Haplotyping of Heterozygous SNPs in Genomic DNA Using Long-Range PCR

Abstract

To study meiotic recombination products, cis- or trans-association of disease polymorphisms, or allele-specific expression patterns, it is necessary to phase heterozygous polymorphisms separated by several kilobases. Haplotyping using long-range polymerase chain reaction (PCR) is a powerful, cost-effective method to directly obtain the phase of multiple heterozygous sites with standard laboratory equipment in a handful of loci for many samples. The method is based on the amplification of large genomic DNA regions (up to ~40 kb) with a reaction mixture that combines a proofreading polymerase with allele-specific primer pairs that preferentially amplify matched templates. The analysis of two heterozygous SNPs requires four reactions, each containing one of the four possible allele-specific primer combinations (two forward and two reverse primers), with the mismatches occurring at the 3' ends of the primers. The two correct primer combinations will more efficiently elongate the matching alleles than the alternative alleles, and the difference in amplification efficiency can be monitored with real-time PCR.

Keywords: Allele-specific amplification; Allelic phase; Genotype; Haplotype; Heterozygous SNP; Long-range PCR; Proof-reading polymerase; Real-time PCR; Single nucleotide polymorphism.