Mapping DNA replication origins on human chromosome 22

描述(申请人提供)：我将绘制人类22号染色体上DNA复制的起始点。斯奈德实验室构建了一个DNA微阵列，它包含了大约85%的22号染色体的非重复DNA，片段大小在0.3-1.4千碱基之间。我将利用复制计时技术将DNA起源与现有的22号染色体微阵列进行映射。此外，我还将构建一个非对称22号染色体DNA微阵列，通过对微阵列中的每个DNA片段进行链特异性扩增来实现。由于起始处DNA复制的双向性质，领先和滞后链合成的极性变化表明存在一个中间的复制起点。我将利用以前发表的起源作图协议来分离领先链和滞后链(冈崎片段)的合成。分离的近亲链将被标记并与不对称阵列杂交。DNA复制的起始点将在复制分叉极性转换的位置确定。这两种技术是互补的，因为复制定时技术识别包含起始点的区域，而分叉极性切换更准确地定义起始点位置。

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.