FastPCR Software for PCR, In Silico PCR, and Oligonucleotide Assembly and Analysis
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 http://primerdigital.com/fastpcr.html and online version at http://primerdigital.com/tools/pcr.html.
Key wordsPCR primer design Primer linguistic complexity Sequence assembly Software probe design Ligase chain reaction DNA primers
Overlap extension PCR
Polymerase chain reaction
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.
- 2.Kalendar R, Lee D, Schulman AH (2009) FastPCR software for PCR primer and probe design and repeat search. Genes, Genomes and Genomics 3:1–14Google Scholar
- 10.National Center for Biotechnology Information, National Library of Medicine, Building 38A, Bethesda, MD, 20894. http://blast.ncbi.nlm.nih.gov/blastcgihelp.shtml
- 11.Nomenclature for incompletely specified bases in nucleic acid sequences (1984) http://www.chem.qmul.ac.uk/iubmb/misc/naseq.html
- 18.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 47:1956–1961Google Scholar
- 26.Il’icheva IA, Florent’ev VL (1992) Four-stranded complexes of oligonucleotides-quadruplexes. Mol Biol (Mosk) 26:512–531Google Scholar
- 36.TREP, the Triticeae Repeat Sequence Database (2008) http://wheat.pw.usda.gov/ITMI/Repeats/
- 40.Cao YF et al (2005) Information theory-based algorithm for in silico prediction of PCR products with whole genomic sequences as templates. BMC Bioinformatics 6:190. doi: 10.1186/ 1471-2105-6-190