. Carryover Contamination The major problem of false-positive signals in PCR occurs frequently even though all individual steps are physically separated. In case all necessary reagents are aliqouted, the easiest way is to take a new aliquot and repeat the analysis. In a few cases this is not enough; false-positives still occur. Then the scientist has two possibilities for continuing with detection of the target DNA: 1. If the DNA sequence is known, an alternative primer pair can be designed, where at least one the primer anneal outside the previous amplification product. Subsequently, PCR can be optimized and applied again. 2. As a prevention tool, thymidine phosphate as part of the dNTP mix can be partially sustituted for by uracyl prior to the PCR. The amplification efficiency of PCR will not be affected. In case carryover contamination occurs, an additional pre-PCR step can be carried out: applying the enzyme uracyl-N-glycosyslate (UNG) at 50 C for 30 min. UNG identifies uracyl incorporated into the DNA and modifies the carbon bonds, resulting in DNA degradation during the initial denaturation step at 95 C. The UNG is degraded, too; thus all contaminating amplified products are destroyed. The addition of UNG is necessary only if contamination occurs. The addition of dUTP is the prerequisite to use of this approach. It should be noted that dUTP hinders subsequent steps, such as cloning or restriction analysis.

REFERENCES Breslauer KJ, et al. (1986). Predicting DNA duplex stability from the base sequence. Proc. Natl. Acad. Sci. USA, 83:3746–3750. Byrappa S, et al. (1995). A highly efficient procedure for site-specific mutagenesis of full-length plasmids using Vent DNA polymerase. Genome Res., 5:404–407. Chamberlain JS, et al. (1988). Deletion screening of the Duchenne muscular dystrophy locus via multiplex DNA amplification. Nucleic Acids Res., 16:11141–11156. Clark JM (1988). Novel non-templated nucleotide addition reactions catalyzed by procaryotic and eucaryotic DNA polymerases. Nucleic Acids Res., 16:9677–9686. Cline J, et al. (1996). PCR fidelity of Pfu DNA polymerase and other thermostable DNA polymerases. Nucleic Acids Res., 24:3546–3551. Dang C, Jayasena SD (1996). Oligonucleotide inhibitors of Taq DNA polymerase facilitate detection of low copy number targets by PCR. J. Mol. Biol., 264:268–278. Eckert KA, Kunkel TA (1990). High fidelity DNA synthesis by the Thermus aquaticus DNA polymerase. Nucleic Acids Res., 18:3739–3744. Eckert KA, Kunkel TA (1991). DNA polymerase fidelity and the polymerase chain reaction. PCR Methods Appl., 1:17–24. Ehrlich HA, et al. (1991). Recent advances in the polymerase chain reaction. Science, 252: 1643–1651.

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Geiduschek EP, Herskovits TT (1961). Nonaqueous solutions of DNA. Reversible and irreversible denaturation in methanol. Arch. Biochem. Biophys., 95:114–129. Gelfand DH (1989). Taq DNA polymerase. In Erlich HA (ed.), PCR Technology: Principles and Applications of DNA Amplifications. Stockton Press, New York, pp. 17–22. Hu G (1993). DNA polymerase-catalyzed addition of non-templated extra nucleotides to the 30 end of a DNA fragment. DNA Cell Biol., 12:763–770. Hu CY, et al. (1992). Effect of freezing of the PCR buffer on the amplification specificity: allelic exclusion and preferential amplification of contaminating molecules. PCR Methods Appl., 2:182–183. Lin Y, Jayasena SD (1997). Inhibition of multiple thermostable DNA polymerases by a heterodimeric aptamer. J. Mol. Biol., 271:100–111. Myers TW, Gelfand DH (1991). Reverse transcription and DNA amplification by a Thermus thermophilus DNA polymerase. Biochemistry, 30:7661–7666. Rychlik W, et al. (1990). Optimization of the annealing temperature for DNA amplification in vitro. Nucleic Acids Res., 18:6409–6412. Saiki RK, et al. (1988). Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science, 239:487–491. Saiki R, et al. (1985). Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science, 230:1350–1354. Sambrook J, Russell DW (2001). Molecular Cloning: A Laboratory Manual, 3rd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Scalice E, et al. (1994). Monoclonal antibodies prepared against the DNA polymerase from Thermus aquaticus are potent inhibitors of enzyme activity. J. Immunol. Methods, 172:147–163. Zhou MY, et al. (1995). Universal cloning method by TA strategy. Biotechniques, 19:34–35.

CHAPTER 3

Quantitative Real-Time PCR HERMANN BROLL € r Risikobewertung, Berlin, Germany Bundesinstitut fu

3.1

INTRODUCTION

Invention of the polymerase chain reaction (PCR) by Kary Mullis in the 1980s revolutionized almost everything in molecular biology. PCR is a fairly standard procedure now, and its use is extremely wide ranging: from basic research up to clinical or other diagnostic purposes. At its most basic application, PCR can amplify a small amount of template DNA into large quantities in a few minutes or hours. This is carried out by mixing the DNA with primers on both sides of the DNA (forward and reverse), Taq polymerase (of the species Thermus aquaticus, a thermophile bacterium whose polymerase is able to withstand extremely high temperatures), free nucleotides [deoxynucleotide triphosphates (dNTPs) for DNA], and buffer containing MgCl2. Applying a specific temperature–time program with alternating hot and cold conditions to denature and reanneal the DNA, the polymerase synthesizes new complementary strands each time. PCR is an integral addition to the molecular biologist’s toolbox, and the method has been improved continually over the years. Fairly recently, a new method of PCR quantification was invented called real-time PCR. It allows scientists to actually view the increase in the amount of DNA as it is amplified. Several different types of real-time PCR are being marketed to the scientific community at this time, each with its own advantages. In this chapter we provide a more detailed view of one of these, TaqMan real-time PCR, and provide an overview of three other types of real-time PCR: molecular beacons, scorpions, and Sybr Green. Basic PCR is an endpoint application in which the amplified target is identified only after the last cycle of the PCR process has finished and a gel electrophoresis is carried out separating the amplified product according to its molecular weight. Because PCR is an exponential procedure in which small variations in the amount of target

Molecular Biological and Immunological Techniques and Applications for Food Chemists Edited by Bert Popping, Carmen Diaz-Amigo, and Katrin Hoenicke Copyright  2010 John Wiley & Sons, Inc.

59

60

QUANTITATIVE REAL-TIME PCR

molecules introduced into the PCR result in big differences in the amount of final products, it is not considered to be quantitative. Real-time PCR has the capability to monitor the progress of the PCR as it occurs (i.e., in real time). Therefore, data are collected throughout the entire PCR process rather than at the end of the PCR. It thereby revolutionizes PCR-based quantitation of DNA and RNA. In real-time PCR, reactions are characterized by the point in time during cycling when amplification of a target is first detected rather than by the amount of target accumulated after a fixed number of cycles. The higher the starting copy number of the nucleic acid target, the sooner a significant increase in fluorescence is observed. In contrast, an endpoint assay (also called a plate-read assay) measures the amount of PCR product accumulated at the end of the PCR cycle. Basic PCR and real-time PCR are generally used in a qualitative format to evaluate biological samples. However, a wide variety of applications, such as determining microbiological presence and characterizing gene expression, are improved by quantitative determination of target abundance. Theoretically, it is quite easy to achieve, based on the exponential nature of PCR. There is a linear relationship between the number of amplification cycles and the logarithm of the number of molecules, at least in theory. In practice, however, the efficiency of amplification is usually lower because of the presence of inhibitors, competitive components, substrate exhaustion, inactivation of the polymerase, and target reannealing. As the number of cycles increases, the amplification efficiency decreases, usually resulting in a plateau effect (Figure 3.1). The primary advantage of real-time PCR quantification includes a broad dynamic range and capability for high-throughput applications. Current detection platforms are able to detect less than 10 pg of DNA and process up to approximately 350 individual samples in a standard format in less than 2 h. In addition, real-time PCR

0.9 Plateau

0.8

Fluorescence

0.7 0.6

Linear

0.5 0.4

Threshold

0.3

Exponential

0.2

Baseline

Ct value

0.1 0

0

10

20 30 PCR cycle

40

50

FIGURE 3.1 Real-time PCR plot. Four distinct phases during real-time PCR can be observed: background without increasing fluorescence, the exponential phase, followed by a shorter linear phase, and the final plateau phase.

