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

ABSTRACT: Linkage and association analyses were performed to identify loci affecting disease susceptibility by scoring previously characterized sequence variations such as microsatellites and single nucleotide polymorphisms. Lack of markers in regions of interest, as well as difficulty in adapting various methods to high-throughput settings, often limits the effectiveness of the analyses. We have adapted the Escherichia coli mismatch detection system, employing the factors MutS, MutL and MutH, for use in PCR-based, automated, high-throughput genotyping and mutation detection of genomic DNA. Optimal sensitivity and signal-to-noise ratios were obtained in a straightforward fashion because the detection reaction proved to be principally dependent upon monovalent cation concentration and MutL concentration. Quantitative relationships of the optimal values of these parameters with length of the DNA test fragment were demonstrated, in support of the translocation model for the mechanism of action of these enzymes, rather than the molecular switch model. Thus, rapid, sequence-independent optimization was possible for each new 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 strategies, which can be easily adapted to automation, for limiting the analysis to intersample heteroduplexes. Thus, the principal barriers to the use of this methodology, which we have designated PCR candidate region mismatch scanning, in cost-effective, high-throughput settings have been removed.

SUBMITTER: Beaulieu M

PROVIDER: S-EPMC29718 | biostudies-literature | 2001 Mar

REPOSITORIES: biostudies-literature

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

Beaulieu M M Larson G P GP Geller L L Flanagan S D SD Krontiris T G TG

Nucleic acids research 20010301 5

Linkage and association analyses were performed to identify loci affecting disease susceptibility by scoring previously characterized sequence variations such as microsatellites and single nucleotide polymorphisms. Lack of markers in regions of interest, as well as difficulty in adapting various methods to high-throughput settings, often limits the effectiveness of the analyses. We have adapted the Escherichia coli mismatch detection system, employing the factors MutS, MutL and MutH, for use in ...[more]

PMID: 11222761

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Project description:BackgroundWhile improvements in genotyping technology have allowed for increased throughput and reduced time and expense, protocols remain hindered by the slow upstream steps of isolating, purifying, and normalizing DNA. Various methods exist for genotyping samples directly through blood, without having to purify the DNA first. These procedures were designed to be used on smaller throughput systems, however, and have not yet been tested for use on current high-throughput real-time (q)PCR based genotyping platforms. In this paper, a method of quantitative qPCR-based genotyping on blood without DNA purification was developed using a high-throughput qPCR platform.FindingsThe performances of either DNA purified from blood or the same blood samples without DNA purification were evaluated through qPCR-based genotyping. First, 60 different mutations prevalent in the Ashkenazi Jewish population were genotyped in 12 Ashkenazi Jewish individuals using the QuantStudio™12K Flex Real-Time PCR System. Genotyping directly from blood gave a call rate of 99.21%, and an accuracy of 100%, while the purified DNA gave a call rate of 92.49%, and an accuracy of 99.74%. Although no statistical difference was found for these parameters, an F test comparing the standard deviations of the wild type clusters for the two different methods indicated significantly less variation when genotyping directly from blood instead of after DNA purification. To further establish the ability to perform high-throughput qPCR based genotyping directly from blood, 96 individuals of Ashkenazi Jewish decent were genotyped for the same 60 mutations (5,760 genotypes in 5 hours) and resulted in a call rate of 98.38% and a diagnostic accuracy of 99.77%.ConclusionThis study shows that accurate qPCR-based high-throughput genotyping can be performed without DNA purification. The direct use of blood may further expedite the entire genotyping process, reduce costs, and avoid tracking errors which can occur during sample DNA purification.

Dataset Information

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

Publications

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

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