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Springer Protocols

Design of Primers and Probes for Quantitative Real-Time PCR Methods

Authors:

Alicia Rodríguez 1 ,

Mar Rodríguez 1 ,

Juan J. Córdoba 1 ,

María J. Andrade Author Email 1

Alicia Rodríguez 1 ,

Mar Rodríguez 1 ,

Juan J. Córdoba 1 ,

María J. Andrade Author Email 1

Overview | DOI: 10.1007/978-1-4939-2365-6_3

Affiliations:

  1. Food Hygiene and Safety, Meat and Meat Products Research Institute, Faculty of Veterinary Science, University of Extremadura, Cáceres, Spain

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Full Text Entitlement Icon Access enabled via: An Institution

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

…more

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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Keywords

Techniques:

Real-time PCR, Primer And Probe Design, Quantitative Reverse Transcription PCR

Others:

Validation, Primers, Probes, Software and databases

Citations (33)

Related articles

References

  1. Invitrogen (2008) Real-time PCR: from theory to practice. . Accessed 6 Nov 2013http://corelabs.cgrb.oregonstate.edu/sites/default/files/Real Time PCR.From Theory to Practice.pdf
  2. Rodríguez-Lázaro D, Hernández M (2013) Real time PCR in food science: introduction. Curr Issues Mol Biol 15:25–38
  3. 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
  4. 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
  5. Heid CA, Stevens J, Livak KJ et al (1996) Real time quantitative PCR. Genome Res 6:986–994
  6. Thornton B, Basu C (2011) Real-time PCR (qPCR) primer design using free online software. Biochem Mol Biol Educ 39:145–154
  7. Nolan T, Hands RE, Bustin SA (2006) Quantification of mRNA using real-time RT-PCR. Nat Protoc 1:1559–1582
  8. Qiagen (2010) Critical factors for successful real-time PCR. . Accessed 9 Nov 2013http://www.qiagen.com/es/resources/resourcedetail?id=f7efb4f4-fbcf-4b25-9315-c4702414e8d6&lang=en
  9. 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
  10. 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
  11. 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
  12. Chuang LY, Cheng YH, Yang CH (2013) Specific primer design for the polymerase chain reaction. Biotechnol Lett 35:1541–1549
  13. 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
  14. 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
  15. McConlogue L, Brow MA, Innis MA (1988) Structure-independent DNA amplification by PCR using 7-deaza-20-deoxyguanosine. Nucleic Acids Res 16:9869
  16. Mitsuhashi M (1996) Technical report: Part 1. Basic requirements for designing optimal oligonucleotide probe sequences. J Clin Lab Anal 10:277–284
  17. Wittwer CT, Herrmann MG, Moss AA et al (1997) Continuous fluorescence monitoring of rapid cycle DNA amplification. Biotechniques 22:130–131
  18. 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
  19. Wu JS, Lee C, Wu CC et al (2004) Primer design using genetic algorithm. Bioinformatics 20:1710–1717
  20. 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
  21. Simonsson T, Pecinka P, Kubista M (1998) DNA tetraplex formation in the control region of c-myc. Nucleic Acids Res 26:1167–1172
  22. 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
  23. 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
  24. 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
  25. Kubista M, Andrade JM, Bengtsson M et al (2006) The real-time polymerase chain reaction. Mol Asp Med 27:95–125
  26. Polz MF, Cavanaugh CM (1998) Bias in template-to-product rations in multitemplate PCR. Appl Environ Microbiol 64:3724–3730
  27. Linhart C, Shamir R (2005) The degenerate primer design problem: theory and applications. J Comput Biol 12:431–456
  28. Biorad (2013) qPCR assay design and optimization. . Accessed 24 Oct 2013http://www.bio-rad.com/en-es/applications-technologies/qpcr-assay-design-optimization
