PCR Troubleshooting and Optimization

PCR Troubleshooting and OptimizationThe Essential Guide

Ec Si niickCaister Academic Press

Copyright 201 1Caister Academic Press Norfolk, UKwww.caister.comBritish Library Cataloguing-in-Publication Data A catalogue record for tlis book is available from the British LibraryISBN: 978-1-904455-72-1Description or mention of instrumentation, sotvvare, or other Products in this book does not imply endorsement by the author or publisher. The author and publisher do not assume responsibility for the validity of any products or procedures mentioned or described in this book or for the consequences of their use.All rights reserved. No part of this publication may be reproduced, stored in a retrieval System, or transmitted, in any orm or by any means, electronic, mechanical, photocopying, recording or othervvise, vvithout the prior permission of the publisher. No caim to original u.s. Government works.Cover image adapted hom Pigure 1.4Printed and bound in Great Britain

List of Contributors_V1 Magic in Solution: an Introduction and Brief History ofPCR 1Carl T. VVittvver and Jared s. Farrar2 qPCR Inhibition and Amplitication of Difficult Templates23Jack M. Gailup3 Signiticance of Controls and Standard Curves in PCR67lan Kavanagh, Gervvyn Jones and Saima N. Nayab4 Obtaining Maximum PCR Sensitivity and Speciticity79Cameron N. Gundry and Matthevv D. Poulson5 RT-PCR Optimization Strategies976 Real-Time PCR Instrumentation: an Instrument Selection Guide 119Sandrine Javorschi-Miller and Ivan Delgado Orlic7 Quantitative PCR Data Analysis - Unlockinq the Secretto Successtul Results139Jan Hellemans and Jo Vandesompee8 The MIQE Guidelines Uncloaked151Greg Shiplev9 PCR Applications for Epigenetics Research167Gavin Meredith, Miroslav Dudas, Mark Landers, Vasiliki Anest,Jonathan Wang, Caitu Chen, Peter Jozsi and Christopher Adams10 High-Resolution Melting Analysis201John F. Mackay and Carl T. Wittwer11 Microtluidic Emulsion PCR217N. Reginald Beer and John H. LeamonIndex233Colour platesAlCurrent Books of InterestViruses and Intereron: Current Research2011Essentials of Veterinary Parasitology2011Hepatitis C: Antiviral Drug Discovery and Development2011Streptomyces: Molecular Biology and Biotechnology2011Alphaherpesviruses: Molecular Virology2011Recent Advances in Plant Virology2011Vaccine Design: Innovative Approaches and Novel Strategies2011Salmonella'. From Genome to Function2011PCR Troubleshooting and Optimization: The Essential Guide2011Insect Virology2010Environmental Microbiology: Current Technology and VVater Applications 2011 Sensory Mechanisms in Bacteria2010Bitidobacteria: Genomics and Molecular Aspects2010Molecular Phylogeny of Microorganisms2010Nanotechnology in Water Treatment Applications2010Iron Uptake and Homeostasis in Microorganisms2010Caliciviruses: Molecular and Cellular Virology2010Epstein-Barr Virus: Latency and Transtormation2010Anaerobic Parasitic Protozoa: Genomics and Molecular Biology2010Lentiviruses and Macrophages: Molecular and Cellular Interactons2010Microbial Population Genetics2010Borrelia'. Molecular and Cellular Biology2010lnfluenza: Molecular Virology2010RNA Interterence and Viruses: Current Innovations and Future Trends 2010 Retroviruses: Molecular Biology, Genomics and Pathogenesis2010Metagenomics: Theory, Methods and Applications2010Aspergillus: Molecular Biology and Genomics2010Environmental Molecular Microbiology2010Neisseria: Molecular Mechanisms of Pathogenesis2010Frontiers in Dengue Virus Research2010ABC Transporters in Microorganisms2009Pili and Flagella: Current Research and Future Trends2009Lab-on-a-Chip Technology: Biomolecular Separation and Analysis2009Lab-on-a-Chip Technology: Fabrication and Microtluidics2009Bacterial Polysaccharides: Current Innovations and Future Trends2009Microbial Toxins: Current Research and Future Trends2009Acanthamoeba2009Bacterial Secreted Proteins2009Lactobacillus Molecular Biology: From Genomics to Probiotics2009Mycobacteriunr. Genomics and Molecular Biology2009Real-Time PCR: Current Technology and Applications2009