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enables target-specific quantification (e.g., gender determination, pathological diagnosis, determination of GMO presence, and species identification). Real-time PCR template DNA quantification estimates are derived from measured fluorescence accumulation, which is correlated directly with the amount of amplified PCR products produced as the reaction progresses (Heid et al., 1996). Fluorescence is generated either by intercalating dyes that are specific for double-stranded DNA (Wittwer et al., 1997a,b) or by sequence-specific oligonucleotide probes (Holland et al., 1991; Livak et al., 1997). The real-time PCR sequence detection system measures the reporter signal (R) and normalizes it to a passive reference dye. Normalizing accounts for minor well-to-well variations in signal strength, allowing for more accurate sample-to-sample comparisons. The progressive cleavage of the probe at each PCR cycle leads to an increase in normalized reporter signal (Rn) which is proportional to the initial PCR cycles. Reporter fluorescence values are below the baseline detection capabilities of current real-time PCR systems, resulting in stochastic fluctuations in fluorescence (i.e., background fluorescence). To minimize this stochastic effect, normalized reporter signal is subtracted from background noise in the fluorescence signal. Normalized reporter signal minus the background fluorescence signal (DRn) is then plotted against cycle number (Figure 3.1). The real-time PCR fluorescence curve generated by the sequence detection system is composed of four distinct phases. When PCR product and reporter signal accumulate beyond background fluorescence levels, the reaction enters the exponential detection phase. At this point the amplification plot crosses a user-defined detection threshold which is set above the background fluorescence noise, preferable at the beginning of the exponential phase. The fractional cycle number at which the reaction crosses the threshold (Ct) is related inversely to the initial template DNA concentration. As PCR products continues to accumulate, the ratio of Taq DNA polymerase to amplified products decreases, resulting in nonexponential accumulation of amplicons. At this point the reaction enters the linear phase. Once PCR product ceases to accumulate due to assay depletion, DRn values remain relatively constant and the reaction enters the plateau phase. PCR amplification efficiency is the rate at which a PCR amplicon is generated, generally expressed as a percentage. If a particular PCR product doubles in quantity during the geometric phase of its PCR amplification, the PCR assay has 100% efficiency. The slope of a standard curve is commonly used to estimate the PCR amplification efficiency of a real-time PCR. A real-time PCR standard curve is represented graphically as a semi log regression line plot of Ct value versus the log of input nucleic acid. A standard curve slope of 3.32 indicates a PCR with 100% efficiency (Table 3.1). Slopes that are more negative than 3.32 (e.g., 3.9) indicate reactions that are less than 100% efficient. Slopes more positive than 3.32 (e.g.,2.5) probably indicate poor sample quality or pipetting problems. A 100% efficient PCR will yield a 10-fold increase in amplified products every 3.32 cycles during the exponential phase of amplification (log 10 ¼ 3.3219). However, it is not often the case that this value is met exactly. Calculating amplification efficiencies therefore allows early detection of nonoptimal assay conditions and will facilitate troubleshooting problematic samples prior to

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TABLE 3.1 Link Between Slope, Amplification, and Efficiency a Slope

Amplification

Efficiency

3.60 3.55 3.50 3.45 3.40 3.35 3.30 3.25 3.20 3.15 3.10

1.8957 1.9129 1.9307 1.9492 1.9684 1.9884 2.0092 2.0309 2.0535 2.0771 2.1017

0.8957 0.9129 0.9307 0.9492 0.9684 0.9884 1.0092 1.0309 1.0535 1.0771 1.1017

a

Given are various values of the slope and calculated amplification efficiencies. The optimal amplification efficiency is highlighted in boldface type.

sequence analysis. Several strategies have been developed. The mechanisms of PCR inhibition can be grouped into three categories based on the point of action during sample preparation and amplification. Inhibitors can interfere with cell lysis during DNA extraction, degrade or capture nucleic acids, or inhibit Taq DNA polymerase (according to the author’s experience, the most frequent case). Although inhibitory mechanisms may vary, the outcome is a general reduction in amplification efficiency. Probably the most elegant way to identify reduced amplification efficiency is the statistical calculation based on the regression analysis of four to six data points within the window of linearity that have the highest coefficient of determination and slope closest to the maximum (Kontanis and Reed, 2006). These authors reported that the anchor of Ct þ 1 is the best point for starting the analysis. This approach does have the great advantage of avoiding any additional laboratory work, because the calculation is based on the results of the unknown sample. An alternative inhibitor detection strategy is to create a serial dilution of the suspect template and construct an intrinsic calibration curve from which the efficiency can be estimated. Because of the exponential nature of PCR amplification, only a small number of template molecules are required to generate a PCR product. Thus, samples can often be diluted to a point where inhibitors are ineffective at preventing amplification of the remaining template DNA. As a result, diluted assays will cross the detection threshold earlier, decreasing the slope of the linear regression curve generated using the suspect sample dilution series. Efficiency is then calculated from the slope of the linear regression line: E ¼ 101=slope : Low efficiency values suggest that dilution has reduced the effects of amplification inhibitors. Although potentially useful, this alternative approach requires extensive sample manipulation and multiple PCR assays, increasing workloads and financial expenditures substantially. In addition, if template DNA concentrations are low, as is often the case in food analysis context, dilution may result in template depletion and no amplification products. Consequently, this method is of limited utility with challenging

INTRODUCTION

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template samples. In GMO analysis, a monitor run is often carried out prior to the quantification assay. Usually, 1: 10 and a 1: 40 dilutions of the sample are analyzed in parallel. Only when the Ct difference between reactions (E ¼ 10) is approximately 2 is the sample is counted as “free of any inhibitor”. Internal positive controls (IPCs) can also be used to identify the presence of PCR inhibitors. IPCs are definitively useful for detecting false-negative results; however, they cannot be used to determine a precise measure of inhibition strength when template samples are marginally compromised. Comparing the amplification efficiencies of clean standards with those of unknown samples is a statistically sound method that can be used in conjunction with IPCs when amplifications are successful but are compromised, producing erroneous quantification results. In general, quantitative PCR requires that the measurement be completed before the plateau phase, so the relationship between the number of cycles and molecules is probably linear. This point must always be determined empirically for different reactions because of the various factors that can affect the amplification efficiency. Because the measurement is taken prior to the reaction plateau, quantitative PCR uses fewer amplification cycles than does basic PCR. The amount of final product can cause problems because there is less to detect. To monitor the efficiency of amplification, many applications are designed to include an internal standard in the PCR. One such approach includes a second primer pair that is specific for a housekeeping gene (i.e., a gene that has constant expression levels among the samples compared) in the reaction. Amplification of housekeeping genes verifies that the target nucleic acid and reaction components were of acceptable quality but does not account for differences in amplification efficiencies due to differences in product size or primer annealing efficiency between the internal standard and the target being quantified. The concept of competitive PCR, a variation of quantitative PCR, is a response to this limitation. In competitive PCR, a known amount of a control template is added to the reaction. This template is amplified using the same primer pair as the experimental target molecule but yields a distinguishable product (e.g., different size, restriction digest pattern). The fluorescence signals (i.e., amounts, intensity) of control and test product are compared after amplification. Although these approaches control for the quality of the target nucleic acid, buffer components, and primer annealing efficiencies, they have their own limitations (Siebert and Larrick, 1993; McCulloch et al., 1995), including the fact that it depends finally on the analysis of products by electrophoresis. Numerous fluorescent solution and solid-phase assays have been described to measure the amount of amplification product generated in each reaction, but they can fail to discriminate amplified DNA of interest from nonspecific amplification products due to the missing final confirmation step. Some of these analyses rely on blotting techniques, which introduce another variable due to nucleic acid transfer efficiencies, while other assays have been developed to eliminate the need for gel electrophoresis, yet provide the requisite specificity. Real-time PCR, which provides the ability to view the results of each amplification cycle, is a popular way to overcome the need for post-PCR analysis by electrophoresis.

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Real-time PCR using labeled oligonucleotide probes or primers employs two different fluorescent reporters and relies on the transfer of energy from one reporter (the donor) to the second reporter (the acceptor) when the reporters are in close proximity. The second reporter can be a quencher or a fluorescence dye. If the second reporter is a quencher, the energy from the first reporter is absorbed but reemitted as heat rather than light, leading to a decrease in the fluorescent signal. Alternatively, if the second reporter is a fluorescence dye, the energy can be absorbed and reemitted at another wavelength through fluorescent resonance energy transfer (FRET; reviewed in Didenko, 2001), and the progress of the reaction can be monitored by the decrease in fluorescence of the energy donor or the increase in fluorescence of the energy acceptor. During the exponential phase of PCR, the change in fluorescence is proportional to the accumulation of PCR product. To simplify quantitation, specific instruments perform both the thermal cycling steps to amplify the target and the fluorescence detection to monitor the change in fluorescence in real time during each PCR cycle. There are several general categories of real-time PCR probes, including hydrolysis, hairpin, and simple hybridization probes. These probes contain a complementary sequence that allows the probe to anneal to the accumulating PCR product, but probes can differ in the number and location of the fluorescent reporters.

