Abstract:
Polymerase chain reaction (PCR) is a simple and rapid method that can detect nucleotide
polymorphisms and sequence variation in basic research applications, agriculture, and
medicine. Variants of PCR, collectively known as allele-specific PCR (AS-PCR), use a
competitive reaction in the presence of allele-specific primers to preferentially amplify only
certain alleles. This method, originally named by its developers as Kompetitive Allele
Specific PCR (KASP), is an AS-PCR variant adapted for fluorescence-based detection of
amplification results. We developed a bioinformatic tool for designing probe sequences for
PCR-based genotyping assays. Probe sequences are designed in both directions, and
both single nucleotide polymorphisms (SNPs) and insertion-deletions (InDels) may be
targeted. In addition, the tool allows discrimination of up to four-allelic variants at a single
SNP site. To increase both the reaction specificity and the discriminative power of SNP
genotyping, each allele-specific primer is designed such that the penultimate base before
the primer’s 3′ end base is positioned at the SNP site. The tool allows design of custom
FRET cassette reporter systems for fluorescence-based assays. FastPCR is a user friendly and powerful Java-based software that is freely available (http://primerdigital.com/
tools/). Using the FastPCR environment and the tool for designing AS-PCR provides
unparalleled flexibility for developing genotyping assays and specific and sensitive
diagnostic PCR-based tests, which translates into a greater likelihood of research
success.