Beaulieu M, Larson G P, Geller L, Flanagan S D, Krontiris T G
Division of Molecular Medicine and Division of Neurosciences, Beckman Research Institute, City of Hope National Medical Center, Duarte, CA 91010, USA.
Nucleic Acids Res. 2001 Mar 1;29(5):1114-24. doi: 10.1093/nar/29.5.1114.
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.
进行连锁分析和关联分析，通过对先前已鉴定的序列变异（如微卫星和单核苷酸多态性）进行评分来确定影响疾病易感性的基因座。感兴趣区域中缺乏标记，以及难以将各种方法应用于高通量检测，常常限制了分析的有效性。我们对大肠杆菌错配检测系统进行了改进，利用MutS、MutL和MutH等因子，用于基于PCR的基因组DNA自动化高通量基因分型和突变检测。由于检测反应主要依赖于单价阳离子浓度和MutL浓度，因此以直接的方式获得了最佳灵敏度和信噪比。证明了这些参数的最佳值与DNA测试片段长度的定量关系，支持了这些酶作用机制的易位模型，而不是分子开关模型。因此，对于每个新的基因组目标区域，都可以进行快速、不依赖序列的优化。还研究了其他可能限制错配扫描灵活性的因素，如目标片段内dam识别位点的定位。我们开发了几种策略，可轻松实现自动化，将分析限制在样本间异源双链体上。因此，我们称为PCR候选区域错配扫描的这种方法在经济高效的高通量检测中使用的主要障碍已被消除。