3.2

REAL-TIME CHEMISTRY

In real-time PCR the fluorescent reporter molecule used can be either a sequencespecific probe comprised of a short oligonucleotide labeled with a fluorescent dye in conjunction with a quencher [e.g., TaqMan probes (hydrolysis probes), molecular beacons, and scorpions] or a nonspecific DNA-binding fluorescence dye such as SybrGreen I, which fluoresces when bound to double-stranded DNA. For selection of the best solution for the individual real-time PCR experiment, the level of sensitivity and accuracy required for the data analysis and the budget available to support the project should be considered. In addition, the solution should be based on the skill and experience of the researcher in designing and optimizing quantitative PCR (qPCR) assays, because primer and probe design is not trivial. Finally, it should be noted whether or not a multiplex assay, in which several targets need to be amplified and subsequently distinguished, is necessary. Almost all chemistries, such as SybrGreen I, TaqMan, LUX primers, FRET probe pairs, or Invader probes will work in all the different types of instruments from various sources. 3.2.1

DNA-Binding Dye Chemistry

In general, small molecules that bind to double-stranded DNA can be divided into two classes: (1) intercalators, and (2) minor-groove binders. DNA binding dyes such as SybrGreen I are cheap and easy to use. Therefore, SybrGreen I is the common choice for optimizing real- time PCR. When free in solution, SybrGreen I displays only a bit

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of background fluorescence, but when bound to double-stranded DNA its fluorescence increases over 1000-fold. The more double-stranded DNA that is present, the more binding sites there are for the dye, so fluorescence increases proportionately to DNA concentration. This property of the dye provides a mechanism that allows it to be used to track the accumulation of PCR product. As the target is amplified, the increasing concentration of double-stranded DNA in the solution can be measured directly by the increase in fluorescence signal (Figure 3.1). The assay design is relatively easy, as well as the run of the PCR compared with probebased methods. All that is needed is the design of a set of primers, optimization of the amplification efficiency and specificity, and running the PCR in the presence of the dye. But one limitation of assays based on DNA-binding dye chemistry is the inherent nonspecificity. Sybr Green I will increase in fluorescence when bound to any doublestranded DNA. Therefore, the reaction specificity is determined solely by the primers. Consequently, the primers should be designed to avoid nonspecific binding (e.g., primer–dimer formation). Otherwise, it is possible that the fluorescence measured may include signal contamination, resulting in artificially early Ct values, giving an inaccurate representation of the true target concentration. A nonspecific signal cannot always be avoided, but its presence can easily be identified by performing melting curve analysis on the PCR products from every run. Immediately following the PCR, amplified products can be melted slowly while the SybrGreen I fluorescence is detected. As the temperature increases, the DNA melts and the fluorescence intensity decreases. The temperature at which a DNA molecule melts depends on its length and sequence composition. If the PCR products consist of molecules of homogeneous length and sequence, a single thermal transition will be detected, resulting in a temperature specific only for this amplicon. If more than one population of PCR products is present, it will be reflected as multiple thermal transitions in the fluorescence intensity. In this way, the fluorescence– temperature curve (also called a dissociation curve) is used to differentiate between specific and nonspecific amplicons based on the Tm (melting temperature) value of the reaction end products. To avoid false-positive signals, it is sometimes also advisable to check for nonspecific product formation using agarose gel electrophoresis. Another aspect of using DNA-binding dyes is that multiple dyes bind to a single amplified molecule. This increases the sensitivity for detecting amplification products. A consequence of multiple dye binding is that the amount of signal is dependent on the mass of double-stranded DNA produced in the reaction. Thus, if the amplification efficiencies are the same, amplification of a longer product will generate more signal than will a shorter one. This is in contrast to the use of a fluorogenic probe, in which a single fluorophore is released from quenching for each amplified molecule synthesized, regardless of its length. DNA-binding dyes are often used for initial optimization of PCR assays for diagnostic purposes. In basic research such as for transcription identification and quantification, real-time PCR using DNA-binding dyes is used much more.

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3.3

QUANTITATIVE REAL-TIME PCR

PROBE-BASED CHEMISTRIES

Compared to nonspecific chemistries such as SybrGreen I dye, a higher level of detection specificity is provided by using an internal probe to detect the real-time PCR product of interest. In the absence of a specific target sequence present in the reaction, the fluorescent probe is not hybridized, remains quenched, and does not result in an increased fluorescence signal. When the probe hybridizes to the target sequence of interest, the reporter dye is no longer quenched, and fluorescence will be detected. The level of fluorescence detected is related directly to the amount of amplified product in each PCR cycle. An important advantage of using probe-based chemistry rather than DNA binding dyes is that multiple probes can be labeled with different reporter dyes and combined to allow detection of more than one target in a single reaction: called multiplex assay. 3.3.1

Hydrolysis Probes

Hydrolysis probes (e.g.,TaqMan probes) are the most widely used and published in detection chemistry literature for probe-based real-time PCR applications. In addition to the primers, it includes a third oligonucleotide, 20 to 26 bases in length, in the reaction known as the probe. A fluorescent reporter dye, most frequently 6-carboxyfluorescein (6-FAM), is attached to the 50 end of the probe and a quencher, generally 6-carboxytetramethylrhodamine (TAMRA) is attached at the 30 end. However, more and more, dark quenchers such as the Black Hole Quenchers (BHQs) are replacing the use of TAMRA because they provide lower background fluorescence. As long as the two molecules (reporter and quencher) are maintained in close proximity, the fluorescence from the reporter is quenched and no fluorescence is detected at the reporter dye’s emission wavelength. TaqMan probes use fluorescence resonance energy transfer (FRET), a quenching mechanism phenomenon first described by in the 1940s (Foerster, 1948), in which quenching can occur over a fairly long distance  (100 A or even more), depending on the fluorescence dye and quencher used, so that as long as the quencher is on the same oligonucleotide as the reporting fluorescence dye, quenching will occur, resulting only in a fluorescent signal of the quencher. The probe is designed to anneal to one strand of the target sequence in close proximity to one of the primers. While the polymerase extends that primer, it will encounter the 50 end of the probe. Taq DNA polymerase has 50 ! 30 nuclease activity, so when Taq DNA polymerase reaches the probe, it displaces and degrades the 50 end, releasing free reporter dye into solution (Holland et al., 1991). Following the separation of reporter dye and quencher, fluorescence can be detected from the reporter dye (Figures 3.2 and 3.3). To optimize probe binding and subsequent cleavage, it is critical to adjust the thermal profile to facilitate both the hybridization of probe and primers and the cleavage of the probe. To meet both of these requirements, TaqMan probes will generally use a two-step thermal profile with a denaturing step (usually at 95 C) and a merged annealing-extension step at 60 C. To guarantee the binding of the TaqMan probe, it is selected with a 7 to 10 C higher Tm value than the annealing temperature. If the temperature in the reaction is too high when Taq DNA polymerase extends

PROBE-BASED CHEMISTRIES

67

FIGURE 3.2 Principle of the widely used TaqMan technology. Shown is only one cycle of the entire PCR amplification process. After denaturation and annealing of primers and probes, the Taq DNA polymerase synthesizes a new DNA strand until the probe is reached. Due to its 50 nuclease activity, the oligonucleotide is cleaved and the probe is released.

Emission intensity

Target is present

Target is absent 500,00

600,00 Reporter dye

wave length

Quencher dye

FIGURE 3.3 Fluorescence signal generation. The reporter and quencher signal have different emission optimums. For quantification purposes only the reporter signal is counting. As long as no target DNA is present in the reaction, only quencher fluorescence is measured. As more and more target is amplified, more reporter molecules are released into the reaction, resulting in an increase in fluorescence signal.

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through the primer (such as at a standard extension temperature of 72 C), the probe will be strand-displaced only, rather than cleaved, resulting in no increase in the fluorescence signal. It should be noted that the increase in fluorescence signal is strongly dependent on the exonuclease activity of the Taq DNA polymerase. It is not totally clear if this activity is 100% or (probably) lower, resulting in decreased PCR efficiency. TaqMan MGB probes consist of an oligonucleotide containing a reporter dye (i.e., FAM or VIC) at the 50 end, a minor-groove-binder moiety, and a nonfluorescent quencher dye at the 30 end. The minor-groove binder acts as a probe Tm enhancer. Probes designed as TaqMan MGB probes have much shorter probes, which enhances the Tm differential between matched and mismatched probes. A probe sequence with a single mismatch is more likely to be displaced by the Taq DNA polymerase than it is to be cleaved by the enzyme during amplification. In addition, TaqMan MGB probes contain a nonfluorescent quencher that provides enhanced spectral resolution when using multiple dyes in a single reaction. It is reported that TaqMan MGB probes enhance specific probe binding and are also more suitable for multiplex assays. 3.3.2

