PHYSICAL MAPPING OF HUMAN CHROMOSOME 17Q23/Q24

人类染色体 17Q23/Q24 的物理图谱

• 批准号: 5211991
• 负责人: KETAN SHAH
• 金额: --
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/5211991
• 关键词: artificial chromosomes; chromosome walking; chromosomes; diagnosis design /evaluation; genetic disorder diagnosis; genetic mapping; genetic markers; human genetic material tag; molecular pathology; nucleic acid sequence; polymerase chain reaction; pulsed field gel electrophoresis; restriction fragment length polymorphism; southern blotting

This is a first time new grant proposal submitted under the MBRS Program of NIH and an expansion of a current pilot project on PCR-based characterization of YAC contigs of 17q22-q23. The long term goal is to carry out physical mapping of q23-q24 region on human chromosome 17. This region consists of several genetic markers such as GHC, CSA, PL, GAA, CSN4A and TK1. A linkage has been shown between some markers. A few internal probes such as pC63, pTHH59, pTh17.12, 128E1 and pRMU1 are available in this region. However, very few YACs (yeast artificial chromosomes) have been isolate from this distal region of the long are of chromosome 17. The specific aims for this project are to begin construction of a physical map with overlapping YACs in the 17q23-q24 region. The physical mapping of this region is important to understand the molecular basis of several genes responsible for different disorders such a placental lactogen deficiency, growth hormone deficiency, acid- maltase deficiency, Pompe disease and hyperkalemic periodic paralysis in humans. In addition, the characterization of the certain genomic sequences will lead in the future to develop early molecular diagnostic tests for the appropriate disorders. For example, acute promyelocytic leukemia can be diagnosed by the detection of rearranged and translocate RARA gene (17q21) into myl locus of chromosome 15q22. It would also help to develop detection of myl-RARA fusion transcript by mRNA-PCR (polymerase chain reaction) amplification. The starting point in construction of overlapping contigs will be either from a known YAC for CSN4A marker (hyperkalemic periodic paralysis) or by isolating a YAC for a known marker {for example GAA marker (Pompe disease, acid-maltase deficiency)}. The strategies for building a physical map in the 17q23-24 region are as follows: chromosome walking will be done by employing (i) 'vectorette'-PCR and/or inverse PCR or Alu- vector PCR techniques to amplify small end-specific fragments in the appropriate YAC clones, (ii) cycle sequencing the fragment by enzymatic method using commercially available kits for either automated or manual DNA sequencer, (iii) designing PCR primers that are unique for the fragment (STS) and testing them for total human and yeast DNAs as template and (iv) submitting the primers to the Genome center, Univ. of Michigan for PCR-based YAC screening to isolate overlapping clone(s). In the course of chromosome walking, the other known genetic markers as discussed above will also be used a sSTSs to isolate overlapping YAC clones in the 17q23-24 region. Analyses of restriction fragments of overlapping YACs by PFGE (pulsed-field gel electrophoresis)/FIGE (field- inversion gel electrophoresis) would generate 'fingerprint' data which will be used in order to determine the direction of chromosome walking. Several probes such a left and right fragments of pBR322, human cot-1 DNA and internal marker will be used in these analyses.

这是第一次根据MBRS计划提交新的赠款提案 和扩大目前的试点项目， 17 q22-q23的YAC重叠群的表征。 长期目标是 对人类17号染色体q23-q24区域进行物理定位。 该区域由几个遗传标记组成，如GHC、CSA、PL， GAA、CSN 4A和TK 1。 一些标记之间存在连锁。 一 一些内部探针如pC63、pTHH 59、pTh17.12、128 E1和pRMU 1， 在这个地区可用。 然而，很少有YAC（酵母人工 染色体）已从这一远端区域的长 第17号染色体 这个项目的具体目标是开始 在17 q23-q24中具有重叠YAC的物理图谱的构建 地区 这个区域的物理地图对于理解 导致不同疾病的几个基因的分子基础 例如胎盘催乳素缺乏症、生长激素缺乏症、酸性- 麦芽糖酶缺乏症、庞贝氏症和高钾性周期性麻痹， 人类 此外，某些基因组的表征 序列将在未来引领开发早期分子诊断 测试适当的疾病。 例如，急性早幼粒细胞性 白血病可以通过检测重排和易位的 RARA基因（17 q21）插入染色体15 q22的myl位点。 这还将有助于 建立myl-RARA融合基因的mRNA-PCR检测方法 （聚合酶链反应）扩增。 构建重叠重叠群的起点将是 来自CSN 4A标志物的已知YAC（高钾性周期性麻痹）或 通过分离已知标志物的YAC（例如GAA标志物（Pompe 疾病，酸性麦芽糖酶缺乏症）。 建立一个 17 q23 -24区域的物理图谱如下：染色体步移 将通过采用（i）“载体”-PCR和/或反向PCR或Alu- 载体PCR技术扩增小的末端特异性片段， （ii）通过酶促PCR对所述片段进行循环测序， 方法使用市售试剂盒进行自动或手动 DNA测序仪，（iii）设计PCR引物，该引物对于DNA测序仪是独特的。 片段（STS），并测试它们的总人类和酵母DNA， 模板和（iv）将引物提交给Genome center，University of Michigan进行基于PCR的YAC筛选，以分离重叠克隆。 在染色体步移的过程中，其他已知的遗传标记， 上面讨论的也将用于sSTS以隔离重叠的YAC 17 q23 -24区域的克隆。 酶切片段分析 通过PFGE（脉冲场凝胶电泳）/FIGE（场电泳）重叠YAC， 反向凝胶电泳）将产生“指纹”数据， 将用于确定染色体步移的方向。 用pBR 322左右片段、人cot-1 DNA等探针， 和内部标记物将用于这些分析。