1. Allele-specific PCR by positioning the variant at the 3′ end of one primer
Allele-specific PCR by positioning the variant at the 3′ end of one primer (A) As with all PCRs, both forward and reverse primers are required, one of which will be a common primer (here the forward primer) and one of which will have specific versions for each allele (here the reverse primer). The specificity is generated by the sequence at the 3′ end of the primer. For assay of a SNP with two alleles, two PCR amplifications are set up, both containing the common primer, but containing the alternate versions of the allele-specific primer. (B) An assay for a T/C SNP. (1) One version of the reverse primer has an A at the 3′ end; this matches the T allele, and extension can occur from the primer when the T allele is present, so PCR products are obtained from homozygotes or heterozygotes for T (TT or TC). (2) The reverse primer with A at the 3′ end does not allow extension, so PCR would fail if only the C allele were present (CC homozygotes). (3) Reverse primer ending in G does not allow PCR amplification when the template contains only the T allele (TT homozygotes). (4) Reverse primer ending in G allows extension if the C allele is present (CC or TC).
Maria Jackson et al. Essays Biochem. 2018;62:
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