STRATEGY FOR THE CHARACTERIZATION OF THE HUMAN GENOME

我们希望使一种新技术得到充分发展 所有或几乎所有单一基地的识别和本地化 人类基因组DNA任何选定区域的变化，相对于 野生型或任何参考序列。这项技术是基于 该实验室的最新工作，以及早期在 变性梯度电泳法。尽管手术过程 需要对基因组区域的克隆探针进行仔细检查，它 不需要确定碱基序列，并且它 只需要很少的时间和精力。我们希望进行完整的 对人类β珠蛋白基因的审查，面临着很大的挑战 携带转录或编码缺陷的基因组样本数量 在基因序列中已被确认的基因。这将需要 不到一打的探测器。然后，它将适合于调查 以确定是否存在多态 或遗传变异的特质，可能会被混淆 重大的序列变化。我们将努力实现全面的 对更大的基因的仔细研究。特别值得关注的是 血友病A-因子基因，激活ras 癌基因、慢性肉芽肿性疾病和阿尔茨海默病。 我们计划全面开发一种新的梯度仪器， 将既简化变性梯度程序，又将 大幅提高数据质量。我们计划探索 这个系统所依赖的DNA融化的某些方面，以及 可以诱导的熔化顺序依赖性的变化 通过不同的环境来提供替代方法 对所有序列变化的全面检测。我们会 继续热力学研究对热稳定性的影响 DNA螺旋中的非沃森-克里克缺陷。 这项工作可望提供一种有效、简单、广泛的 遗传病诊断的适用手段。会的 通过直接识别缺陷来帮助进行基因诊断 在一个个体的基因中，他可能已经或可能还没有表现出 问题的生理迹象，或在未来的父母。

We wish to bring to full development a new technique for recognition and localization of all or nearly all single base changes in any selected region of human genomic DNA, relative to wild-type or any reference sequence. The technique is based on recent work in this laboratory, together with earlier advances in denaturing gradient electrophoresis. Although the procedure requires cloned probes of the genomic region to be scrutinized, it does not require that the base sequence be determined, and it requires little time and effort. We wish to carry out complete scrutiny of the human beta globin gene, with challenge by a large number of genomic samples carrying transcription or coding defects that have been identified in the gene sequence. This will require fewer than one dozen probes. It will then be appropriate to survey of a small population to determine the presence of polymorphisms or idiosyncrasies of genetic variation that might be confused with significant sequence changes. We will work toward comprehensive scrutiny of much larger genes. Of particular interest are hemophilia A - the factor VIII gene, activation of the ras oncogene, chronic granulomatous disease, and Alzheimer's Disease. We plan to bring to full development a new gradient instrument that will both simplify the denaturing gradient procedure and will improve the quality of the data substantially. We plan to explore certain aspects of DNA melting, on which this system depends, and changes in the sequence-dependence of melting that can be induced by different environments to provide an alternative approach for the comprehensive detection of all sequence changes. We will continue thermodynamic studies on the effects on thermal stability of non-Watson-Crick defects in the DNA helix. This work is expected to provide an effective, simple, and widely applicable means for the diagnosis of genetic disease. It will contribute to genetic diagnosis by direct recognition of defects in the genes of an individual who may or may not already display physiological signs of the problem, or in prospective parents.