COMPUTER-ASSISTED NON-RADIOACTIVE MULTIPLEX GENOTYPING

这项研究的目标是建立一种快速、非放射性的 人微卫星遗传标记多重基因分型系统。 该系统将包括一个用于自动等位基因调用的计算机程序 和数据库交互。大约300个信息量很大的 相距约10 cM的微卫星标记 覆盖了整个人类基因组，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，五个不同的标记将在 每个人的DNA都有相同的聚合酶链式反应。池化将根据以下条件确定 常见的聚合酶链式反应条件。第二，来自10个这样的集合的PCR产物(即， 来自50个基因座的聚合酶链式反应产物将混合在一起，变性后分离 聚丙烯酰胺凝胶的直接转移电泳法(DTE)。聚合酶链式反应 产品将被转移到尼龙膜上，由 杂交。第三，解码步骤将使用五个 每一次杂交都有寡核苷酸探针。这五个探测器将 揭示五个大小不同的单个基因座。此外， 用于自动等位基因调用的计算机程序将以空间为特征 基于多路复用特定内部标准的标准化，自动 改进了从同一膜派生的多个图像的对齐 算法实现了等位基因的自动调用，并且直观、简单 便携用户界面。自动分析的结果将是 存储在与用于链接的常用程序兼容的文件中 分析。完成的系统将只需要6个人月的时间来 大约200个个体的300个基因标记。建制派 非放射性检测方法对于减少健康是必不可少的 对替补科学家和消除放射性废物处置的风险 有问题。

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.