Abstract
Design of primers and probes is one of the most crucial factors affecting the success and quality of quantitative real-time PCR (qPCR) analyses, since an accurate and reliable quantification depends on using efficient primers and probes. Design of primers and probes should meet several criteria to find potential primers and probes for specific qPCR assays. The formation of primer-dimers and other non-specific products should be avoided or reduced. This factor is especially important when designing primers for SYBR® Green protocols but also in designing probes to ensure specificity of the developed qPCR protocol. To design primers and probes for qPCR, multiple software programs and websites are available being numerous of them free. These tools often consider the default requirements for primers and probes, although new research advances in primer and probe design should be progressively added to different algorithm programs. After a proper design, a precise validation of the primers and probes is necessary. Specific consideration should be taken into account when designing primers and probes for multiplex qPCR and reverse transcription qPCR (RT-qPCR).
This chapter provides guidelines for the design of suitable primers and probes and their subsequent validation through the development of singlex qPCR, multiplex qPCR, and RT-qPCR protocols.
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References
Invitrogen (2008) Real-time PCR: from theory to practice. http://corelabs.cgrb.oregonstate.edu/sites/default/files/Real%20Time%20PCR.From%20Theory%20to%20Practice.pdf. Accessed 6 Nov 2013
Rodríguez-Lázaro D, Hernández M (2013) Real time PCR in food science: introduction. Curr Issues Mol Biol 15:25–38
Rosadas C, Cabral-Castro MJ, Vicente AC et al (2013) Validation of a quantitative real-time PCR assay for HTLV-1 proviral load in peripheral blood mononuclear cells. J Virol Methods 193:536–541
Holland PM, Abramson RD, Watson R et al (1991) Detection of specific polymerase chain reaction product by utilizing the 50–30 exonuclease activity of Thermus aquaticus DNA polymerase. Proc Natl Acad Sci U S A 88: 7276–7280
Heid CA, Stevens J, Livak KJ et al (1996) Real time quantitative PCR. Genome Res 6:986–994
Thornton B, Basu C (2011) Real-time PCR (qPCR) primer design using free online software. Biochem Mol Biol Educ 39:145–154
Nolan T, Hands RE, Bustin SA (2006) Quantification of mRNA using real-time RT-PCR. Nat Protoc 1:1559–1582
Qiagen (2010) Critical factors for successful real-time PCR. http://www.qiagen.com/es/resources/resourcedetail?id=f7efb4f4-fbcf-4b25-9315-c4702414e8d6&lang=en. Accessed 9 Nov 2013
Yu Y, Lee C, Kim J et al (2005) Group-specific primer and probe sets to detect methanogenic communities using quantitative real-time polymerase chain reaction. Biotechnol Bioeng 89:670–679
Raymaekers M, Smets R, Maes B et al (2009) Checklist for optimization and validation of real-time PCR assays. J Clin Lab Anal 23:145–151
Lim J, Shin SG, Lee S et al (2011) Design and use of group-specific primers and probes for real-time quantitative PCR. Front Environ Sci Eng 5:28–39
Chuang LY, Cheng YH, Yang CH (2013) Specific primer design for the polymerase chain reaction. Biotechnol Lett 35:1541–1549
Hanna SE, Connor CJ, Wang HH (2005) Real-time polymerase chain reaction for the food microbiologist: technologies, applications, and limitations. J Food Sci 70:49–53
Toouli CD, Turner DR, Grist SA et al (2000) The effect of cycle number and target size on polymerase chain reaction amplification of polymorphic repetitive sequences. Anal Biochem 280:324–326
McConlogue L, Brow MA, Innis MA (1988) Structure-independent DNA amplification by PCR using 7-deaza-20-deoxyguanosine. Nucleic Acids Res 16:9869
Mitsuhashi M (1996) Technical report: Part 1. Basic requirements for designing optimal oligonucleotide probe sequences. J Clin Lab Anal 10:277–284
