Alternative Approach for Rapid and Reliable Single-Nucleotide Polymorphism Typing with Double Restriction Mutagenesis Primer PCR

Complex disorders such as type 2 diabetes or coronary heart disease are of major public interest because they affect millions of individuals. To unravel the genetic background of those traits, genome-wide linkage scans with microsatellite markers have been performed, followed by association studies of single-nucleotide polymorphisms (SNPs) in the identified candidate regions. High-throughput SNP typing technologies such as matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (1), pyrosequencing (2), TaqMan-based allelic discrimination (3), and others (4)(5)(6)(7)(8) can be used for that approach. However, many smaller and medium-throughput laboratories have no access to rapid, reliable, universal, and cost-effective SNP typing methods. The establishment of such a technique therefore has a high priority.We have developed a simple and inexpensive two-step method for SNP detection (PCR followed by modified restriction digestion), called double restriction mutagenesis primer PCR (DRMP-PCR). Our approach is based on the introduction of two restriction sites in one of the two PCR primers (mutagenesis reverse primer). This method appears to represent an improvement over older techniques such as PCR–primer-introduced restriction analysis (PIRA) (9) with respect to both specificity and reliability. In addition, we have generated a PERL-based computer program that provides the restriction enzymes and necessary mutagenesis sequences for primer design.Our method allows detection of SNPs by use of fluorescence scanning sequencers and automated automatic allele calling. The forward primer must therefore have a 5′ fluorescence label (hexachloro-6-carboxyfluorescein, 6-carboxyfluorescein, or others) but does not undergo any additional changes. The 3′ end of the reverse primer is positioned next to the SNP and forms a restriction site together with one of the two SNP alleles (Fig. 1A⇓ ). In most cases, one or two bases of the 3′ end of the primer must be changed to generate a restriction site. For …