DEVELOP PCRS TO DETECT POLYMORPHISMS AT MAPPED RFLP LOCI

已被遗传定位的人类RFLP探针将被转化为 基于PCR的标记。 作图RFLP探针是研究的重要工具 在许多领域。 已经选择了约150个RFLP探针的子集， 目前用于绘制遗传疾病基因和研究 肿瘤细胞中的杂合性。 将子集探针转换为基于PCR的 标记物将极大地促进这些研究，因为基于PCR的基因分型 方法是对目前使用的基因组Southern的重大改进 杂交方法 本研究开发的寡核苷酸 也将是构建物理地图的重要研究成果 for human人类chromosomes染色体. 在某些情况下，它们也可以用于 遗传疾病和肿瘤的诊断，以及身份检测。 在I期可行性研究中，两个涉及CA二核苷酸的PCR标记物 重复，这是在原来映射的RFLP基因座附近，将 发展。 此外，二态性RFLP侧翼的区域将是 用随机方法进行亚克隆和测序。 在第二阶段， 为RFLP探针的整个子集开发。 其他方法，如DGGE （变性梯度凝胶电泳），SSCP（单链 构象多态性），反向PCR和载体PCR也将被 用于实现总体目标。

Human RFLP probes which have been genetically mapped will be converted into PCR-based markers. Mapped RFLP probes are an important tool for research in many areas. A subset of about 150 RFLP probes has been selected and is currently being used for mapping genetic disease genes and studying loss of heterozygosity in tumor cells. Converting the subset probes into PCR-based markers will greatly facilitate these studies because PCR-based genotyping methods are a significant improvement over currently used genomic Southern hybridization methods. The oligonucleotides developed in this research will also be an important research product for constructing physical maps for human chromosomes. In some cases they can also be used for the diagnosis of genetic diseases and tumors, and for identity testing. In phase I feasibility studies, two PCR-markers involving CA dinucleotide repeats, which are in the vicinity of the originally mapped RFLP loci, will be developed. In addition, the region flanking a dimorphic RFLP will be subcloned and sequenced using a random approach. In phase II, PCRs will be developed for the entire subset of RFLP probes. Other methods such as DGGE (denaturing gradient gel electrophoresis), SSCP (single-stranded conformation polymorphism), inverse PCR and vectorette PCR will also be used to achieve the overall goal.