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

为了构建染色体区域或带特异性DNA文库，我们发展了一种染色体显微切割/微克隆的方法。简而言之，一个确定的染色体区域或条带是在显微镜下被解剖的，显微镜上配备了一根细小的玻璃针，用来操作显微操纵器。从分离和收集的多条染色体片段中提取DNA，在显微镜下连接到10聚体DNA(接头)/24聚体DNA(引物)。以24聚体DNA为引物，分30个循环进行聚合酶链式反应。将PCR扩增的DNA连接到pUC19上，从8q23.3-q24.11区域获得3.5万个克隆，从2q33-qTER区域获得5万个克隆。以扩增产物为探针池，通过荧光染色体原位抑制杂交(染色体涂染)证实扩增产物来自解剖区域。从8q文库中选择的60个克隆中，有12个(20%)是唯一序列(单拷贝)，而从2q文库中选择的88个克隆中，有15个是唯一序列(17%)。在来自8q文库的12个独特克隆中，有9个在两个三鼻指趾综合征(TRPS)和del(8)(q23.3q24.12)患者中显示了单拷贝密度，这表明它们被映射到这两个患者中缺失的区域。用2q-特异性微克隆对噬菌体文库进行筛选，得到6个克隆，并定位在解剖区域。在6个克隆中，有2个克隆具有RFLP。这些克隆可用于TRPS或Waardenburg综合征I型的分析。显微切割/聚合酶链式反应用于诊断来源不明的染色体异常。利用这项技术，成功地追踪到了一个微小的额外标记染色体，它是从Y染色体衍生出来的。同样，17P+的另一个片段被追踪到来自15qter。

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.