COMPUTER-ASSISTED NON-RADIOACTIVE MULTIPLEX GENOTYPING

这项研究的目标是建立一个快速，非放射性的 人类微卫星遗传标记的多重基因分型系统 该系统将包括用于自动等位基因识别的计算机程序 数据库交互。 约300个高度信息化的 微卫星标记，它们相距约10 cM， 覆盖他们的整个人类基因组，将被确定为多重 发展 我们建议的多路传输系统将涉及多路传输， 三个层次。 首先，五种不同的标记物将在 每个人的DNA都有相同的PCR 合并将根据以下因素确定 常见的PCR条件 第二，来自10组这样的PCR产物（即， 将来自50个基因座的PCR产物合并并在变性时分离 聚丙烯酰胺凝胶直接转移电泳（DTE）。 的PCR 产品将被转移到尼龙膜上进行“解码”， 杂交方法 第三，解码步骤将使用五个 在每次杂交中使用寡核苷酸探针。 这五个探测器将 揭示了五个具有可区分大小的个体基因座。 此外该 用于自动等位基因识别的计算机程序将以空间 基于多路特异性内标物的标准化，自动 来自同一膜的多个图像的对齐，改进 算法自动等位基因调用，和一个直观的，容易 便携式用户界面。 自动分析的结果将是 存储在与常用程序兼容的文件中，用于链接 分析. 完成后的系统只需要六个人工月， 基因型300标记在大约200个人。 建立 非放射性检测方法对于减少健康至关重要 对实验室科学家和消除放射性废物处置的风险 问题

The goal of this research is to establish a rapid, non-radioactive multiplex genotyping system for human microsatellite genetic markers. This system will include a computer program for automated allele calling and database interaction. Approximately 300 highly informative microsatellite markers, which are approximately 10 cM apart and which cover their entire human genome, will be identified for multiplex development. Our proposed multiplex system will involve multiplexing at three levels. First, five different markers will be amplified in the same PCR for each individual's DNA. Pooling will be determined based on common PCR conditions. Second, the PCR products from 10 such sets (i.e, the PCR products from 50 loci) will be pooled and separated on denaturing polyacrylamide gels by Direct Transfer Electrophoresis (DTE). The PCR products will be transferred onto nylon membranes for "decoding" by hybridization. Third, the decoding step will be performed using five oligonucleotide probes at each hybridization. These five probes will reveal five individual loci with distinguishable sizes. In addition, the computer program for automated allele calling will feature spatial normalization based on multiplex-specific internal standards, automatic alignment of several images derived form the same membrane, improved algorithms for automatic allele calling, and an intuitive, easily portable user interface. The results of the automatic analysis will be stored in files compatible with commonly used programs for linkage analysis. The finished system will require only six person-months to genotype 300 markers on approximately 200 individuals. The establishment of non-radioactive detection methods is essential for reducing health risks to bench scientists and for eliminating radioactive waste disposal problems.