PCR pp 1-31 | Cite as

In Silico PCR Tools for a Fast Primer, Probe, and Advanced Searching

Part of the Methods in Molecular Biology book series (MIMB, volume 1620)


The polymerase chain reaction (PCR) is fundamental to molecular biology and is the most important practical molecular technique for the research laboratory. The principle of this technique has been further used and applied in plenty of other simple or complex nucleic acid amplification technologies (NAAT). In parallel to laboratory “wet bench” experiments for nucleic acid amplification technologies, in silico or virtual (bioinformatics) approaches have been developed, among which in silico PCR analysis. In silico NAAT analysis is a useful and efficient complementary method to ensure the specificity of primers or probes for an extensive range of PCR applications from homology gene discovery, molecular diagnosis, DNA fingerprinting, and repeat searching. Predicting sensitivity and specificity of primers and probes requires a search to determine whether they match a database with an optimal number of mismatches, similarity, and stability. In the development of in silico bioinformatics tools for nucleic acid amplification technologies, the prospects for the development of new NAAT or similar approaches should be taken into account, including forward-looking and comprehensive analysis that is not limited to only one PCR technique variant. The software FastPCR and the online Java web tool are integrated tools for in silico PCR of linear and circular DNA, multiple primer or probe searches in large or small databases and for advanced search. These tools are suitable for processing of batch files that are essential for automation when working with large amounts of data. The FastPCR software is available for download at and the online Java version at

Key words

Polymerase chain reaction Isothermal amplification of nucleic acids DNA primers nucleic acid hybridization Primer binding site PCR primer and probe analysis Degenerate PCR Probe Genetic engineering tools DNA fingerprints 



Java Web tools are publicly available. They may not be reproduced or distributed for commercial use. This work was supported by the companies Primer Digital Ltd.


