PCR candidate region mismatch scanning: adaptation to quantitative, high-throughput genotyping

Linkage:and association analyses were performed to identify loci affecting disease susceptibility by scoring previously characterized sequence variati ons such as microsatellites and single nucleotide polymorphisms. Lack of:ma rkers in regions of interest, as well as difficulty in adapting various met hods to high-throughput settings, often limits the effectiveness of the ana lyses. We have adapted the Escherichia coli mismatch detection system, empl oying the factors MutS, MutL and MutH, for use in PCR-based, automated, hig h-throughput genotyping and mutation detection of genomic DNA. Optimal sens itivity and signal-to-noise ratios were obtained in a straightforward fashi on because the detection reaction proved to be principally dependent upon m onovalent cation concentration and MutL concentration. Quantitative relatio nships of the optimal values of these parameters with length of the DNA tes t fragment were demonstrated, in support of the translocation model for the mechanism of action of these enzymes, rather than the molecular switch mod el. Thus, rapid, sequence-independent optimization was possible for each ne w genomic target region. Other factors potentially limiting the flexibility of mismatch scanning, such as positioning of dam recognition sites within the target fragment, have also been investigated. We developed several stra tegies, which can be easily adapted to automation, for limiting the analysi s to intersample heteroduplexes. Thus, the principal barriers to the use of this methodology, which we have designated PCR candidate region mismatch s canning, in cost-effective, high-throughput settings have been removed.