GENETIC MAPPING AND CLONING OF THE HD GENE USING YACS

使用 YACS 进行 HD 基因的遗传图谱和克隆

• 批准号: 2208442
• 负责人: Danilo A. Tagle
• 金额: $ 2.86万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-04-01 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/2208442
• 关键词: Huntington's disease; artificial chromosomes; complementary DNA; dinucleotide; genetic mapping; genetic polymorphism; human genetic material tag; molecular cloning; nucleic acid probes; nucleic acid repetitive sequence; nucleic acid sequence; polymerase chain reaction; southern blotting

The Huntington's disease gene (HD) lies in the telomeric chromosome band 4p16.3, within a 2.5 megabase region flanked by genetic markers D4S125 and D4S168 (Whaley et al., 1991; Bates et al., 1991). The enormity of this region suggests the need for further refinement of the candidate region through linkage disequilibrium studies, which for example have been invaluable in the cloning of the cystic fibrosis gene. It is expected that nonrandom association of certain marker alleles with the HD mutation may provide the genetic information necessary for more precise placement of the HD gene. The first part of this proposal will seek to identify highly polymorphic (dC-dA).(dG-dT) dinucleotide repeats in the candidate region. At least four such repeats have already been identified and are being subcloned from phages and cosmids that have been mapped in this region. A PCR-based assay is further proposed to facilitate the identification of these (CA)n markers directly from yeast artificial chromosomes (YACs) isolated for the candidate region; (CA)n containing inter-Alu PCR amplification products will be identified by Southern hybridization to a (CA)n probe and the sequences flanking the (CA)n repeat block will be directly determined by sequencing the gel- purified PCR product using Alu primers or (CA)n and (GT)n oligomers. The second part of the proposal will seek to identify and characterize candidate genes for HD. This will entail several approaches: a) utilizing gel-purified YACs as probes to directly screen cDNA libraries, b) immobilizing gel-purified YACs onto membrane filters and applying PCR- amplified cDNA inserts as probes.

亨廷顿舞蹈病基因位于染色体端粒区 4p16.3，在2.5兆碱基区域内，两侧是遗传标记D4 S125 和D4 S168（Whaley等人，1991; Bates等人，1991年）。 的艰巨性 该区域表明需要进一步完善候选人 通过连锁不平衡研究，例如， 在囊性纤维化基因的克隆中发挥了不可估量的作用。 是 预期某些标记等位基因的非随机关联 HD突变可以提供更多必要的遗传信息， HD基因的精确定位 该提案的第一部分将 寻求鉴定高度多态性（dC-dA）。（dG-dT）二核苷酸重复序列 在候选人区域。 至少有四个这样的重复已经被 鉴定并正在从已被 在这个地区的地图。 进一步提出了基于PCR的测定， 有助于直接从酵母中鉴定这些（CA）n标记 为候选区域分离的人工染色体（YAC）;（CA）n 将通过以下方法鉴定含有Alu间PCR扩增产物的 与（CA）n探针的Southern杂交和与（CA）n探针的侧翼序列的Southern杂交是通过DNA序列的Southern杂交来进行的。 (CA)n将通过对凝胶进行测序直接确定重复阻断。 使用Alu引物或（CA）n和（GT）n寡聚物纯化PCR产物。 的 建议的第二部分将设法查明和描述 HD的候选基因 这将需要采取几种办法： 利用凝胶纯化的YAC作为探针直接筛选cDNA文库， B）将凝胶纯化的YAC固定在膜滤器上，并应用PCR-PCR技术， 扩增的cDNA插入片段作为探针。