FastPCR Software for PCR, In Silico PCR, and Oligonucleotide Assembly and Analysis

  • Ruslan Kalendar
  • David Lee
  • Alan H. Schulman
Part of the Methods in Molecular Biology book series (MIMB, volume 1116)


This chapter introduces the software FastPCR as an integrated tools environment for PCR primer and probe design. It also predicts oligonucleotide properties based on experimental studies of PCR efficiency. The software provides comprehensive facilities for designing primers for most PCR applications and their combinations, including standard, multiplex, long-distance, inverse, real-time, group-specific, unique, and overlap extension PCR for multi-fragment assembly in cloning, as well as bisulphite modification assays. It includes a program to design oligonucleotide sets for long sequence assembly by the ligase chain reaction. The in silico PCR primer or probe search includes comprehensive analyses of individual primers and primer pairs. It calculates the melting temperature for standard and degenerate oligonucleotides including LNA and other modifications, provides analyses for a set of primers with prediction of oligonucleotide properties, dimer and G/C-quadruplex detection, and linguistic complexity, and provides a dilution and resuspension calculator. The program includes various bioinformatics tools for analysis of sequences with CG or AT skew, of CG content and purine–pyrimidine skew, and of linguistic sequence complexity. It also permits generation of random DNA sequence and analysis of restriction enzymes of all types. It finds or creates restriction enzyme recognition sites for coding sequences and supports the clustering of sequences. It generates consensus sequences and analyzes sequence conservation. It performs efficient and complete detection of various repeat types and displays them. FastPCR allows for sequence file batch processing, which is essential for automation. The FastPCR software is available for download at and online version at

Key words

PCR primer design Primer linguistic complexity Sequence assembly Software probe design Ligase chain reaction DNA primers 



Overlap extension PCR


Polymerase chain reaction


Real-time PCR


Simple sequence repeat



Web tools are available free to academic institutions, provided that they are used for noncommercial research and education only. They may not be reproduced or distributed for commercial use. This work was partially supported by the companies PrimerDigital Ltd. and Oligomer Ltd. and by the Academy of Finland, Project 134079.


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Copyright information

© Springer Science+Business Media, New York 2014

Authors and Affiliations

  • Ruslan Kalendar
    • 1
    • 2
  • David Lee
    • 3
  • Alan H. Schulman
    • 1
    • 4
  1. 1.MTT/BI Plant Genomics Laboratory, Institute of BiotechnologyUniversity of HelsinkiHelsinkiFinland
  2. 2.PrimerDigital LtdHelsinkiFinland
  3. 3.John Bingham LaboratoryNational Institute of Agricultural BotanyCambridgeUK
  4. 4.Biotechnology and Food Research, MTT Agrifood Research FinlandJokioinenFinland

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