Construction of DNA Libraries Specific for Chromosomal Regions or Bands by Chromosome Microdissection, and Its Application to Medical Genetics

染色体显微切割技术构建染色体区域或条带特异性DNA文库及其在医学遗传学中的应用

基本信息

批准号：
02454493
负责人：
NIIKAWA Norio
金额：
$ 4.35万
依托单位：
Nagasaki University School of Medicine
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (B)
财政年份：
1990
资助国家：
日本
起止时间：
1990 至 1991
项目状态：
已结题

项目摘要

In order to construct chromosomal region or band-specific DNA libraries, we developed a method of chromosome microdissection/microcloning. In short, a defined chromosomal region or band was dissected under a microscope equipped with a fine glass-needle handling a micromanipulator. From several chromosome pieces dissected and collected, DNA was extracted and ligated to a 10mer DNA (linker)/24mer DNA (primer) under a microscope. PCR was performed by 30 cycles, using the 24mer DNA as a primer. PCR amplified DNA was ligated to pUC19 and cloned.A total of 35, 000 clones were obtained from a region 8q23.3-q24.11, and 50, 000 from a region 2q33-qter. The confirmation that the PCR product was derived from the dissected regions was done by fluorescence chromosome in situ suppression hybridization (chromosome painting) using the product as a probe pool. Of 60 clones selected from the 8q-library, 12 (20%) were unique (single-copy) sequences, while 15 (17%) of 88 clones from the 2q-library were unique sequences. Nine of the 12 unique clones from the 8q-library showed a one-copy density in two patients with tricho-rhino-phalangeal syndrome (TRPS) and del (8) (q23.3q24.12), an indication that they are mapped to the region deleted in both patients. Screening of a phage-libraty with the 2q-specific microclones revealed 6 clones and they are mapped at the dissected region. Of the 6 clones, 2 revealed RFLPs. These clones are useful for analaysis of TRPS or Waardenburg syndrome type 1.The microdissection/PCR was applied to the diagnosis of chromosome abnormalities of which origin was unknown. With this technique, an minute additional marker chromosome was successfully traced ; it was derived from Y chromosome. Likewise, an additional segment of 17p+ was traced to be derived from 15qter.

为了构建染色体区域或带特异性DNA文库，我们开发了一种染色体显微解剖/微分子的方法。简而言之，在配备细小玻璃针的显微镜下剖定了定义的染色体区域或条带，以处理微型手持。从剖析和收集的几个染色体片中，将DNA提取并连接到显微镜下的10mer DNA（接头）/24mer DNA（底漆）。使用24mer DNA作为底漆，通过30个周期进行PCR。将PCR扩增的DNA连接至PUC19，并克隆。总共从8Q223.3-Q24.11区的区域获得了35、000个克隆，并从2q33-Q的区域2q33-qter获得了50,000个克隆。证实PCR产物是从解剖区域得出的，该荧光染色体以原位抑制杂交（染色体涂料）为探针库来完成。在从8Q图书馆中选择的60个克隆中，有12个（20％）是唯一的（单拷贝）序列，而来自2q-library的88个克隆中有15个（17％）是独特的序列。来自8Q图书馆的12个独特克隆中有9个显示了两名毛 - rhino-phlangeal综合征（TRP）和DEL（8）患者的单拷贝密度（Q23.3Q24.12），这表明它们被映射到该地区在两名患者中均删除。用2Q特异性微球筛选了噬菌体 - 上线显示6个克隆，并在解剖区域映射。在6个克隆中，有2个揭示了RFLP。这些克隆对于TRP或Waardenburg综合征的分析很有用。微分解/PCR用于诊断起源不明的染色体异常。通过这种技术，成功追踪了一个分钟的其他标记染色体。它源自Y染色体。同样，将额外的17p+段追溯到从15qter得出。