Other Probe Systems

Instead of only one single oliginucleotide, the LightCycler hybridization probe method uses two DNA probes that hybridize, in a head-to-tail arrangement, in close proximity to the target DNA. Usually, it should not more than 2 to 5 bases space between the 30 end of probe 1 (donor probe) and the 50 end of probe 2 (acceptor probe). This leaves space for the fluorescence dyes at the ends of the probes. Each probe is labeled with a different fluorescence dye. Interaction of the two dyes occurs when both are bound to their target at the same time. When the two probes are hybridized to their target sequences, the fluorescence dyes are in close proximity and (FRET) can occur between them (Figure 3.4). The choice of hybridization probes should take into consideration a “balanced” sequence region because a sequence region that is nearly equal tends to bind probes tightly, but not too tightly. It should not contain monotonous or repetitive sequences because such sequences can form hybrids and the sequence should not be selfcomplementary. DNA sequences can form loops and therefore be less accessible to hybridization. In principle, those probes can be cheaper than probes bearing two fluorescent labels. Structured probes contain stem–loop structure regions that give enhanced target specificity compared with traditional linear probes. This characteristic enables a higher level of discrimination between similar sequences and makes these chemistries specifically well suited for clinical purposes such as SNP and allele discrimination applications. The widely used molecular beacons include a hairpin loop structure, where the central loop sequence is complementary to the target of interest and the stem arms are complementary to each other. One end, typically the 50 end of the stem, is modified with a reporter fluorescence molecule, and the other end carries a quencher. Rather than using a FRET-quenching mechanism similar to TaqMan probes, molecular beacons are based on static quenching, which requires the fluorescence dye and

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FIGURE 3.4 LightCycler hybridization probes. During the PCR annealing step, the two oligonucleotides hybridize, head to tail, to adjacent regions of the target DNA. The fluorescence dyes, which are coupled directly to the oligonucleotides, are very close in the hybrid structure. The F1 donor fluorophore (e.g., fluorescein) is excited by an external light source, then passes on part of its excitation energy to the adjacent F2 acceptor (e.g., LightCycler-Red 640) FRET via dipole–dipole interactions. The excited F2 fluorescence dye emits light, which can be measured by the instrument.

quencher to be in very close proximity for quenching to occur. Historically, DABCYL or methyl red has been used for molecular beacons; more common today is the use of the Black Hole Quencher, which reduces background noises. In the absence of a specific target, the molecular beacon’s thermodynamic properties favor the formation of the hairpin over mismatched binding. This places the fluorescence dye and quencher immediately adjacent to each other so that quenching will occur. In the presence of the target sequence, the annealing of the loop sequence to the target is the preferred conformation. When the probe is annealed to the target, the fluorescence dye and quencher are separated, and the reporter fluorescence can be detected. Since molecular beacon chemistry is not based on the 50 ! 30 exonuclease activity of Taq DNA polymerase, it can be used in a traditional three-step thermal profile. When the thermal cycling ramps up to 72 C and the Taq DNA polymerase extends to where the molecular beacon probe is annealed, the probe will simply be strand-displaced, and it will again form the hairpin loop conformation. Because formation of the molecular beacon hairpin loop is a reversible process, the probe will be recycled with each PCR cycle. Proper design of the molecular beacon stem is crucial to ensuring optimized performance of the reaction. If the stem structure is too stable, target hybridization can be inhibited. Additionally, if the molecular beacon probe does not fold in the expected stem loop conformation, it will not quench in the manner expected. It is quite easy to verify after synthesis that the proper design is behaving as wanted before it will be used in any real-time PCR assay. By melting the molecular beacon alone, in the presence of its perfect complement, and/or of a mismatched sequence, the dynamics of the reaction can easily be compared and used to determine the optimal temperature for fluorescence measurement and mismatch discrimination. One advantage of this technique is that hairpin probes are less likely to mismatch than are hydrolysis probes (Tyagi and Kramer, 1996). However, preliminary experiments

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must be performed to show that the signal is specific for the desired PCR product and that nonspecific amplification does not occur. Scorpion probe chemistry is to a certain extent similar to molecular beacons, but rather than having a separate probe, the hairpin structure is incorporated onto one of the primers. The fluorescence dye is attached to the 50 end of the primer, whereas the 30 end is complementary to the target and functions as a site for extension initiation. The quencher is located between the primer and probe regions of the oligonucleotide, so that if the probe is in the hairpin configuration, the reporter dye is located in close proximity to the quencher. After amplification and incorporation of the hairpin probe, the newly created strand is able to adopt a new structure. The loop sequence in the hairpin is complementary to the extension product of the probe/primer. During further rounds of denaturation and annealing, the loop sequence will anneal to the newly formed complementary sequence within the same strand of DNA. In this conformation, the fluorescence dye is separated from the quencher, resulting in an increased fluorescence signal. As is also the case for other types of probes, the primer also contains a “PCR preventing system” in the hairpin, which prevents the stem–loop structure from being copied during PCR by extension from the other primer. Since annealing of the loop sequence with the downstream PCR product is an intramolecular interaction, it is kinetically more favorable than probe systems that require two separate molecules to interact. Therefore, scorpions usually result in a stronger fluorescence signal than those associated with TaqMan and molecular beacons. Like molecular beacons, scorpions are not based on the 50 ! 30 exonuclease activity of Taq DNA polymerase, so the reaction can be performed using a typical three-step thermal profile with the optimal extension temperature for the polymerase (72 C). However, a disadvantage of scorpion chemistry is that the design and optimization of the probe structure are often much more challenging than with either molecular beacons or TaqMan probes, and as a result, scorpions are not generally suggested for those researchers who are new to the real-time technology. The Plexor qPCR and qRT-PCR systems require no probes, only two PCR primers, one of which is labeled fluorescently. These systems take advantage of the specific interaction between two modified nucleotides (Moser and Prudent, 2003; Johnson et al., 2004; Sherrill et al., 2004). The two novel bases, isoguanine (iso-dG) and 50 -methylisocytosine (iso-dC), form a unique base pair in double-stranded DNA (Johnson et al., 2004). To perform fluorescent quantitative PCR using this new technology, one primer is synthesized with an iso-dC residue as the 50 -terminal nucleotide and a fluorescent label at the 50 end; the second primer is unlabeled. During PCR, the labeled primer is annealed and extended, becoming part of the template used during subsequent rounds of amplification, and the complementary iso-dGTP, which is available in the nucleotide mix as dabcyl-iso-dGTP, pairs specifically with iso-dC. When dabcyl-iso-dGTP is incorporated, the close proximity of DABCYL and the fluorescent label on the opposite strand effectively quenches the fluorescent signal. It is important to realize that the initial fluorescence level of the labeled primers is high, which is the opposite for all other real-time systems described here. As amplification product accumulates, the signal decreases. However, in general the system is suitable for quantitative purposes as well as all others.

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The fluorescence dye ROX is used as an internal reference by some sequence detection instruments to normalize fluorescence. ROX reference dye is available from various suppliers: included in the mix or as a separate vial for optimization.

3.4

REAL-TIME PCR PRIMER AND PROBE DESIGN

There are numerous primer and probe design software programs on the market, and coupled with a set of easy-to-follow design rules, the process is made relatively simple and reliable. However, the first step in primer and probe design is to acquire the sequence of gene or simple DNA sequence of interest. The primary choice to acquire the publicly available sequences is the open-access NCBI database (www.ncbi.nlm. nih.gov). After the sequence is obtained, a primer and probe design software program should be used to simplify and maximize success for the design process. Moreover, such programs have the capacity to check for hundreds of parameters at the same time. Designer software packages are available both as freeware on the Internet and through many oligonucleotide vendors. When using a software program to design primers and probes, it is important to set the concentration of monovalent ions (Na þ /Kþ ) and divalent ions (Mg2 þ ) to those that are used in your reaction for accurate melting temperature prediction. (The buffer conditions will generally be in the range of 50 to 100 mM monovalent cation and 1.5 to 5.5 mM Mg2þ ). The region of the template sequence to be used for detection must be considered carefully. The region of interest should be compared to the entire genome to ensure that the target sequence is unique, and potential secondary structures should be identified and avoided. In addition, it should be checked that no similarity to other cross-contaminating species is occurring. To identify a coding sequence specific to RNA targets, it is most advisable to design the probe to span exon–exon boundaries (excluding intron sequences), thus preventing the detection of sequences from residual genomic DNA in the RNA sample. A DNase I step prior to the reverse transcription (RT) reaction could then be skipped. This is an efficient approach and results in minimal loss of sample when carried out on a column-based purification system. In qRT-PCR, the method of cDNA synthesis needs to be considered when designing primers if oligo-dT priming is used. It is generally safe to assume that the RT reaction has transcribed between 500 and 1000 bases from the polyA site with quantitative linearity, so it is best to design the assay to target a sequence for amplification toward the 30 end of the gene. The presence of SNPs and splice variants within a sequence should also be considered, as these must either be avoided or targeted as required according to the goal of the experiment. For optimal performance, the region spanned by the primers (measured from the 50 end of each primer) should be between 70 and 150 bp in length for probe-based chemistries, and between 100 and 300 bp in length if SybrGreen I will be used. To maximize the efficiency of the PCR amplification, it is generally best to keep the target length relatively short. However, with SybrGreen I it could be advantageous to use a slightly longer target so that more of the dye molecules can bind to the amplified product and produce a higher fluorescence signal. When designing for SybrGreen I with the intention of moving later to a probe-based chemistry, keep in mind using the lower