  29. Kalendar R, Lee D, Schulman AH (2011) Java web tools for PCR, in silico PCR, and oligonucleotide assembly and analysis. Genomics 98:137–144
  30. Abd-Elsalam KA (2003) Bioinformatic tools and guideline for PCR primer design. Afr J Biotechnol 2:91–95
  31. 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
  32. 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
  33. 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
  34. 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
  35. Rozen S, Skaletsky H (2000) Primer3 on the WWW for general users and for biologist programmers. Methods Mol Biol 132:365–386
  36. Untergasser A, Cutcutache I, Koressaar T et al (2012) Primer3: new capabilities and interfaces. Nucleic Acids Res 40:e115
  37. Untergasser A, Nijveen H, Rao X et al (2007) Primer3Plus, an enhanced web interface to Primer3. Nucleic Acids Res 35:W71–W74
  38. Marshall OJ (2004) PerlPrimer: cross-platform, graphical primer design for standard, bisulphite and real-time PCR. Bioinformatics 20:2471–2472
  39. 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
  40. Boutros PC, Okey AB (2004) PUNS: transcriptomic- and genomic-in silico PCR for enhanced primer design. Bioinformatics 20:2399–2400
  41. Altschul SF, Gish W, Miller W et al (1990) Basic local alignment search tool. J Mol Biol 215:403–410
  42. 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
  43. Ziesel AC, Chrenek MA, Wong PW (2008) MultiPriDe: automated batch development of quantitative real-time PCR primers. Nucleic Acids Res 36:3095–3100
  44. 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
  45. Brosseau JP, Lucier JF, Lapointe E et al (2010) High-throughput quantification of splicing isoforms. RNA 16:442–449
  46. Sobhy H, Colson P (2012) Gemi: PCR primers prediction from multiple alignments. Comp Funct Genomics 2012:783138. doi:10.1155/2012/783138
  47. 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
  48. Applied Biosystems (2004) Primer Express software version 3.0. getting started guide. . Accessed 10 Jan 2005http://www.bu.edu/picf/files/2010/11/Primer-express-30.pdf
  49. Zuker M (2003) Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res 31:3406–3415
  50. 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
  51. 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
  52. 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
  53. Wu X, Munroe DJ (2006) EasyExonPrimer: automated primer design for exon sequences. Appl Bioinformatics 5:119–120
  54. Cao Y, Sun J, Zhu J et al (2010) PrimerCE: designing primers for cloning and gene expression. Mol Biotechnol 46:113–117
  55. 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
  56. Fredslund J (2008) DATFAP: a database of primers and homology alignments for transcription factors from 13 plant species. BMC Genomics 9:140
  57. 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
  58. Kalendar R, Lee D, Schulman AH (2009) FastPCR software for PCR primer and probe design and repeat search. Genes Genomes Genomics 3:1–14
  59. 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
  60. 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
  61. 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
  62. 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
  63. 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
  64. Mallona I, Weiss J, Egea-Cortines M (2011) pcrEfficiency: a web tool for PCR amplification efficiency prediction. BMC Bioinformatics 12:404
  65. 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
  66. Applied Biosystems (2010) Real-time PCR systems. Reagent guide. . Accessed 7 Jul 2010https://www3.appliedbiosystems.com/cms/groups/mcb_support/documents/generaldocuments/cms_052263.pdf
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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

