PCR pp 33-64 | Cite as

Introduction on Using the FastPCR Software and the Related Java Web Tools for PCR and Oligonucleotide Assembly and Analysis

  • Ruslan Kalendar
  • Timofey V. Tselykh
  • Bekbolat Khassenov
  • Erlan M. Ramanculov
Part of the Methods in Molecular Biology book series (MIMB, volume 1620)


This chapter introduces the FastPCR software as an integrated tool environment for PCR primer and probe design, which predicts properties of oligonucleotides based on experimental studies of the PCR efficiency. The software provides comprehensive facilities for designing primers for most PCR applications and their combinations. These include the standard PCR as well as the multiplex, long-distance, inverse, real-time, group-specific, unique, overlap extension PCR for multi-fragments assembling cloning and loop-mediated isothermal amplification (LAMP). It also contains a built-in program to design oligonucleotide sets both for long sequence assembly by ligase chain reaction and for design of amplicons that tile across a region(s) of interest. The software calculates the melting temperature for the standard and degenerate oligonucleotides including locked nucleic acid (LNA) and other modifications. It also provides analyses for a set of primers with the prediction of oligonucleotide properties, dimer and G/C-quadruplex detection, linguistic complexity as well as a primer dilution and resuspension calculator. The program consists of various bioinformatical tools for analysis of sequences with the GC or AT skew, CG% and GA% content, and the purine–pyrimidine skew. It also analyzes the linguistic sequence complexity and performs generation of random DNA sequence as well as restriction endonucleases analysis. The program allows to find or create restriction enzyme recognition sites for coding sequences and supports the clustering of sequences. It performs efficient and complete detection of various repeat types with visual display. The FastPCR software allows the sequence file batch processing that is essential for automation. The program is available for download at, and its online version is located at

Key words

PCR primer design Isothermal amplification of nucleic acids Software probe design DNA primers DNA primers nucleic acid hybridization Degenerate PCR Tiling arrays Primer linguistic complexity Ligase chain reaction 



Web tools are available free, provided for noncommercial research and education use only. They may not be reproduced or distributed for commercial use. This work was supported by the companies PrimerDigital Ltd.