  1. 1.
    Walker-Daniels J (2012) Current PCR methods. Mat Methods 2:119. doi: 10.13070/mm.en.2.119 Google Scholar
  2. 2.
    Tisi LC et al. (2010) Nucleic acid amplification. Canada Patent CA2417798Google Scholar
  3. 3.
    Notomi T et al (2000) Loop-mediated isothermal amplification of DNA. Nucleic Acids Res 28(12):e63. doi: 10.1093/nar/28.12.e63 CrossRefGoogle Scholar
  4. 4.
    Walker GT et al (1992) Strand displacement amplification—an isothermal, in vitro DNA amplification technique. Nucleic Acids Res 20(7):1691–1696. doi: 10.1093/nar/20.7.1691 CrossRefGoogle Scholar
  5. 5.
    Banér J et al (1998) Signal amplification of padlock probes by rolling circle replication. Nucleic Acids Res 26(22):5073–5078. doi: 10.1093/nar/26.22.5073 CrossRefGoogle Scholar
  6. 6.
    Tatsumi K et al (2008) Rapid screening assay for KRAS mutations by the modified smart amplification process. J Mol Diagn 10(6):520–526. doi: 10.2353/jmoldx.2008.080024 CrossRefGoogle Scholar
  7. 7.
    Kwoh DY et al (1989) Transcription-based amplification system and detection of amplified human immunodeficiency virus type 1 with a bead-based sandwich hybridization format. Proc Natl Acad Sci U S A 86(4):1173–1177CrossRefGoogle Scholar
  8. 8.
    Fahy E et al (1991) Self-sustained sequence replication (3SR): an isothermal transcription-based amplification system alternative to PCR. PCR Methods Appl 1(1):25–33. doi: 10.1101/gr.1.1.25 CrossRefGoogle Scholar
  9. 9.
    Vincent M et al (2004) Helicase-dependent isothermal DNA amplification. EMBO Rep 5(8):795–800. doi: 10.1038/sj.embor.7400200 CrossRefGoogle Scholar
  10. 10.
    Kurn N et al (2005) Novel isothermal, linear nucleic acid amplification systems for highly multiplexed applications. Clin Chem 51(10):1973–1981. doi: 10.1373/clinchem.2005.053694 CrossRefGoogle Scholar
  11. 11.
    Fang R et al (2009) Cross-priming amplification for rapid detection of Mycobacterium tuberculosis in sputum specimens. J Clin Microbiol 47(3):845–847. doi: 10.1128/JCM.01528-08 CrossRefGoogle Scholar
  12. 12.
    Zhao Y et al (2015) Isothermal amplification of nucleic acids. Chem Rev 115(22):12491–12545. doi: 10.1021/acs.chemrev.5b00428 CrossRefGoogle Scholar
  13. 13.
    Katja Niemann VT (2015) Isothermal amplification and quantification of nucleic acids and its use in microsystems. J Nanosci Nanotechnol 06(03). doi: 10.4172/2157-7439.1000282
  14. 14.
    Fakruddin M et al (2013) Nucleic acid amplification: alternative methods of polymerase chain reaction. J Pharm Bioallied Sci 5(4):245–252. doi: 10.4103/0975-7406.120066 CrossRefGoogle Scholar
  15. 15.
    Liu W et al (2015) Polymerase spiral reaction (PSR): a novel isothermal nucleic acid amplification method. Sci Rep 5:12723. doi: 10.1038/srep12723 CrossRefGoogle Scholar
  16. 16.
    Smykal P et al (2009) Evolutionary conserved lineage of Angela-family retrotransposons as a genome-wide microsatellite repeat dispersal agent. Heredity (Edinb) 103(2):157–167. doi: 10.1038/hdy.2009.45 CrossRefGoogle Scholar
  17. 17.
    Kalendar R, Schulman AH (2014) Transposon-based tagging: IRAP, REMAP, and iPBS. Methods Mol Biol 1115:233–255. doi: 10.1007/978-1-62703-767-9_12
  18. 18.
    Kalendar R et al (2011) Analysis of plant diversity with retrotransposon-based molecular markers. Heredity 106(4):520–530. doi: 10.1038/hdy.2010.93 CrossRefGoogle Scholar
  19. 19.
    Hosid E et al (2012) Diversity of long terminal repeat retrotransposon genome distribution in natural populations of the wild diploid wheat Aegilops speltoides. Genetics 190(1):263–274. doi: 10.1534/genetics.111.134643 CrossRefGoogle Scholar
  20. 20.
    Belyayev A et al (2010) Transposable elements in a marginal plant population: temporal fluctuations provide new insights into genome evolution of wild diploid wheat. Mobile DNA 1(6):1–16. doi: 10.1186/1759-8753-1-6 Google Scholar
  21. 21.
    Kalendar R et al (2014) FastPCR software for PCR, in silico PCR, and oligonucleotide assembly and analysis. In: Valla S, Lale R (eds) DNA cloning and assembly methods, Methods in molecular biology, vol 1116. Humana, New York, NY, pp 271–302. doi: 10.1007/978-1-62703-764-8_18 CrossRefGoogle Scholar
  22. 22.
    Kalendar R et al (2011) Java web tools for PCR, in silico PCR, and oligonucleotide assembly and analysis. Genomics 98(2):137–144. doi: 10.1016/j.ygeno.2011.04.009 CrossRefGoogle Scholar
  23. 23.
    Lexa M et al (2001) Virtual PCR. Bioinformatics 17(2):192–193. doi: 10.1093/bioinformatics/17.2.192 CrossRefGoogle Scholar
  24. 24.
    Yu B, Zhang C (2011) In silico PCR analysis. Methods Mol Biol 760:91–107. doi: 10.1007/978-1-61779-176-5_6 CrossRefGoogle Scholar
  25. 25.
    Salinas NR, Little DP (2012) Electric LAMP: virtual loop-mediated isothermal AMPlification. ISRN Bioinform 2012:696758. doi: 10.5402/2012/696758 CrossRefGoogle Scholar
  26. 26.
    Johnson M et al (2008) NCBI BLAST: a better web interface. Nucleic Acids Res 36(Web Server issue):5–9. doi: 10.1093/nar/gkn201 CrossRefGoogle Scholar
  27. 27.
    Boutros PC, Okey AB (2004) PUNS: transcriptomic- and genomic-in silico PCR for enhanced primer design. Bioinformatics 20(15):2399–2400. doi: 10.1093/bioinformatics/bth257 CrossRefGoogle Scholar
  28. 28.
    Bikandi J et al (2004) In silico analysis of complete bacterial genomes: PCR, AFLP–PCR and endonuclease restriction. Bioinformatics 20(5):798–799. doi: 10.1093/bioinformatics/btg491 CrossRefGoogle Scholar
  29. 29.
    Rotmistrovsky K et al (2004) A web server for performing electronic PCR. Nucleic Acids Res 32(Suppl 2):W108–W112. doi: 10.1093/nar/gkh450 CrossRefGoogle Scholar
  30. 30.
    Gardner SN, Slezak T (2014) Simulate_PCR for amplicon prediction and annotation from multiplex, degenerate primers and probes. BMC Bioinformatics 15(1):1–6. doi: 10.1186/1471-2105-15-237 CrossRefGoogle Scholar
  31. 31.
    Ye J et al (2012) Primer-BLAST: a tool to design target-specific primers for polymerase chain reaction. BMC Bioinformatics 13:134. doi: 10.1186/1471-2105-13-134 CrossRefGoogle Scholar
  32. 32.
    Peyret N et al (1999) Nearest-neighbor thermodynamics and NMR of DNA sequences with internal A.A, C.C, G.G, and T.T mismatches. Biochemistry 38(12):3468–3477. doi: 10.1021/bi9825091 CrossRefGoogle Scholar
  33. 33.
    SantaLucia J Jr et al (1996) Improved nearest-neighbor parameters for predicting DNA duplex stability. Biochemistry 35(11):3555–3562. doi: 10.1021/bi951907q CrossRefGoogle Scholar
  34. 34.
    Lane AN et al (2008) Stability and kinetics of G-quadruplex structures. Nucleic Acids Res 36(17):5482–5515. doi: 10.1093/nar/gkn517 CrossRefGoogle Scholar
  35. 35.
    Shing Ho P (1994) The non-B-DNA structure of d(CA/TG)n does not differ from that of Z-DNA. Proc Natl Acad Sci U S A 91(20):9549–9553CrossRefGoogle Scholar
  36. 36.
    Nomenclature for incompletely specified bases in nucleic acid sequences (1984)

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© Springer Science+Business Media LLC 2017

Authors and Affiliations

  1. 1.National Center for BiotechnologyAstanaKazakhstan
  2. 2.Laboratory of Plant Molecular Genetics and CytogeneticsThe Federal Research Center Institute of Cytology and GeneticsNovosibirskRussia
  3. 3.Institute of Plant Biology and BiotechnologyAlmatyKazakhstan
  4. 4.PrimerDigital LtdHelsinkiFinland

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