Mapping DNA replication origins on human chromosome 22

绘制人类 22 号染色体上的 DNA 复制起点

• 批准号: 6584868
• 负责人: ERIC J WHITE
• 金额: $ 4.16万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-06-01 至 2006-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6584868
• 关键词: DNA replication origin; HeLa cells; chromosomes; functional /structural genomics; genetic mapping; genetic regulatory element; human genetic material tag; microarray technology; polymerase chain reaction; postdoctoral investigator; tissue /cell culture

DESCRIPTION (provided by applicant): I will map origins of DNA replication on human chromosome 22. The Snyder laboratory constructed a DNA microarray that contains approximately 85% of the nonrepetitive DNA of chromosome 22 in fragments ranging from 0.3-1.4 kilobases. I will utilize a replication timing technique to map DNA origins with the existing chromosome 22 microarray. In addition, I will construct an assymetric chromosome 22 DNA microarray by strand specific amplification of each of the DNA fragments from the microarray. Due to the bidirectional nature of DNA replication at origins, the change in polarity of leading and lagging strand synthesis indicates the presence of an intervening origin of replication. I will utilize previously published origin mapping protocols for the isolation of both leading and lagging strand (Okazaki fragment) synthesis. The isolated nacent strands will be labeled and hybridized to the assymetric array. Origins of DNA replication will be identified at sites of replication fork polarity switching. The two techniques are complementary since the replication timing technique identifies regions containing origins while fork polarity switching more accurately defines origin location.

描述（由申请人提供）：我将绘制人类22号染色体DNA复制的起源。Snyder实验室构建了一个DNA微阵列，其中包含约85%的22号染色体非重复DNA，片段范围为0.3-1.4千碱基。我将利用复制定时技术用现有的22号染色体微阵列绘制DNA起源图。此外，我将构建一个不对称的22号染色体DNA微阵列通过特定链扩增每个DNA片段从微阵列。由于DNA复制起点的双向性，前导链和后导链合成极性的变化表明存在一个中间的复制起点。我将利用先前发表的起源映射协议来分离前导链和滞后链（冈崎片段）合成。分离的近链将被标记并杂交到不对称阵列。DNA复制的起源将在复制叉极性转换的位点被识别。这两种技术是互补的，因为复制定时技术识别包含起源的区域，而叉极性切换更准确地定义起源位置。