…more

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.

less

Related articles

References

  1. Invitrogen (2008) Real-time PCR: from theory to practice. . Accessed 6 Nov 2013http://corelabs.cgrb.oregonstate.edu/sites/default/files/Real Time PCR.From Theory to Practice.pdf
  2. Rodríguez-Lázaro D, Hernández M (2013) Real time PCR in food science: introduction. Curr Issues Mol Biol 15:25–38
  3. 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
  4. 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
  5. Heid CA, Stevens J, Livak KJ et al (1996) Real time quantitative PCR. Genome Res 6:986–994
  6. Thornton B, Basu C (2011) Real-time PCR (qPCR) primer design using free online software. Biochem Mol Biol Educ 39:145–154
  7. Nolan T, Hands RE, Bustin SA (2006) Quantification of mRNA using real-time RT-PCR. Nat Protoc 1:1559–1582
  8. Qiagen (2010) Critical factors for successful real-time PCR. . Accessed 9 Nov 2013http://www.qiagen.com/es/resources/resourcedetail?id=f7efb4f4-fbcf-4b25-9315-c4702414e8d6&lang=en
  9. 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
  10. 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
  11. 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
  12. Chuang LY, Cheng YH, Yang CH (2013) Specific primer design for the polymerase chain reaction. Biotechnol Lett 35:1541–1549
  13. 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
  14. 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
  15. McConlogue L, Brow MA, Innis MA (1988) Structure-independent DNA amplification by PCR using 7-deaza-20-deoxyguanosine. Nucleic Acids Res 16:9869
  16. Mitsuhashi M (1996) Technical report: Part 1. Basic requirements for designing optimal oligonucleotide probe sequences. J Clin Lab Anal 10:277–284
  17. Wittwer CT, Herrmann MG, Moss AA et al (1997) Continuous fluorescence monitoring of rapid cycle DNA amplification. Biotechniques 22:130–131
  18. 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
  19. Wu JS, Lee C, Wu CC et al (2004) Primer design using genetic algorithm. Bioinformatics 20:1710–1717
  20. 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
  21. Simonsson T, Pecinka P, Kubista M (1998) DNA tetraplex formation in the control region of c-myc. Nucleic Acids Res 26:1167–1172
  22. 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
  23. 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
  24. 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
  25. Kubista M, Andrade JM, Bengtsson M et al (2006) The real-time polymerase chain reaction. Mol Asp Med 27:95–125
  26. Polz MF, Cavanaugh CM (1998) Bias in template-to-product rations in multitemplate PCR. Appl Environ Microbiol 64:3724–3730
  27. Linhart C, Shamir R (2005) The degenerate primer design problem: theory and applications. J Comput Biol 12:431–456
  28. Biorad (2013) qPCR assay design and optimization. . Accessed 24 Oct 2013http://www.bio-rad.com/en-es/applications-technologies/qpcr-assay-design-optimization
  29. Kalendar R, Lee D, Schulman AH (2011) Java web tools for PCR, in silico PCR, and oligonucleotide assembly and analysis. Genomics 98:137–144
  30. Abd-Elsalam KA (2003) Bioinformatic tools and guideline for PCR primer design. Afr J Biotechnol 2:91–95
  31. 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
  32. 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
  33. 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
  34. 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
  35. Rozen S, Skaletsky H (2000) Primer3 on the WWW for general users and for biologist programmers. Methods Mol Biol 132:365–386
  36. Untergasser A, Cutcutache I, Koressaar T et al (2012) Primer3: new capabilities and interfaces. Nucleic Acids Res 40:e115
  37. Untergasser A, Nijveen H, Rao X et al (2007) Primer3Plus, an enhanced web interface to Primer3. Nucleic Acids Res 35:W71–W74
  38. Marshall OJ (2004) PerlPrimer: cross-platform, graphical primer design for standard, bisulphite and real-time PCR. Bioinformatics 20:2471–2472
  39. 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
  40. Boutros PC, Okey AB (2004) PUNS: transcriptomic- and genomic-in silico PCR for enhanced primer design. Bioinformatics 20:2399–2400
  41. Altschul SF, Gish W, Miller W et al (1990) Basic local alignment search tool. J Mol Biol 215:403–410
  42. 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
  43. Ziesel AC, Chrenek MA, Wong PW (2008) MultiPriDe: automated batch development of quantitative real-time PCR primers. Nucleic Acids Res 36:3095–3100
  44. 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
  45. Brosseau JP, Lucier JF, Lapointe E et al (2010) High-throughput quantification of splicing isoforms. RNA 16:442–449
  46. Sobhy H, Colson P (2012) Gemi: PCR primers prediction from multiple alignments. Comp Funct Genomics 2012:783138. doi:10.1155/2012/783138
  47. 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
  48. Applied Biosystems (2004) Primer Express software version 3.0. getting started guide. . Accessed 10 Jan 2005http://www.bu.edu/picf/files/2010/11/Primer-express-30.pdf
  49. Zuker M (2003) Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res 31:3406–3415
  50. 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
  51. 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
  52. 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
  53. Wu X, Munroe DJ (2006) EasyExonPrimer: automated primer design for exon sequences. Appl Bioinformatics 5:119–120
  54. Cao Y, Sun J, Zhu J et al (2010) PrimerCE: designing primers for cloning and gene expression. Mol Biotechnol 46:113–117
  55. 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
  56. Fredslund J (2008) DATFAP: a database of primers and homology alignments for transcription factors from 13 plant species. BMC Genomics 9:140
  57. 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
  58. Kalendar R, Lee D, Schulman AH (2009) FastPCR software for PCR primer and probe design and repeat search. Genes Genomes Genomics 3:1–14
  59. 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
  60. 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
  61. 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
  62. 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
  63. 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
  64. Mallona I, Weiss J, Egea-Cortines M (2011) pcrEfficiency: a web tool for PCR amplification efficiency prediction. BMC Bioinformatics 12:404
  65. 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
  66. Applied Biosystems (2010) Real-time PCR systems. Reagent guide. . Accessed 7 Jul 2010https://www3.appliedbiosystems.com/cms/groups/mcb_support/documents/generaldocuments/cms_052263.pdf
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Citations (33)

Keywords

Techniques:

Real-time PCR, Primer And Probe Design, Quantitative Reverse Transcription PCR

Others:

Validation, Primers, Probes, Software and databases

Taqman Probe Design Tool Free

Source: https://experiments.springernature.com/articles/10.1007/978-1-4939-2365-6_3

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