Single Nucleotide Polymorphism (SNP) Genotyping in Unprocessed Whole Blood and Serum by Real-Time PCR

Abstract

Several single nucleotide polymorphisms (SNPs) have been identified that affect folate and homocysteine metabolism, which in turn are implicated in the pathogenesis of cardiovascular disease (1), neural tube defects (2), and colorectal cancer (3). These SNPs include methylenetetrahydrofolate ( MTHFR ) C677T (1), MTHFR A1298C (4), methionine synthase ( MTR ) A2756G (5)(6), and methionine synthase reductase ( MTRR ) A66G (7). To determine the relationships of these and other SNPs with the risk of chronic diseases, along with possible interactions with environmental factors, large population-based epidemiologic studies are necessary. This requires high-throughput methodologies for SNP determination. SNP genotyping can generally be divided into two steps: sample preparation, e.g., purification of DNA from blood, and allele detection. To date, great advances have been made in allele detection because novel technologies for DNA analysis have been developed (8). The DNA purification step is required because the enzymes used for manipulation of DNA (e.g., polymerases) are susceptible to inhibition by substrate impurities. Purification of DNA is often labor-intensive, time-consuming, and costly and enhances the risk of back- or cross-contamination of samples. The development of automated DNA purification methods has been aimed at alleviating some of these problems (9), but may increase the need for expensive equipment. Other researchers have investigated the feasibility of PCR amplification without prior DNA purification (10)(11)(12). Allele-detection methods based on real-time PCR are attractive because signal amplification and allele detection are accomplished in a single, closed tube (13). For this type of assay, DNA purification seems mandatory because blood constituents such …