  1. 1.
    Walker-Daniels J (2012) Current PCR methods. Mater Methods 2:119. doi: 10.13070/mm.en.2.119 Google Scholar
  2. 2.
    Tisi LC, Gandelman O, Kiddle G, Mcelgunn C (2010) Nucleic acid amplification. Canada Patent CA2417798Google Scholar
  3. 3.
    Notomi T, Okayama H, Masubuchi H, Yonekawa T, Watanabe K, Amino N, Hase T (2000) Loop-mediated isothermal amplification of DNA. Nucleic Acids Res 28(12):e63. doi: 10.1093/nar/28.12.e63 CrossRefGoogle Scholar
  4. 4.
    Rozen S, Skaletsky H (2000) Primer3 on the WWW for general users and for biologist programmers. Methods Mol Biol 132:365–386Google Scholar
  5. 5.
    Kalendar R, Lee D, Schulman AH (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, pp 271–302. doi: 10.1007/978-1-62703-764-8_18
  6. 6.
    Kalendar R, Lee D, Schulman AH (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
  7. 7.
    Marshall OJ (2004) PerlPrimer: cross-platform, graphical primer design for standard, bisulphite and real-time PCR. Bioinformatics 20(15):2471–2472. doi: 10.1093/bioinformatics/bth254 CrossRefGoogle Scholar
  8. 8.
    Owczarzy R, Tataurov AV, Wu Y, Manthey JA, McQuisten KA, Almabrazi HG, Pedersen KF, Lin Y, Garretson J, McEntaggart NO, Sailor CA, Dawson RB, Peek AS (2008) IDT SciTools: a suite for analysis and design of nucleic acid oligomers. Nucleic Acids Res 36:163–169. doi: 10.1093/nar/gkn198 CrossRefGoogle Scholar
  9. 9.
    Bekaert M, Teeling EC (2008) UniPrime: a workflow-based platform for improved universal primer design. Nucleic Acids Res 36(10):e56. doi: 10.1093/nar/gkn191 CrossRefGoogle Scholar
  10. 10.
    Ye J, Coulouris G, Zaretskaya I, Cutcutache I, Rozen S, Madden TL (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
  11. 11.
    Smykal P, Kalendar R, Ford R, Macas J, Griga M (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
  12. 12.
    Giegerich R, Meyer F, Schleiermacher C (1996) GeneFisher--software support for the detection of postulated genes. Proc Int Conf Intell Syst Mol Biol 4:68–77Google Scholar
  13. 13.
    Gadberry MD, Malcomber ST, Doust AN, Kellogg EA (2005) Primaclade--a flexible tool to find conserved PCR primers across multiple species. Bioinformatics 21(7):1263–1264. doi: 10.1093/bioinformatics/bti134 CrossRefGoogle Scholar
  14. 14.
    Nomenclature Committee of the International Union of Biochemistry (NC-IUB) (1984) Nomenclature for incompletely specified bases in nucleic acid sequences.
  15. 15.
    Allawi HT, SantaLucia J Jr (1997) Thermodynamics and NMR of internal G.T mismatches in DNA. Biochemistry 36(34):10581–10594. doi: 10.1021/bi962590c CrossRefGoogle Scholar
  16. 16.
    SantaLucia J (1998) A unified view of polymer, dumbbell, and oligonucleotide DNA nearest-neighbor thermodynamics. Proc Natl Acad Sci U S A 95(4):1460–1465CrossRefGoogle Scholar
  17. 17.
    Le Novere N (2001) MELTING, computing the melting temperature of nucleic acid duplex. Bioinformatics 17(12):1226–1227. doi: 10.1093/bioinformatics/17.12.1226 CrossRefGoogle Scholar
  18. 18.
    Bolton ET, McCarthy BJ (1962) A general method for the isolation of RNA complementary to DNA. Proc Natl Acad Sci U S A 48(8):1390–1397CrossRefGoogle Scholar
  19. 19.
    Guedin A, Gros J, Alberti P, Mergny JL (2010) How long is too long? Effects of loop size on G-quadruplex stability. Nucleic Acids Res 38(21):7858–7868. doi: 10.1093/nar/gkq639 CrossRefGoogle Scholar
  20. 20.
    Wallace RB, Shaffer J, Murphy RF, Bonner J, Hirose T, Itakura K (1979) Hybridization of synthetic oligodeoxyribonucleotides to ΦX 174 DNA: the effect of single base pair mismatch. Nucleic Acids Res 6(11):3543–3558. doi: 10.1093/nar/6.11.3543 CrossRefGoogle Scholar
  21. 21.
    von Ahsen N, Wittwer CT, Schutz E (2001) Oligonucleotide melting temperatures under PCR conditions: nearest-neighbor corrections for Mg2+, deoxynucleotide triphosphate, and dimethyl sulfoxide concentrations with comparison to alternative empirical formulas. Clin Chem 47(11):1956–1961Google Scholar
  22. 22.
    Gabrielian A, Bolshoy A (1999) Sequence complexity and DNA curvature. Comput Chem 23(3–4):263–274. doi: 10.1016/S0097-8485(99)00007-8 CrossRefGoogle Scholar
  23. 23.