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range (e.g., 100 to 200 bp) for primer design. General rules for primers used in all chemistries are that each primer should be between 15 and 30 bp in length, and the theoretical Tm value of the two primers should be within 2 C of each other. It is best trying to avoid G/C clamps at the 30 ends of the primers to prevent these oligos from folding on themselves or annealing nonspecifically. The five bases at the 50 terminal end generally should contain no more than two guanines and cytosines, although it is acceptable to have three in the final five bases if no two are adjacent. Since thymidine tends to misprime more readily than the other bases, a 30 terminal T should also be avoided. The 50 end of the primers should not contain an inverted repeat sequence that would allow it to fold on itself. In general, the Gibbs free enthalpy (DG) of primer–dimer and cross-primer–dimer formation should be greater than 4 kcal/mol to ensure that primers do not form stable dimers. For multiplex reactions it may be necessary to loosen the free enthalpy specification to allow for the design of the oligos required to work together in the same reaction. It is best to restrict the DG between each oligo pair to greater than 6 kcal/mol in a triplex reaction, and greater than 8 kcal/mol in a quadruplex reaction. Probes should not contain runs of the same base (avoid more than three of the same base), and optimally should contain more C than G nucleotides. Guanine is an effective fluorescence quencher and should not be adjacent to the reporter dye. Historically, TaqMan probes were situated 3 to 12 bp downstream of the primer on the same strand, but recent evidence suggests that the distance from the upstream primer to the probe is less important than thought previously. TaqMan probes are generally between 20 and 30 bp in length. Ideally, they should have balanced GC content, although probes with varying content (20 to 80% GC) can still be effective. The Tm requirements of the probe will most often dictate the specific percentage of GC. TaqMan assays are conventionally performed as a two-step PCR consisting of a product melt at 95 C, followed by primer annealing and Taq DNA polymerase extension at 60 C (Figure 3.1). For these assays the probe is designed with a Tm value 8 to 10 C higher than the primer Tm values. Using the higher Tm value for the probe ensures hybridization to the target before extension can occur from the primer, so there will always be a corresponding increase in fluorescence signal for every amplified copy that is produced. Since TaqMan chemistry requires using the same thermal profile for each reaction, primers should always be designed with a Tm value of approximately 60 C, and the hydrolysis probe with a Tm value around 70 C. Optimization of the assay is accomplished by adjusting primer concentration rather than optimizing annealing temperatures. Molecular beacon probes should be designed to anneal at 7 to 10 C higher than the primers, to allow hybridization before primer extension. For molecular beacons, the stem sequence should be designed to be 5 to 7 bp in length and should have a Tm value similar to that of the melting temperature of the probe region in the loop. As a general rule, stem sequences that are 5 bp long will have a Tm value of 55 to 60 C, stems that are 6 bp long will have a Tm value of 60 to 65 C, and stems that are 7 bp long will have a Tm value of 65 to 70 C. Unlike TaqMan probes, molecular beacons are usually designed so that the probe is annealed closer to the midpoint between the two primers rather than adjacent to the upstream primer. This should ensure that any low-activity extension by the polymerase at the annealing temperature will not displace the probe before the fluorescence reading is

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73

taken. A scorpion probe sequence should be approximately 17 to 30 bp in length. It is best to place the probe no more than 11 bp upstream of the complementary target sequence. The farther downstream this complementary sequence is, the lower the probe efficiency will be. The stem sequence should be about 6 to 7 bp in length and contain sufficient pyrimidines so that the Tm value of the stem loop structure is 5 to 10 C higher than the Tm value of the primer sequence to the target, and the DG value for the stem loop confirmation is negative. The more negative the DG value, the more likely it is that folding will occur. When designing probes, the combination and positioning of reporter dyes and quenchers is important. Make sure that the quencher chosen will efficiently suppress the fluorescence of your chosen reporter dye to ensure a low background. Information on the quenchers recommended for each fluorophore is generally available from companies that synthesize these probes. When designing primers and probes for multiplex reactions, adhere to the following additional rules: All amplicons should be of similar length (5 bp) as well as similar GC content (3%), and the primer set Tm and probe Tm values used in a multiplex assay should be within 1 C of each other. For all real-time PCRs, it is a good idea to verify that all of the oligos (primers and probe) that will be used together in the same reaction will not form dimers, particularly at the 30 ends. The 30 complementarity can be checked by scanning the sequences manually. If you are using primer design software, the program itself may run a check to make sure that the sequence choices it picks are not complementary to each other.

3.4.1

Dye and Quencher Choice

When designing a fluorescent probe it is necessary to ensure that the fluorophore and quencher pair are compatible given the type of detection chemistry. In addition, when designing multiplexed reactions, the spectral overlap between the fluorophores and quenchers for the different targets should be minimized to avoid possible crosstalk issues (see Table 3.1). For TaqMan probes, the most historically common dye–quencher combination is a FAM fluorophore with a TAMRA quencher. This combination will certainly work well, but in recent years dark quenchers have become more popular. Dark quenchers emit the energy they absorb from the fluorophore as heat rather than light of a different wavelength. They tend to give results with lower background and are especially useful in a multiplex reaction, where it is important to avoid light emitted from the quencher, giving a crosstalk signal with one of the reporters.

3.5

TYPES OF MACHINES

Many real-time machines are on the market today. In general, they could be divided into two distinct categories, depending on the technique used to heat and cool the reactions.

74

QUANTITATIVE REAL-TIME PCR

3.5.1

Block Thermal Cyclers

Block thermal cyclers are based on a conventional 96-well-plate format in a metal block combined with either a laser-induced fluorescence system or a LED/halogen lamp–based optical excitation source. Due to the usual thermal block time– temperature profiles, the ramp rates for cooling and heating are approximately 1.5 to 3 C/s, which results in an overall time of about 2 h for a typical PCR run of 45 cycles. Due to the 96-well-plate format, the usual pipetting and equipment can be used. Of particular interest for food analysis, in which relative quantification is often used, the numbers of reactions per run are of great importance. Therefore, those types of machines may have an advantage over the machine types described below. 3.5.2

Rotor-Based Systems

Rotor-based systems are available from only two companies: Roche Diagnostics and Corbett Lifescience. Tubes are arranged in a circular rotor that spins continuously at a low rpm value in a chamber of moving air. Consequently, temperature shifting can be done much faster up to 20 C/s, thus facilitating a significant short-time PCR. However, the usual standard lab format of plates and well cannot be used in those types of instruments. This seems to be a drawback; nevertheless, formats are available in the range of 36 to 100 individual reactions. Thus, there is no temperature variation between tubes, due to positional effects such as the recognized edge effect reported in block thermal cyclers. Importantly, reactions also remain isothermal during programmed temperature transition steps. So there is no equilibration time difference between wells; in other words, every tube changes temperature at the same rate. This eliminates another well-to-well variable normally affecting real-time reaction kinetics. In short, Corbett’s Rotor-Gene has the best thermal characteristics yet developed. Detection is carried out using a photomultiplier detector with variable or automatic gain setting filter sets and a CCD (charge-coupled device) camera. Major differences exist in the software developed and used for the various types of machines. However, all current real-time PCR machines are based on the Windows operating system. A partial list of suppliers and real-time PCR instruments is given in Table 3.2. 3.6

REAL-TIME PCR APPLICATIONS

Real-time PCR can be used in traditional PCR applications as well as new applications that would have been less effective with traditional PCR. With the ability to collect data in the exponential growth phase, the power of PCR has been expanded into such applications as: . . . .

Viral quantification Quantification of gene expression Array verification Drug therapy efficacy

REAL-TIME PCR APPLICATIONS

TABLE 3.2

75

Suppliers and Types of Real-Time Instruments on the Market

Supplier

Real-Time Instrument

Homepage

Applied Biosystems

ABPrism (5700, 7000, 7300, 7500, and 7900) iQ5 real-time PCR detection system DNA Engine Opticon 2 system MiniOpticon real-time PCR detection system Rotor-Gene 6000 LightCycler 2.0 System LightCycler 480 MX4000 SmartCycler system Mastercycler ep realplex

www.appliedbiosystems.com

Bio-Rad Laboratories Bio-Rad Laboratories Bio-Rad Laboratories Corbett Lifescience Roche Diagnostics Stratagene Cepheid Eppendorf

. . . . .

www.bio-rad.com www.bio-rad.com www.bio-rad.com www.corbettlifescience.com www.roche.com www.stratagene.com www.cepheid.com www.eppendorf.com

DNA damage measurement Quality control and assay validation Pathogen detection Genotyping Food control

The advantages of using real-time PCR include the following: . . . . . . .