miro.dudas(tDlifetech.comChristopher AdamsLife Technologies Carsbad, CA USAchristopher.adams(a)lifetech.comVasiliki AnestLife Technologies Carlsbad, CA USAvasiliki.anest(a)lifetech.comN. Reginald BeerLawrence Livermore National LaboratoryLivermore, CAUSAbeer2() llnl.govCaifu ChenGenomic Assays R&D Molecular Biology Systems Division Life Technologies Corporation Foster City, CA USAcaifu.chen(d)lifetech.comMiroslavDudasLife Technologies Carlsbad, CA USAJared s. FarrarDepartment of PathologyUniversity of Utah Health Sciences CenterSalt Lake City, UTUSAj.farrar(d)utah.eduJackM. GallupDepartment of Veterinary Pathology College of Veterinary Medicine Iowa State University Ames, A USAeag(a) iastate.eduCameron N. GundryIdaho Technology, Inc.Salt Lake City, UT USAcameron_gundry(d)idahotech.com Jan HellemansGhent University Center for Medical Genetics Ghent;BiogazelleZwijnaardeBelgiumjan.hellemans(DUG ent.be jan.hellemans(d)b iogazelle.com

SandrineJavorschi-MillerIlumina Inc.San Diego, CA USAGavin MeredithLife Technologies Carlsbad, CA USA

smiller(d)illumina.comgavin.meredith(d)lifetech.com

Gerwyn JonesThermo Fisher ScientihcEpsomSurreyKSaima N. NayabThermo Eisher ScientihcEpsomSurreyUK

gerwyn.jones(d)thermosher.comsaima.nayab(a)thermofisher.com

Peter JozsiEpigenie LLC Delmar, CA USAIvan Delgado OrlicMouse Genotype Carlsbad; CA USA

pete(d)epigenie.comivan(d)m ousegenotype.com

lan KavanaghThermo Eisher ScienticEpsomSurreyUKMichael w. PfafflPhysiology Weihenstephan Technical University Munich Freising Germany

ian.kavanagh()thermofisher.commichael.pfatl(d) wzw.tum.de

MarkLandersLife Technologies Carlsbad, CA USAMatthew D. PoulsonIdaho Technology, Inc. Salt Lake City, UT USA

mark.landers(d)l ifetech.commatthew_poulson(d)idahotech.com

John H. Leamonlon Torrent Systems Guilord, CT USAMartna ReiterBioEPS GmbHFreisingGermany

jleamon(d)iontorrents.commartina.reiter(d)bioeps.com

John F. Mackaydnature diagnostics and research LtdGisborneNew ZealandGregShipleyUT Health Science Center - HoustonHouston, TXUSA

john()dnature.co.nzGregory.L.Shipley(d)uth. tmc.edu

vi I Contributors

Contributors I

Contributors I

jonathan.wang(d)lifetech.comJo VandesompeleGhent University Center for Medical Genetics Ghent;BiogazeleZwijnaardeBelgiumjoke.vandesompele()UGent.bejo.vandesompele(d)biogazelle.comJonathan WangPCR Systems R & DLife Technologies CorporationFoster City, CAUSACarl T. WittwerDepartment of Pathology niversity of Utah School ofMedicine; ARUP Institute for Clinica and Experimental Pathology Salt Lake City, UT USAcarl.wittwer()path.utah.edu

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5.) VVhat m.thod do you u.a to ONaa .at your RNA (or RNa.e-treat your DNA)7 (u. " ") AmbtonABI TURBO DNaa. (2 'uLJ? th.r DMa*. Na. (1 UuL)?#0th.r?(1 Unha/ulNon. Iprim.rs and/or prob.a span g.nomtc invonslor oth.r rtnon?

