Differential haplotype amplification leads to misgenotyping of heterozygote as homozygote when using single...

Mismatches at the 3'end of /or within a primer are reported to affect the efficiency of PCR and cause allele drop. Here, we report preferential amplification of one haplotype and misgenotyping, when double heterozygotes at NAT1 (rs1057126 and rs15561) were genotyped by sequencing and PCR-RFLP methods using mismatch reverse primers located next to the target SNP. Detailed study revealed highest (100%) and lowest (0%) misgenotyping when the mismatch was at the 3rd and 15th nucleotide positions from 3' end of the primer, respectively. But, the same primers, without any mismatch genotyped heterozygotes correctly. Homozygotes can always be detected correctly irrespective of mismatch position in the primer. Similar results were observed for two SNPs (rs12947788 and rs 12951053) at TP53. Using mismatch NAT1 reverse primers, located three nucleotides away from the target SNP, both TaqMan and sequencing methods showed preferential synthesis of one haplotype strand and misgenotyping in heterozygotes, respectively. So, mismatch primer, located next to target SNP, should be avoided to genotype heterozygotes, since, PCR and sequencing based genotyping methods may lead the investigators to report faulty allelic and genotypic frequencies. This study mimics a situation when an unknown variation is present in the primer-binding sites of both chromosomes.