A novel assay for allelic discrimination that combines the fluorogenic 5′ nuclease polymerase chain reaction (TaqMan®) and mismatch amplification mutation assay

Abstract

Recently much attention has been focused on single nucleotide polymorphisms (SNPs) within fundamentally important genes, such as those involved in metabolism, cell growth regulation, and other disease-associated genes. Methodologies for discriminating different alleles need to be specific (robust detection of an altered sequence in the presence of wild-type DNA) and preferably, amenable to high throughput screening. We have combined the fluorogenic 5′ nuclease polymerase chain reaction (TaqMan®) and the mismatch amplification mutation assay (MAMA) to form a novel assay, TaqMAMA, that can quickly and specifically detect single base changes in genomic DNA. TaqMan® chemistry utilizes fluorescence detection during PCR to precisely measure the starting template concentration, while the MAMA assay exploits mismatched bases between the PCR primers and the wild-type template to selectively amplify specific mutant or polymorphic sequences. By combining these assays, the amplification of the mutant DNA can be readily detected by fluorescence in a single PCR reaction in 2 hours. Using the human TK6 cell line and specific HPRT-mutant clones as a model system, we have optimized the TaqMAMA technique to discriminate between mutant and wild-type DNA. Here we demonstrate that appropriately designed MAMA primer pairs preferentially amplify mutant genomic DNA even in the presence of a 1000-fold excess of wild-type DNA. The ability to selectively amplify DNAs with single nucleotide changes, or the specific amplification of a low copy number mutant DNA in a 1000-fold excess of wild-type DNA, is certain to be a valuable technique for applications such as allelic discrimination, detection of single nucleotide polymorphisms or gene isoforms, and for assessing hotspot mutations in tumor-associated genes from biopsies contaminated with normal tissue.