Wittwer CT, Herrmann MG, Moss AA et al (1997) Continuous fluorescence monitoring of rapid cycle DNA amplification. Biotechniques 22:130–131
Ririe KM, Rasmussen RP, Wittwer CT (1997) Product differentiation by analysis of DNA melting curves during the polymerase chain reaction. Anal Biochem 245:154–160
Wu JS, Lee C, Wu CC et al (2004) Primer design using genetic algorithm. Bioinformatics 20:1710–1717
Marchesi JR (2001) Primer design for PCR amplification of environmental DNA targets. In: Rochelle PA (ed) Environmental molecular microbiology: protocols and applications. Horizon Scientific Press, Wymondham, pp 43–54
Simonsson T, Pecinka P, Kubista M (1998) DNA tetraplex formation in the control region of c-myc. Nucleic Acids Res 26:1167–1172
Giulietti A, Overbergh L, Valckx D et al (2001) An overview of real-time quantitative PCR: applications to quantify cytokine gene expression. Methods 25:386–401
Gunson RN, Collins TC, Carman WF (2006) Practical experience of high throughput real time PCR in the routine diagnostic virology setting. J Clin Virol 35:355–367
Saiki RK (1989) The design and optimization of the PCR. In: Erlich HA (ed) PCR technology: principles and applications for DNA amplification. McMillan Publishers (Stockton Press), New York, NY, pp 7–22
Kubista M, Andrade JM, Bengtsson M et al (2006) The real-time polymerase chain reaction. Mol Asp Med 27:95–125
Polz MF, Cavanaugh CM (1998) Bias in template-to-product rations in multitemplate PCR. Appl Environ Microbiol 64:3724–3730
Linhart C, Shamir R (2005) The degenerate primer design problem: theory and applications. J Comput Biol 12:431–456
Biorad (2013) qPCR assay design and optimization. http://www.bio-rad.com/en-es/applications-technologies/qpcr-assay-design-optimization. Accessed 24 Oct 2013
Kalendar R, Lee D, Schulman AH (2011) Java web tools for PCR, in silico PCR, and oligonucleotide assembly and analysis. Genomics 98:137–144
Abd-Elsalam KA (2003) Bioinformatic tools and guideline for PCR primer design. Afr J Biotechnol 2:91–95
Fredman D, Jobs M, Strömqvist L et al (2004) DFold: PCR design that minimizes secondary structure and optimizes downstream genotyping applications. Hum Mutat 24:1–8
Nonis A, Scortegagna M, Nonis A et al (2011) PRaTo: a web-tool to select optimal primer pairs for qPCR. Biochem Biophys Res Commun 415:707–708
Gubelmann C, Gattiker A, Massouras A et al (2011) GETPrime: a gene- or transcript-specific primer database for quantitative real-time PCR. Database 2011:bar040. doi:10.1093/database/bar040
Rychlik W (2007) OLIGO 7 primer analysis software. In: Yuryev A (ed) Methods in molecular biology, vol 402, PCR primer design. Humana, Totowa, NJ, pp 35–59
Rozen S, Skaletsky H (2000) Primer3 on the WWW for general users and for biologist programmers. Methods Mol Biol 132:365–386
Untergasser A, Cutcutache I, Koressaar T et al (2012) Primer3: new capabilities and interfaces. Nucleic Acids Res 40:e115
Untergasser A, Nijveen H, Rao X et al (2007) Primer3Plus, an enhanced web interface to Primer3. Nucleic Acids Res 35:W71–W74
Marshall OJ (2004) PerlPrimer: cross-platform, graphical primer design for standard, bisulphite and real-time PCR. Bioinformatics 20:2471–2472
Marshall OJ (2007) Graphical design of primers with PerlPrimer. In: Yuryev A (ed) Methods in molecular biology, vol 402, PCR primer design. Humana, Totowa, NJ, pp 403–414
Boutros PC, Okey AB (2004) PUNS: transcriptomic- and genomic-in silico PCR for enhanced primer design. Bioinformatics 20:2399–2400
Altschul SF, Gish W, Miller W et al (1990) Basic local alignment search tool. J Mol Biol 215:403–410
Arvidsson S, Kwasniewski M, Riaño-Pachón DM et al (2008) QuantPrime: a flexible tool for reliable high-throughput primer design for quantitative PCR. BMC Bioinformatics 9:465
Ziesel AC, Chrenek MA, Wong PW (2008) MultiPriDe: automated batch development of quantitative real-time PCR primers. Nucleic Acids Res 36:3095–3100