Traditional PCR is measured at the endpoint (plateau), whereas real-time PCR collects data in the exponential growth phase. An increase in reporter fluorescent signal is directly proportional to the number of amplified products generated. The cleaved probe provides a permanent record of the amplification of an PCR product (assay). The dynamic range of detection is increased. There is no post-PCR processing. Detection can be carried out down to a twofold change. Potential cross-contamination is reduced due to elimination of post-PCR analysis because there is no need to open vials following the PCR process.

However, real-time PCR is not a quantitative method per se. As a starting point for real-time PCR, the following description provides suggestions for PCR conditions and a temperature–time profile that should give reasonable results. Specifically, for TaqMan assays the following procedure is recommended to optimize the primer– probe concentration; it is known as a primer–probe matrix. At the beginning the probe

76

QUANTITATIVE REAL-TIME PCR

TABLE 3.3

PCR Parameters for Various Types of Real-Time PCR Approaches a

Reaction with SybrGreen I 0 to 30 ng template–DNA 6 mM MgCl2 0.5 g/L bovine serum albumin 0.5 mM forward primer 0.5 mM reverse primer 200 mM dNTP-mix 1:30,000  SybrGreen I 0.5 to 1 U DNA polymerase in (1x)-PCR buffer

Reaction with TaqMan Probe

Reaction with Hybridization Probes

0 to 30 ng template–DNA 6 mM MgCl2 —

0 to 30 ng template–DNA 6 mM MgCl2 0.5 g/L BSA

0.5 mM forward primer 0.5 mM reverse primer 0.2 mM TaqMan probe 200.0 mM dNTP-mix 0.5 to 1 U DNA– polymerase in (1x)-PCR buffer

0.5 mM forward primer 0.5 mM reverse primer 0.2 mM donor probe 0.4 mM acceptor probe 200 mM dNTP-mix, 0.5 to 1 U DNA–polymerase in (1x)-PCR buffer

a The total volume varies between 25 – 50 mL. Sometimes it is reduced to only 10 mL to save reagents and therefore reduce significantly the cost related to real-time analysis.

concentration should be kept constant at 200 mL, whereas the primer should be varied between 150, 300, and 900 nmol/mL. Nine individual reactions are necessary to determine the most appropriate primer concentration for both primers. A typical reaction composition for PCR is shown in Table 3.3; a temperature–time profile that usually results in a satisfactory amplification is given in Table 3.4. The primer concentration is optimal where the amplification curve first exits the background: consequently, the reaction with the smallest Ct value. By varying the concentration, a shift in Tm value is mimicked which could adjust for a Tm difference between the theoretical calculated and the true Tm in the reaction. Afterward the probe concentration can be varied as well, usually not below 50 nm/mL and not above 250 nm/mL.

TABLE 3.4

PCR Temperature–Time Profile for Block and LightCycler Machines

Pre-PCR: decontamination (optional) Pre-PCR: activation of DNA polymerase and denaturation of template DNA PCR (45 cycles) Step 1: denaturation Step 2: annealing elongation c a

Time (s)

Temperature ( C)

Acquisition Mode

120 600

50 95

None None

15 a/5 b 60 a/25 b

95 60

None Single

Typical for block thermal cyclers. Optimized for the LightCycler system. c Depending on the nature of the application used, the acquisition of the fluorescence is either during the annealing phase or at the end of the amplification/elongation phase. b

ABSOLUTE VS. RELATIVE QUANTITATION

3.7

77

ABSOLUTE VS. RELATIVE QUANTITATION

When setting up and/or calculating the results of quantification assays, it is possible to use either absolute or relative quantification. 3.7.1

Absolute Quantitation

The absolute quantification assay is used to quantify unknown samples by interpolating their quantity from a standard curve. Absolute quantification might be used to correlate a viral copy number with a disease status. It is of interest to the scientist to know the exact copy number of the target DNA in a given biological sample in order to monitor the progress of the disease. Absolute quantification can be performed with data from all real-time PCR instruments; however, knowledge of the absolute quantities of the standards is an absolute prerequisite in order to run the analysis, and most appropriate by some independent means. The most direct and precise approach to the analysis of quantitative data is use of a standard curve that is prepared from a dilution series of template of known concentration. This is called a standard curve or absolute quantification. The standard curve approach is used when it is important to the experimental design and objective of the project to measure the exact level of template in the samples (e.g., monitoring the viral load in a sample). A variety of sources can be used as standard templates. Examples include a plasmid containing a cloned gene of interest or sequence representing the target of interest, genomic DNA, cDNA, synthetic oligos, or in vitro transcripts. After amplification of the standard dilution series, the standard curve is generated by plotting the log of the initial template copy number against the Ct values generated for each dilution. If the aliquoting of the standard solution was accurate and the efficiency of the amplification does not change over the range of template concentrations being used, the plot of these points should generate a linear regression line. This line represents the standard curve (Figure 3.5). Comparing the Ct values of the unknown samples to this standard curve allows the quantification of initial copy numbers. Ideally, a standard curve will consist of at least four points, and each concentration should be analyzed at least in duplicate (the more points the better). The range of concentrations in the standard curve must cover the entire range of concentrations that will be measured in the assay (this may be several orders of magnitude). Extrapolating of data for unknown samples beyond the lowest and highest standards is totally forbidden because it is not known if the curve will succeed as it was calculated for the range within the standards. In addition, the curve must be linear over the entire concentration range. The linearity is denoted by the R squared value (r2; the Pearson correlation coefficient) and should be ideally 1 or very close to 1 (but at least above 0.985). A linear standard curve also indicates that the efficiency of amplification of real-time PCR is consistent at varying template concentrations. The validity of the standard curve method is based on an assumption of equal amplification efficiencies between DNA samples used as quantification standards and unknown test samples under investigation. If the standard

QUANTITATIVE REAL-TIME PCR

Cycle no. 0

10

20

1 30

40

0.1

0.01

Threshold

Fluorescence

78

0.001 45

Ct value

40 35 30 25 20 15 10 10

100

1000

10000

Genome copies

FIGURE 3.5 Absolute quantification using the standard curve approach. The upper part shows the amplification plot of 1 : 4 dilution series. The crossing points are then plotted against the initial starting copy number of the known standard. The regression of the standard curve is finally used to determine the DNA copies of an unknown sample.

curve becomes nonlinear at a very low template concentration, it is probably approaching the limit of detection for that assay. Usually, Ct values above 35 are no more stable from a statistical point of view, resulting in Ct differences above 0.5 between the repeated standards and unknown samples. Unknown samples of which the Ct values fall within a nonlinear section of the standard curve cannot be quantified accurately. Ideally, the efficiency of both the standard curve and sample reactions should be between 90 and 110%. If the efficiency is significantly less, it implies a nonoptimal reaction, due either to inhibitors present in the reaction mix or suboptimal primer sets or reaction conditions. Efficiencies significantly above 100% typically indicate experimental error (e.g., PCR inhibitors, miscalibrated pipetting devices, probe degradation, formation of nonspecific products, formation of primer–dimers). Primer–dimer formation is typically of greatest concern with SybrGreen I assays, where any double-stranded product will be detected. However, a significant amount of primer–dimer implies a suboptimal real-time PCR system. To identify the formation of primer–dimer, the best way is to check it by gel electrophoresis after conventional or real-time PCR.

ABSOLUTE VS. RELATIVE QUANTITATION

79

Deviations in efficiency can also be due to poor serial dilution preparation as well as extreme ranges of concentrations that either inhibit PCR (high template amounts) or exceed the sensitivity of that particular assay (very low amounts). The most important aspect is to have the efficiencies of standards and targets within about 5% of each other if possible, with both near 100%. Once the reactions for the standard curve and the samples have been optimized, Ct values can be compared to each other and an initial template quantity can be estimated. It is important that for this type of quantification a standard curve must be run on the same sample plate as the unknown samples. To date it has never been demonstrated that sample analysis for initial template DNA based on standard curves run on different plates can be calculated reliably. Replicates can vary in Ct values when run at different times or on different plates, and thus are not directly comparable to other runs. Nevertheless, replicates on the same plate should not vary more than 0.5 Ct below Ct values of 35. If they do, it is advisable to retrain the personnel running the analysis. It should also be kept in mind that the “absolute” quantity obtained from the standard curve is only as good as the DNA quantification methods used to measure the standards, so considerable care should be taken to use a very clean template and to perform replicate measurements (whether using gel-based estimation of the DNA concentration, UV spectrophotometry, or nucleic acid–binding dyes such as PicoGreen). At least two or three no template control (NTC) wells should be included for each individual real-time PCR run. In addition, independent positive controls of known quantities within the range of the standard curve are necessary to ensure that the initial copy numbers calculated are accurate. It is also very advisable to use control charts to get the overall range of measurement uncertainty over time periods. It is therefore necessary to use the same known sample in each individual PCR run and to record the quantity determined. 3.7.2

