Molecular haplotyping of tandem single nucleotide polymorphisms by allele-specific PCR

Abstract

BARD1 Val507Met (1592 A>G) is an interesting marker for association studies on cancer risk. However, studies are scarce in the literature, probably reflecting the methodological problem imposed by the fact that next to the 1592 A>G stands the 1591 C>T single nucleotide polymorphism (SNP). We have designed an allele-specific PCR method capable of molecular haplotyping tandem SNPs. In the tandem SNPs haplotyping assay (tSNPh), four reverse primers are designed to be perfect matches of each potential haplotype. The forward primer is labeled with a fluorochrome. PCR products are analyzed by capillary electrophoresis. Haplotyping is performed by size calling. To ascertain the accuracy and reproducibility of the assay, we measured the level of concordance with sequencing data in 124 samples. In vitro-generated templates have been used for further testing. We developed a novel and reliable assay that permits typing two SNPs directly adjacent to each other, avoiding mutual interferences. The method is amenable to automation and high throughput. We expect that this assay will contribute to clarifying the role of BARD1 in cancer susceptibility. In addition, we suggest that tandem SNPs are potentially interesting polymorphic markers in which molecular haplotyping can be performed easily.