Allele-specific PCR in SNP genotyping.
The increasing need for large-scale genotyping applications of single nucleotide polymorphisms (SNPs) in model and nonmodel organisms requires the development of low-cost technologies accessible to minimally equipped laboratories. The method presented here allows efficient discrimination of SNPs by allele-specific PCR in a single reaction with standard PCR conditions. A common reverse primer and two forward allele-specific primers with different tails amplify two allele-specific PCR products of different lengths, which are further separated by agarose gel electrophoresis. PCR specificity is improved by the introduction of a destabilizing mismatch within the 30 end of the allele-specific primers. This is a simple and inexpensive method for SNP detection that does not require PCR optimization.
Gaudet
Muriel
M
Department of Forest Resources and Environment (DiSAFRi), University of Tuscia, Viterbo, Italy.
Fara
Anna-Giulia
AG
Beritognolo
Isacco
I
Sabatti
Maurizio
M
D000483:Alleles; D001483:Base Sequence; D017931:DNA Primers / Q000378:metabolism; D004587:Electrophoresis, Agar Gel; D005838:Genotype; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D020641:Polymorphism, Single Nucleotide / Q000235:genetics*
D017931:DNA Primers