Relative Quantification

In general, two different strategies are possible for relative quantification: (1) the relative standard curve method and (2) the comparative Ct method (DDCt). Depending on various factors, one method may be more appropriate than the other. The advantages of each method and factors to be considered are described below. Relative Standard Curve Method An advantage of this approach is that it requires the least validation because the PCR efficiencies of the target and reference control do not have to be equivalent. The procedure is based on the use of two independent standard curves. One is established for the reference DNA sequence. This could be a housekeeping gene in case the transcription of a certain gene will be analyzed under various conditions or over a period of time. Or it could be a single-copy DNA sequence in order to determine the relative presence of another DNA sequence, as described in Chapter 6. In all cases, the reference sample is used for both real-time PCR systems as a standard, and the unknown sample needs to be analyzed in both systems. If the same amount of DNA

80

QUANTITATIVE REAL-TIME PCR

from the sample is used in the reference and the target real-time PCR systems, the respective copy numbers calculated can be used directly to determine the percentage. Again, it is very important to use copy numbers for the unknown sample only within the range of standard curves. Extrapolating beyond the lowest and highest standards is forbidden because it is not known whether or not the shape of the standard curves will change. This method requires that each 96-well reaction plate contain the standard curves for both PCR systems. It requires more reagents and more space on a single reaction plate than does the alternative approach, but it gives highly accurate quantitative results because unknown sample quantitative values are interpolated from the standard curve. This method should be considered when low numbers of targets and of samples are tested (Figure 3.6). Comparative Ct Method (DDCt) In contrast to the relative standard curve method, this approach does not require that standard curves run on each plate. This can result in reduced reagent use and therefore contribute to less expensive experiments. The comparative Ct method is useful when a high number of targets and/or number of samples are analyzed. Specifically, for high-throughput analyses and when validating microarray results, for example, the DDCt method is an appropriate strategy, as it avoids a lot of individual reactions. The method is similar to the relative standard curve method except that it uses arithmetic formulas to achieve results for relative quantification. It is possible to eliminate the use of standard curves and to use the DDCt method for relative quantification as long as the PCR efficiencies between the target sequence and reference control sequence are relatively equivalent. The amount of target, normalized to a reference sequence and relative to a calibrator, is given by 2DDCt . For a valid DDCt calculation, the PCR efficiency of the target amplification and the PCR efficiency of the reference amplification must be almost the same. To determine if the two amplification reactions have the same amplification efficiency, it is necessary to look at how the DCt (Ct target  Ct reference) varies with template dilution. The sample in the validation experiment must contain both the target and reference genes. For example, a sample that ultimately is evaluated during the experiment (such as the calibrator) could be used. The Ct values generated from equivalent standard curve mass points (target vs. reference) are used in the DCt calculation (Ct target  Ct reference). These DCt values are plotted vs. log input amount to create a semilog regression line. The slope of the resulting semilog regression line is used as a general criterion for passing a validation experiment. In a validation experiment that passes, the absolute value of the slope of DCt vs. log input should be smaller than 0.1. However, in an ideal situation, if the efficiencies of the two PCRs are the same, the plot of log input amount vs. DCt has a slope of approximately zero, which means the two regression lines are running absolutely parallel. An example of the parallel shape of two independent regression curves established for the same sample is given in Figure 3.7. From an equation of both standard curves it could be deduced that the two real-time PCR systems have very similar amplification efficiency.

81

FIGURE 3.6 Quantification of a selected gene (target sequence) relative to a reference gene sequence. Two independent standard curves need to be established for a sample under investigation. The copy numbers measured for the unknown sample DNA are obtained by interpolation from standard curves. By dividing the copy number calculated for the target sequence by the reference sequence and subsequent multiplication by 100, a percentage value for the target relative to the reference gene sequence can be determined. Due to the fact that only DNA copy numbers are quantified, the percentage cannot be transferred to any other unit.

82

QUANTITATIVE REAL-TIME PCR

45

Ct value

40 y = -1.4172 ln(x) + 37.744

35 30 25 20

y = -1.3509 ln(x) + 36.217

15 10 10

100

1000 10000 100000 Genome copy number

1000000

FIGURE 3.7 Comparison of the efficiency of two real-time PCR systems. The regression curves for both systems show parallel behavior, also demonstrated by the equation given for each regression curve. Applying the DDCt approach is possible only if the parallel shape can be demonstrated.

Today, real-time PCR is one of the most popular molecular biology methods in laboratories dealing with different issues, such as diagnostics and basic genetic research. It opened totally new areas of research, and real-time PCR applications are already included in global standard procedures (e.g., International Standards Organization) in the field of human diagnostics, identification of pathogens, and the authenticity of food and feed products (Codex Alimentarius).Therefore, it can be expected that further modifications of general principles such as real-time PCR will be developed in the future.

REFERENCES Didenko V (2001). DNA probes using fluorescence resonance energy transfer (FRET): designs and applications. Biotechniques, 31:1106–1121. Foerster T (1948). Zwischenmolekulare energiewanderung and fluoreszenz. Annalen der Physik, 437(1–2):55–75. Heid CA, Stevens J, Livak KJ, Williams PM (1996). Real time quantitative PCR. Genome Res., 6(10):986–994. Holland PM, Abramson RD, Watson R, Gelfand DH (1991). Detection of specific polymerase chain reaction product by utilizing the 50 -30 exonuclease activity of Thermus aquaticus DNA polymerase. Proc. Natl. Acad. Sci. USA, 88:357–362. Johnson SC, et al. (2004). A third base pair for the polymerase chain reaction: inserting isoC and isoG. Nucleic Acids Res., 32:1937–1941. Kontanis EJ, Reed FA (2006). Evaluation of real-time PCR amplification efficiencies to detect PCR inhibitors. J Forensic Sci., 51(4):795–804. Livak KJ, Flood SJ, Marmaro J, Giusti W, Deetz K (1997). Oligonucleotides with fluorescent dyes at opposite ends provide a quenched probe system useful for detecting PCR product and nucleic acid hybridization. PCR Methods Appl., June, 4(6):357–362.

REFERENCES

83

McCulloch RK, Choong CS, Hurley DM (1995). An evaluation of competitor type and size for use in the determination of mRNA by competitive PCR. PCR Methods Appl., 4(4):219–226. Moser MJ, Prudent JR (2003). Enzymatic repair of an expanded genetic information system. Nucleic Acids Res., 31:5048–5053. Sherrill CB, et al. (2004). Nucleic acid analysis using an expanded genetic alphabet to quench fluorescence. J. Am. Chem. Soc., 126:4550–4556. Siebert PD, Larrick JW (1993). PCR MIMICS: competitive DNA fragments for use as internal standards in quantitative PCR. Biotechniques, 14(2):244–249. Tyagi S, Kramer FR. (1996). Molecular beacons: probes that fluoresce upon hybridization. Nat. Biotechnol., 14:303–308. Wittwer CT, Ririe KM, Andrew RV, David DA, Gundry RA, Balis UJ (1997a). The LightCycler: a microvolume multisample fluorimeter with rapid temperature control. Biotechniques, 22(1):176–181. Wittwer CT, Herrmann MG, Moss AA, Rasmussen RP (1997b). Continuous fluorescence monitoring of rapid cycle DNA amplification. Biotechniques, 22(1):130–131, 134–138.

CHAPTER 4

Polymerase Chain Reaction–Restriction Fragment Length Polymorphism Analysis KLAUS PIETSCH and HANS-ULRICH WAIBLINGER €runtersuchungsamt Freiburg, Freiburg, Germany Chemisches und Veterina

4.1

INTRODUCTION

Polymerase chain reaction–restriction fragment length polymorphism analysis (PCR-RFLP) is a technique in which species may be differentiated by analysis of restriction fragments derived from cleavage of their DNA after PCR. DNA from a species is first extracted and purified. DNA is amplified by PCR and cut into restriction fragments using suitable restriction endonucleases, which digest the DNA molecules only when specific DNA sequences are present, termed recognition sequences. The restriction fragments are separated according to length by agarose gel electrophoresis. If two meat species differ in the distance between sites of cleavage of a particular restriction endonuclease, the length of the restriction fragments produced will be different. Comparison of the patterns generated can be used to differentiate between species. 4.1.1

Restriction Endonucleases

Restriction endonucleases are enzymes that digest DNA molecules at specific DNA sequences, depending on the particular enzyme used. Enzyme recognition sites are usually 4 to 6 bp in length. Generally, the shorter the recognition sequence, the larger the number of fragments generated. If molecules differ in nucleotide sequence, fragments of different sizes may be obtained. Restriction enzymes are isolated from a wide variety of bacterial genera and are thought to be part of the cell’s defenses

Molecular Biological and Immunological Techniques and Applications for Food Chemists Edited by Bert Popping, Carmen Diaz-Amigo, and Katrin Hoenicke Copyright  2010 John Wiley & Sons, Inc.

