STRATEGY FOR THE CHARACTERIZATION OF THE HUMAN GENOME

我们希望充分发展一种新技术， 所有或几乎所有单碱基的识别和定位 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。 该技术是基于 该实验室最近的工作，以及早期的进展， 变性梯度电泳。 虽然程序 需要仔细检查基因组区域的克隆探针， 不需要确定碱基序列， 需要很少的时间和精力。 我们希望进行全面的 对人类β珠蛋白基因的审查， 携带转录或编码缺陷的基因组样本数量 已经在基因序列中被识别出来。 这将需要 少于一打的探测器 这样就可以调查 以确定多态性的存在 或遗传变异的特质， 重大的序列变化。 我们将致力于全面 对更大基因的审视 特别感兴趣的是 血友病A -因子VIII基因，ras激活 癌基因、慢性肉芽肿病和阿尔茨海默病。 我们计划全面开发一种新的梯度仪器， 既简化了变性梯度过程， 大大提高数据质量。 我们计划探索 该系统所依赖的DNA解链的某些方面，以及 可诱导的解链序列依赖性的变化 不同的环境，以提供一种替代方法， 全面检测所有序列变化。 我们将 继续对热稳定性的影响进行热力学研究 DNA螺旋中的非沃森-克里克缺陷。 这项工作有望提供一个有效的，简单的，广泛的 用于诊断遗传性疾病的适用手段。 它将 通过直接识别缺陷有助于基因诊断 在一个个体的基因中， 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.