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

Recently much attention has been focused on single nucleotide polymorphisms (SNPs) within fundamentally important genes, such as those involved in met abolism, cell growth regulation, and other disease-associated genes. Method ologies for discriminating different alleles need to be specific (robust de tection of an altered sequence in the presence of wild-type DNA) and prefer ably, amenable to high throughput screening. We have combined the fluorogen ic 5' nuclease polymerase chain reaction (TaqMan(R)) and the mismatch ampli fication mutation assay (MAMA) to form a novel assay, TaqMAMA, that can qui ckly and specifically detect single base changes in genomic DNA. TaqMan(R) chemistry utilizes fluorescence detection during PCR to precisely measure t he starting template concentration, while the MAMA assay exploits mismatche d bases between the PCR primers and the wild-type template to selectively a mplify specific mutant or polymorphic sequences. By combining these assays, the amplification of the mutant DNA can be readily detected by fluorescenc e in a single PCR reaction in 2 hours. Using the human TK6 cell line and sp ecific HPRT-mutant clones as a model system, we have optimized the TaqMAMA technique to discriminate between mutant and wild-type DNA. Here we demonst rate that appropriately designed MAMA primer pairs preferentially amplify m utant genomic DNA even in the presence of a 1000-fold excess of wild-type D NA. The ability to selectively amplify DNAs with single nucleotide changes, or the specific amplification of a low copy number mutant DNA in a 1000-fo ld excess of wild-type DNA, is certain to be a valuable technique for appli cations such as allelic discrimination, detection of single nucleotide poly morphisms or gene isoforms, and for assessing hotspot mutations in tumor-as sociated genes from biopsies contaminated with normal tissue. (C) 1999 Else vier Science B.V. All rights reserved.