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Methods Mol Biol. 2009;578:415-24. doi: 10.1007/978-1-60327-411-1_26.

Allele-specific PCR in SNP genotyping.

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Department of Forest Resources and Environment (DiSAFRi), University of Tuscia, Viterbo, Italy.


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.

[Indexed for MEDLINE]

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