Abstract
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 http://primerdigital.com/fastpcr.html and the online Java version at http://primerdigital.com/tools/pcr.html.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Walker-Daniels J (2012) Current PCR methods. Mat Methods 2:119. doi:10.13070/mm.en.2.119
Tisi LC et al. (2010) Nucleic acid amplification. Canada Patent CA2417798
Notomi T et al (2000) Loop-mediated isothermal amplification of DNA. Nucleic Acids Res 28(12):e63. doi:10.1093/nar/28.12.e63
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
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
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
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):1173–1177
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
Vincent M et al (2004) Helicase-dependent isothermal DNA amplification. EMBO Rep 5(8):795–800. doi:10.1038/sj.embor.7400200
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
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
Zhao Y et al (2015) Isothermal amplification of nucleic acids. Chem Rev 115(22):12491–12545. doi:10.1021/acs.chemrev.5b00428
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
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
Liu W et al (2015) Polymerase spiral reaction (PSR): a novel isothermal nucleic acid amplification method. Sci Rep 5:12723. doi:10.1038/srep12723
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
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
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
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
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
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
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
Lexa M et al (2001) Virtual PCR. Bioinformatics 17(2):192–193. doi:10.1093/bioinformatics/17.2.192
Yu B, Zhang C (2011) In silico PCR analysis. Methods Mol Biol 760:91–107. doi:10.1007/978-1-61779-176-5_6
Salinas NR, Little DP (2012) Electric LAMP: virtual loop-mediated isothermal AMPlification. ISRN Bioinform 2012:696758. doi:10.5402/2012/696758
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
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
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
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
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
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
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
SantaLucia J Jr et al (1996) Improved nearest-neighbor parameters for predicting DNA duplex stability. Biochemistry 35(11):3555–3562. doi:10.1021/bi951907q
Lane AN et al (2008) Stability and kinetics of G-quadruplex structures. Nucleic Acids Res 36(17):5482–5515. doi:10.1093/nar/gkn517
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):9549–9553
Nomenclature for incompletely specified bases in nucleic acid sequences (1984) http://www.chem.qmul.ac.uk/iubmb/misc/naseq.html.
Acknowledgments
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.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2017 Springer Science+Business Media LLC
About this protocol
Cite this protocol
Kalendar, R., Muterko, A., Shamekova, M., Zhambakin, K. (2017). In Silico PCR Tools for a Fast Primer, Probe, and Advanced Searching. In: Domingues, L. (eds) PCR. Methods in Molecular Biology, vol 1620. Springer, New York, NY. https://doi.org/10.1007/978-1-4939-7060-5_1
Download citation
DOI: https://doi.org/10.1007/978-1-4939-7060-5_1
Published:
Publisher Name: Springer, New York, NY
Print ISBN: 978-1-4939-7059-9
Online ISBN: 978-1-4939-7060-5
eBook Packages: Springer Protocols