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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条带特异性DNA文库，我们发展了一种染色体微切割/微克隆的方法。简而言之，在配备有细玻璃针的显微镜下解剖确定的染色体区域或带，该细玻璃针操作显微操作器。从解剖和收集的几个染色体片段中提取DNA，并在显微镜下连接到10聚体DNA（接头）/24聚体DNA（引物）。使用24聚体DNA作为引物，通过30个循环进行PCR。将PCR扩增的DNA连接到pUC 19上进行克隆，从8 q23. 3-q24. 11区域共获得35，000个克隆，从2 q33-qter区域共获得50，000个克隆。使用产物作为探针池，通过荧光染色体原位抑制杂交（染色体涂染）确认PCR产物来源于切割区域。从8 q文库中选择的60个克隆中，12个（20%）是独特的（单拷贝）序列，而从2 q文库中选择的88个克隆中，15个（17%）是独特的序列。来自8 q文库的12个独特克隆中的9个在两名患有鼻-指-趾综合征（TRPS）和del（8）（q23.3q24.12）的患者中显示出单拷贝密度，这表明它们被定位到两名患者中缺失的区域。用2 q特异性微克隆筛选噬菌体文库，发现6个克隆，并将它们定位在切割区域。在6个克隆中，2个克隆显示RFLP。这些克隆可用于TRPS或Waardenburg综合征1型的分析。显微切割/PCR用于诊断不明原因的染色体异常。利用这种技术，成功地追踪了一个微小的额外标记染色体;它来自Y染色体。同样，17 p+的另一个片段被追踪到源自15 qter。