85

86

PCR–RESTRICTION FRAGMENT LENGTH POLYMORPHISM ANALYSIS

against invading bacterial viruses. These enzymes are named by using the first letter of the genus, the first two letters of the species, and the order of discovery. 4.1.2

Mitochondrial DNA

An organism inherits its DNA from its parents. Since DNA is replicated with each generation, any given sequence can be passed on to the next generation. Nucleic acid– based species identification and evolutionary analysis based on PCR-RFLP therefore often targets mitochondrial DNA (mtDNA). Mitochondrial DNA is very often used for evolutionary analysis or species identification. Mitochondria have a higher rate of mutations than nuclear DNA, which makes it easier to resolve differences between closely related individuals. MtDNA is inherited only from the mother, without recombination, which allows tracing a direct genetic line. Mitochondria have their own genome of about 16,500 bp, which exists outside the cell nucleus, coding for 13 protein genes, 22 tRNAs, and two rRNAs. One of the genes is coding for the highly conserved enzyme cytochrome b (cytb) (Meyer et al., 1995). Meyer et al. (1995) described a method using PCR primers for amplification of sequences in the conserved areas of the vertebrate mitochondrial cytochrome b gene, which flank sufficient restriction sites for interspecific differentiation of species (Figure 4.1). During the course of evolution, transfer of mtDNA sequences to the nucleus can occur. Therefore, care in interpreting PCR-generated sequences may be necessary, particularly in those produced with universal primers. These nuclear pseudogenes are a potential source of artifact when total DNA is used in PCR-RFLP analyses. Burgener and H€ ubner (1998) described a quick method for the enrichment of mitochondrial DNA which resulted in unambiguous PCR-RFLP patterns. cytb gene cytb-PCR

Mitochondrial DNA

cytb-amplicon Digestion with restriction enzymes cattle 74 bp

pig

285 bp

74 bp 132 bp 153 bp

Hae III Restriction patterns after gel electrophoresis

285 Hae III 153 132 74 cattle

cattle+ pig

pig

FIGURE 4.1 Principle of PCR-RFLP analysis. Amplification of conserved regions of the mitochondrial cytb gene. For the differentiation of cattle and pig, amplicons were digested with Hae III and restriction patterns separated by gel electrophoresis.

APPLICATIONS: MEAT SPECIES IDENTIFICATION

4.2

87

APPLICATIONS: MEAT SPECIES IDENTIFICATION

4.2.1

Method According to Meyer et al. (1995)

One of the first papers describing PCR methods in food analysis was published by Meyer et al. (1995). A conserved 359-bp region, including a variable 307-bp region, is obtained for lots of mammals, poultry, and game meat species. The differentiation of animal species is possible after endonuclease digestion of the PCR amplicons and gel electrophoretical separation of restriction fragments. In a detailed table, the authors gave information on restriction patterns of important animal species based on sequence data and experimental verification (Table 4.1). In our experience this method can be used in a very broad range of animal species differentiation, especially if no other method is available [e.g., for rare or exotic animal species (birds, mammals, and reptiles)]. The method can also be used for fish species differentiation; however, the use of adapted primer pairs is recommended (see Section 4.2.3). The principal applications and limitations of the method are presented below. Raw Meat Figure 4.2 shows the analysis of important meat species: beef, pork, lamb, turkey, chicken, and ostrich. Amplified DNA was digested with Alu I and Hae III. Even using only these two enzymes, the animal species can be distinguished clearly if no other species is expected. Selection of the enzyme depends on the animal species to be differentiated. In practice, we use three different restriction enzymes. Sometimes, additional unexpected (mostly weak) signals can be obtained. For example, in lane 3 an additional 285-bp fragment was obtained for the lamb reference after digestion with Hae III. One explanation of this phenomenon is the presence of nuclear pseudogenes (see Section 4.1). Mitochondrial genes could have been transferred to the nuclear genome during evolution. Due to the fact that total DNA is extracted and analyzed in PCR-RFLP, possible artifacts caused by nuclear DNA have to be taken into consideration. However, in most cases, these additional signals do not affect PCRRFLP analysis. If uncertainties in species identification remain, mitochondrial DNA

TABLE 4.1 PCR-RFLPs for Differentiation of Meat Species According to Meyer et al. (1995) Restriction Enzyme

Species and Size of DNA Fragment After Digestion with Endonuclease Pig

Cattle

Sheep

Goat

Chicken

Turkey

Alu I

244 115 153 132 74

190 169 285 74

359

359

359

359

159 126 74

230 74 55

159 126 74

126 103 74 56

Hae III

88

PCR–RESTRICTION FRAGMENT LENGTH POLYMORPHISM ANALYSIS

FIGURE 4.2 Analysis of meat from important farm animal species. Restriction fragments of cytb amplicons (359 bp) obtained with Alu I (left) and Hae III (right) according to Meyer et al. (1995). Lane 1, beef; lane 2, pork; lane 3, lamb; lane 4, turkey; lane 5, chicken; lane 7, ostrich. M, molecular-weight marker (50-bp ladder).

enrichment would be a possible tool to provide unambiguous cytb PCR-RFLP patterns (Burgener and H€ ubner, 1998). Game Meat An analysis of some game meat species is shown in Figure 4.3. Amplified DNA was digested with Hinf I and Mbo II. Differentiation between European and Siberian roe deer is possible using Mbo II (lanes 1 and 2). Most likely, different fragments originate from nuclear pseudogenes (see above). All four game meat samples clearly differ from

FIGURE 4.3 Analysis of game meat species. Restriction fragments of cytb amplicons (359 bp) obtained with Hinf I (left) and Mbo II (right) according to Meyer et al. (1995). Lane 1, Siberian roe deer (Capreolus pygargus); lane 2, European roe deer (Capreolus capreolus); lane 3, European red deer (Cervus elaphus elaphus); lane 4, fallow deer (Dama dama); lane 5, beef. M, molecularweight marker (50-bp ladder).

APPLICATIONS: MEAT SPECIES IDENTIFICATION

89

FIGURE 4.4 Differentiation between rabbit and hare. Restriction fragments of cytb amplicons (359 bp) obtained with Mbo I (left) and Hinf I (right) according to Meyer et al. (1995). Lane 1, hare; lane 2, rabbit. M, molecular-weight marker (50-bp ladder).

beef (lane 5). In our experience, at least three different enzymes should be used for game meat species analysis (Hinf I, Mbo II, and Mbo I). Restriction patterns obtained with Xba I and Hae III can be used for additional verification of the results (Waiblinger and Weber, 1998). Figure 4.4 illustrates the differentiation of rabbit and hare with the enzymes Mbo I and Hinf I. Mixtures and Heated Materials In general, the PCR-RFLP method described can also be used for the analysis of mixtures of meat. However, if more than two different species are present in the sample, the restriction patterns become too complex and can no longer be evaluated easily. In practice, the analysis of mixtures is relevant in heated and processed materials. Figure 4.5 shows some limitations of the PCR-RFLP method: Restriction patterns of heat-treated reference samples (sausages) with defined amounts of different meat species are separated by gel electrophoresis. In lanes 1 to 5, the RFLP patterns of boiled sausages treated 1 h at 70 to 75 C are shown. Proportions of 1% beef, lamb, turkey, and chicken mixed within pork cannot be detected. Furthermore, traces of pork in the range of 1% are not detectable in chicken or turkey meat. Similar (high) amounts of pork and beef in the range of 50% can both be detected (lane 3). However, traces of sheep (4%) also present in the sample can just be presumed from the undigested fragment after digestion with Alu I (lane 3, left). In lanes 6 to 9, the analysis of preserves with different heat treatments (121 C, F value 20 mg/kg (ppm)]. 9.3.1

Polymerase Chain Reaction

Many PCR assays have been published for the detection of allergen residues in crops, raw ingredients, and processed products (Table 9.1). Although the selection of primers and PCR conditions can be optimized as to both specificity and sensitivity, a second step is sometime suggested to confirm specificity of the amplified PCR product because unrelated products with the same base pair size can lead to false positives. To

178

Soybean

1% protein concentrate NR 0.01% soy flour

414 þ 118 343 208

le1 (lectin gene)

trnL (chloroplast tRNA) Gly m Bd 30K

0.001% peanut DNA in wheat DNA samples

PCR þ nested PCR

95

Peanut agglutinin precursor

PCR

Legumes, cakes, hamburgers, sausage

Commercial processed foods Tree nuts, soybean, potato, corn, rice, wheat, meats, fish, commercial foods Processed meat products Soybeans