FastPCR Software for PCR Primer and Probe Design and Repeat Search

Abstract

Reproducible and target-specific polymerase chain reaction (PCR) amplification relies on several interrelated factors of which primer design is central. Here, we describe new free bioinformatics software, the FastPCR which was developed, and continues to be updated, based on detailed experimental studies of PCR efficiency for the optimal design of primers and probe sequences and for repeat searching. This software forms an environment of integrated tools, which provides comprehensive facilities for designing primers for most PCR applications including multiplex and self-reporting fluorescent systems. FastPCR consists of a data editor, build commands for probe and primer design, and automation tools. The software selects the best primers with the widest range of melting temperatures, which allows designing qualified primers for all PCR tasks. The “in silico” PCR primer or probe searching includes comprehensive individual primers and primer pair analysis tests. FastPCR utilizes combinations of normal and degenerate primers for all tools. The melting temperature calculation is based on nearest neighbour thermodynamic parameters starting with multiple nucleic acid or protein sequences. It performs efficient and complete detection of various repeat types with visual display. FastPCR is able to perform repeat searches for a single sequence or for comparisons of two sequences. The program includes various bioinformatics tools for analysis of sequences with GC or AT skew, GC content, and purine-pyrimidine skew, and considers linguistic sequence complexity. It can generate random DNA sequence, make restriction analysis, and supports the clustering of sequences and consensus sequence generation, as well as sequence similarity and conservancy analyses.