Vijaya SR, Kumar K, Zavaljevski N et al (2010) A high-throughput pipeline for the design of real-time PCR signatures. BMC Bioinformatics 11:340
Brosseau JP, Lucier JF, Lapointe E et al (2010) High-throughput quantification of splicing isoforms. RNA 16:442–449
Sobhy H, Colson P (2012) Gemi: PCR primers prediction from multiple alignments. Comp Funct Genomics 2012:783138. doi:10.1155/2012/783138
Brodin J, Krishnamoorthy M, Athreya G et al (2013) A multiple-alignment based primer design algorithm for genetically highly variable DNA targets. BMC Bioinformatics 14:255
Applied Biosystems (2004) Primer Express software version 3.0. getting started guide. http://www.bu.edu/picf/files/2010/11/Primer-express-30.pdf. Accessed 10 Jan 2005
Zuker M (2003) Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res 31:3406–3415
You FM, Huo N, Gu YQ et al (2009) ConservedPrimers 2.0: a high-throughput pipeline for comparative genome referenced intron-flanking PCR primer design and its application in wheat SNP discovery. BMC Bioinformatics 10:331
You FM, Huo N, Gu YQ et al (2008) BatchPrimer3: a high throughput web application for PCR and sequencing primer design. BMC Bioinformatics 9:253
Riaz T, Shehzad W, Viari A et al (2011) ecoPrimers: inference of new DNA barcode markers from whole genome sequence analysis. Nucleic Acids Res 39:e145
Wu X, Munroe DJ (2006) EasyExonPrimer: automated primer design for exon sequences. Appl Bioinformatics 5:119–120
Cao Y, Sun J, Zhu J et al (2010) PrimerCE: designing primers for cloning and gene expression. Mol Biotechnol 46:113–117
Lefever S, Vandesompele J, Speleman F et al (2009) RTPrimerDB: the portal for real-time PCR primers and probes. Nucleic Acids Res 37:D942–D945
Fredslund J (2008) DATFAP: a database of primers and homology alignments for transcription factors from 13 plant species. BMC Genomics 9:140
Wang X, Spandidos A, Wang H et al (2012) PrimerBank: a PCR primer database for quantitative gene expression analysis, 2012 update. Nucleic Acids Res 40:D1144–D1149
Kalendar R, Lee D, Schulman AH (2009) FastPCR software for PCR primer and probe design and repeat search. Genes Genomes Genomics 3:1–14
Guerrero D, Bautista R, Villalobos DP et al (2010) AlignMiner: a web-based tool for detection of divergent regions in multiple sequence alignments of conserved sequences. Algorithms Mol Biol 5:24
Taylor S, Wkem M, Dijkman G et al (2010) A practical approach to RT-qPCR: publishing data that conform to the MIQE guidelines. Methods 50:S1–S5
Lam CW, Mak CM (2013) Allele dropout caused by a non-primer-site SNV affecting PCR amplification: a call for next-generation primer design algorithm. Clin Chim Acta 421:208–212
Karlin S, Altschul SF (1990) Methods for assessing the statistical significance of molecular sequence features by using general scoring schemes. Proc Natl Acad Sci U S A 87:2264–2268
Bustin SA, Benes V, Garson JA et al (2009) The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin Chem 55:611–622
Mallona I, Weiss J, Egea-Cortines M (2011) pcrEfficiency: a web tool for PCR amplification efficiency prediction. BMC Bioinformatics 12:404
Edwards KJ (2004) Performing real-time PCR. In: Edwards K, Logan J, Saunders N (eds) Real-time PCR, an essential guide. Horizon Bioscience, Norfolk, pp 71–83
Applied Biosystems (2010) Real-time PCR systems. Reagent guide. https://www3.appliedbiosystems.com/cms/groups/mcb_support/documents/generaldocuments/cms_052263.pdf. Accessed 7 Jul 2010
Promega Corporation (2009) Protocols & applications guide. http://www.promega.com/~/media/files/resources/paguide/letter/paguide_us.pdf?la=en. Accessed 21 Oct 2013
Pfaffl MW (2004) Quantification strategies in real-time PCR. In: Bustin SA (ed) A-Z of Quantitative PCR (IUL Biotechnology, No. 5). International University Line (IUL), San Diego, CA, pp 87–112
Lee MA, Squirell DJ, Leslie DL et al (2004) Homogeneous fluorescent chemistries for real-time PCR. In: Edwards K, Logan J, Saunders N (eds) Real-time PCR, an essential guide. Horizon Bioscience, Norfolk, pp 31–70