    Orlov YL, Potapov VN (2004) Complexity: an internet resource for analysis of DNA sequence complexity. Nucleic Acids Res 32(Web Server issue):628–633. doi: 10.1093/nar/gkh466 CrossRefGoogle Scholar
  24. 24.
    Gilson MK, Given JA, Bush BL, McCammon JA (1997) The statistical-thermodynamic basis for computation of binding affinities: a critical review. Biophys J 72(3):1047–1069. doi: 10.1016/S0006-3495(97)78756-3 CrossRefGoogle Scholar
  25. 25.
    Peyret N, Seneviratne PA, Allawi HT, SantaLucia J Jr (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
  26. 26.
    Watkins NE Jr, SantaLucia J Jr (2005) Nearest-neighbor thermodynamics of deoxyinosine pairs in DNA duplexes. Nucleic Acids Res 33(19):6258–6267. doi: 10.1093/nar/gki918 CrossRefGoogle Scholar
  27. 27.
    Sen D, Gilbert W (1992) Guanine quartet structures. Methods Enzymol 211:191–199CrossRefGoogle Scholar
  28. 28.
    Il'icheva IA, Florent'ev VL (1992) Four-stranded complexes of oligonucleotides--quadruplexes. Mol Biol 26(3):512–531Google Scholar
  29. 29.
    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
  30. 30.
    Kypr J, Kejnovska I, Renciuk D, Vorlickova M (2009) Circular dichroism and conformational polymorphism of DNA. Nucleic Acids Res 37(6):1713–1725. doi: 10.1093/nar/gkp026 CrossRefGoogle Scholar
  31. 31.
    SantaLucia J Jr, Hicks D (2004) The thermodynamics of DNA structural motifs. Annu Rev Biophys Biomol Struct 33:415–440. doi: 10.1146/annurev.biophys.32.110601.141800 CrossRefGoogle Scholar
  32. 32.
    Williams JGK, Kubelik AR, Livak KJ, Rafalski JA, Tingey SV (1990) DNA polymorphisms amplified by arbitrary primers are useful as genetic-markers. Nucleic Acids Res 18(22):6531–6535. doi: 10.1093/nar/18.22.6531 CrossRefGoogle Scholar
  33. 33.
    Welsh J, Mcclelland M (1990) Fingerprinting genomes using pcr with arbitrary primers. Nucleic Acids Res 18(24):7213–7218. doi: 10.1093/nar/18.24.7213 CrossRefGoogle Scholar
  34. 34.
    Kalendar R, Schulman A (2006) IRAP and REMAP for retrotransposon-based genotyping and fingerprinting. Nat Protoc 1(5):2478–2484. doi: 10.1038/nprot.2006.377 CrossRefGoogle Scholar
  35. 35.
    Chang RY, O'Donoughue LS, Bureau TE (2001) Inter-MITE polymorphisms (IMP): a high throughput transposon-based genome mapping and fingerprinting approach. Theor Appl Genet 102(5):773–781. doi: 10.1007/s001220051709 CrossRefGoogle Scholar
  36. 36.
    Nelson DL, Ledbetter SA, Corbo L, Victoria MF, Ramirez-Solis R, Webster TD, Ledbetter DH, Caskey CT (1989) Alu polymerase chain reaction: a method for rapid isolation of human-specific sequences from complex DNA sources. Proc Natl Acad Sci U S A 86(17):6686–6690CrossRefGoogle Scholar
  37. 37.
    Sinnett D, Deragon JM, Simard LR, Labuda D (1990) Alumorphs—human DNA polymorphisms detected by polymerase chain reaction using Alu-specific primers. Genomics 7(3):331–334CrossRefGoogle Scholar
  38. 38.
    Jurka J, Kapitonov VV, Pavlicek A, Klonowski P, Kohany O, Walichiewicz J (2005) Repbase update, a database of eukaryotic repetitive elements. Cytogenet Genome Res 110(1–4):462–467. doi: 10.1159/000084979 CrossRefGoogle Scholar
  39. 39.
    Heckman KL, Pease LR (2007) Gene splicing and mutagenesis by PCR-driven overlap extension. Nat Protoc 2(4):924–932. doi: 10.1038/nprot.2007.132 CrossRefGoogle Scholar
  40. 40.
    Higasa K, Hayashi K (2002) Ordered catenation of sequence-tagged sites and multiplexed SNP genotyping by sequencing. Nucleic Acids Res 30(3):E11CrossRefGoogle Scholar
  41. 41.
    Quan J, Tian J (2009) Circular polymerase extension cloning of complex gene libraries and pathways. PLoS One 4(7):e6441. doi: 10.1371/journal.pone.0006441 CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  • Ruslan Kalendar
    • 1
    • 5
  • Timofey V. Tselykh
    • 2
    • 3
  • Bekbolat Khassenov
    • 4
  • Erlan M. Ramanculov
    • 4
  1. 1.National Center for BiotechnologyAstanaKazakhstan
  2. 2.Biochemistry and Developmental Biology, MedicumUniversity of HelsinkiHelsinkiFinland
  3. 3.Minerva Medical Research InstituteHelsinkiFinland
  4. 4.National Center for Biotechnology,AstanaKazakhstan
  5. 5.PrimerDigital LtdHelsinkiFinland

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