Figure 2.5 A partial glimpse of the Questionnaire intertace for the PRExcel-Q program. The screen-capture image here represents only of a small portion of the entire program. The entire P-Q program consists of 35 Excel 2003 tlles which interdependently calculate all set-up parameters for all qPCR approaches. The printouts generated by the program are fail-safe recipes for setting up every part of the qPCR workflow. P-Q is not a qPCR data analysis software.qPCR Inhibition and Amplitication of Difficult Templates I #

qPCR Inhibition and Amplitication of Difficult Templates I 53

sample mixture (termed Stock r) as the Tanguage, gauge or measure by which all sample and Standard dilutions and Standard curve ranges are established for each target of interest within each entire, particular sample set. It turns out - as revealed by using and tweaking the program for several years - that the point or range at (or vvithin) which all samples and standards have been diluted to exist within their valid dynamic ranges, most (if not all) of the qPCRproblems associated with inhibition are, as if by implicit dehnition, gone. In otherwords, dynamically appropriate dilution ranges, it seems, can only be identihed when the qPCR is behaving correctly in the first place. The qPCR simply vvould not be able to ex- hibit any of the healthy maniestations of a good 2-to-the-n-like process (high-amplification ehciency and dilution-responsive LOG-linear-amplification-like-capability) if inhibitory materials or processes were still waging an inluence. The program tells each investigator whether the samples (and targets within the samples) are even worth investigating in the first place on account of pilot results obtained up-front from the initial Test Plate. Given good primer and probe designs to begin with, P-Q^can receive, process and find the useful range for every qPCR sample and target in any qPCR sample set, as long as care has been

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SPUD assay. The SPUD assay involves running a control qPCR using a synthesized 101 bp amplicon (from the potato genome) in parallel with an identical qPCR reaction that contains both the spiked potato amplicon (typically 22,000 copies of it per reaction) and the biological sample or Standard. If the sample contains an inhibitor, it will take more cycles for the potato amplicon luorescence signal to reach Cq. n the presence of inhibi- tion, the Cq will appear later. But, additionally conounding such analyses is the possibility that inhibitory agents may aftect the 3' and the 5' qPCR assay to different degrees, about which Dr Nolan has pointed out that it is never safe to assume that certain inhibitors will always inhibit all qPCR assays to the same degree. Thereore, correcting for inhibitors by quantifying a reerence gene is really not a solution. While the SPD assay can identify the presence of an inhibitor, the results of the 3':5' assay may also reflect the variable eects of an inhibitor, resulting in either more 5' amplicons or more 3' amplicons. As a suggested quality control practice, Dr Nolan has thus recommended using both 3':5' and SPUD as- says on every RNA sample (Nolan et ai, 2006; Glaser, 2007).qPCR and musicqPCR Inhibition and Amplitication of Difficult Templates I #

qPCR Inhibition and Amplitication of Difficult Templates I 57

As a side-note to some of the seriousness that abounds within the world of troubleshoot- ing and properly rmulating good RT, RT-PCR, PCR and qPCR reactions, there arises the entertaining relationship between the classical PCR equation Xn = X0(2)" and the rmula for requency (in Hertz) of musical notes (tones on a Standard size, A440Hz piano), Hzn = Hz0(2) l2. What could possibly be learned from such a relationship? Plenty. In particular, when qPCR reactions are less than 100% ehcient (e.g. when exponential am- plikation or amplicon doubling falls short of a factor of 2 per cycle), its governing rmula becomes XCq = Xc(AMp)Cq. Cq values generated at less than 100% amplihcation ehciency, when compared with the same target concentration dierences represented by Cq values generated at 100% eciency (or AMp = 2), appear irther and further apart. The rate of Cq appearance (tempo) slows down - yet, the successive Cq values are actually signifying or playing the exact same melody, only on ligher keys than the corresponding 100% ef- hcient reaction. The other difference here, to fully accommodate the analogy, is that the piano has been stretched, and the same Taq-hand is merely playing the same tune only over a longer time period using larger physical key distances, due to the stretching of the keyboard (the Cq values and/or piano keys are physically arther apart). The higher the frequency of the note on a piano, the less discerning the human ear is at telling which note is which. The same thing applies to high Cq values generated by qPCR (e.g. Cq values above 38) when