Life Technologies Corporation (2012) Real-time PCR handbook. http://find.lifetechnologies.com/Global/FileLib/qPCR/RealTimePCR_Handbook_Update_FLR.pdf. Accessed 6 Nov 2013
Rajeevan MS, Ranamukhaarachchi DG, Vernon SD et al (2001) Use of real-time quantitative PCR to validate the results of cDNA array and differential display PCR technologies. Methods 25:443–451
Kavanagh I, Jones G, Nayab SN (2011) Significance of controls and standard curves in PCR. In: Kennedy S, Oswald N (eds) PCR troubleshooting and optimization: the essential guide. Caister Academic Press, Norfolk, pp 67–78
Pfaffl MW (2001) A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res 1:29–45
Gadkar VY, Filion M (2013) New developments in quantitative real-time polymerase chain reaction technology. Curr Issues Mol Biol 8:1–6
Ishii T, Sootome H, Shan L et al (2007) Validation of universal conditions for duplex quantitative reverse transcription polymerase chain reaction assays. Anal Biochem 362:201–212
Quellhorst, G., Rulli, S. (2008) A systematic guideline for developing the best real-time PCR primers. SABiosci. http://www.sabiosciences.com/manuals/RT2performanceWhitePaper.pdf. Accessed 26 Aug 2013
Bustin SA, Nolan T (2004) Analysis of mRNA expression by real-time PCR. In: Edwards K, Logan J, Saunders N (eds) Real-time PCR, an essential guide. Horizon Bioscience, Norfolk, pp 125–184
Zhang J, Byrne CD (1999) Differential priming of RNA templates during cDNA synthesis markedly affects both accuracy and reproducibility of quantitative competitive reverse-transcriptase PCR. Biochem J 337:231–241
Lekanne Deprez RH, Fijnvandraat AC, Ruijter JM et al (2002) Sensitivity and accuracy of quantitative real-time polymerase chain reaction using SYBR green I depends on cDNA synthesis conditions. Anal Biochem 307:63–69
VanGuilder HD, Vrana KE, Freeman WM (2008) Twenty-five years of quantitative PCR for gene expression analysis. Biotechniques 44:619–626
Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT method. Methods 25:402–408
Wang X, Seed B (2003) A PCR primer bank for quantitative gene expression analysis. Nucleic Acids Res 31:e154
Applied Biosystems (2008) Guide to performing relative quantitation of gene expression using real-time quantitative PCR. http://www3.appliedbiosystems.com/cms/groups/mcb_support/documents/generaldocuments/cms_042380.pdf. Accessed 2 Jun 2008
Bauer P, Rolfs A, Regitz-Zagrosek V et al (1997) Use of manganese in RT-PCR eliminates PCR artefacts resulting from DNase I digestion. Biotechniques 22:1128–1132
Bustin SA (2000) Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays. J Mol Endocrinol 25:169–193
Rodríguez A (2012) Desarrollo de métodos de PCR en tiempo real para la detección y cuantificación de mohos productores de micotoxinas en alimentos. Doctoral Thesis. University of Extremadura, Spain
Sayers EW, Barrett T, Benson DA et al (2012) Database resources of the National Center for Biotechnology Information. Nucleic Acids Res 40:D13–D25
Cui W, Taub DD, Gardner K (2007) qPrimerDepot: a primer database for quantitative real time PCR. Nucleic Acids Res 35:D805–D809
Acknowledgments
We acknowledge financial support of this work by projects “AGL2010-21623” and “Carnisenusa CSD2007-00016—Consolider Ingenio 2010” of the Spanish Government and GR10162 of the Government of Extremadura and FEDER.
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Rodríguez, A., Rodríguez, M., Córdoba, J.J., Andrade, M.J. (2015). Design of Primers and Probes for Quantitative Real-Time PCR Methods. In: Basu, C. (eds) PCR Primer Design. Methods in Molecular Biology, vol 1275. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2365-6_3
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DOI: https://doi.org/10.1007/978-1-4939-2365-6_3
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