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，我们开发了染色体微解剖/微克隆方法。简而言之，一个确定的染色体区域或带在显微镜下解剖配备了一个精细的玻璃针处理的显微操作器。从解剖和收集的几条染色体片段中提取DNA，并在显微镜下连接到10mer DNA（连接物）/24mer DNA（引物）上。以24mer DNA为引物，PCR 30次。将PCR扩增的DNA连接到pUC19上进行克隆。从8q23.3-q24.11区共获得35000个克隆，从2q33-qter区获得50000个克隆。用荧光染色体原位抑制杂交（染色体染色）作为探针池，证实PCR产物来源于解剖区。从8q文库中筛选的60个克隆中，有12个（20%）是唯一（单拷贝）序列，而从2q文库中筛选的88个克隆中有15个（17%）是唯一序列。来自8q文库的12个独特克隆中，有9个在两名tricho-rhino-phalangeal syndrome （TRPS）和del (8) （q23.3q24.12）患者中显示出1拷贝密度，这表明它们被映射到这两名患者中缺失的区域。用2q特异性微克隆筛选噬菌体文库，发现6个克隆，并在解剖区域定位。6个克隆中有2个显示RFLPs。这些克隆可用于TRPS或Waardenburg综合征1型的分析。应用显微解剖/PCR技术诊断病因不明的染色体异常。利用这种技术，成功地追踪了一分钟额外的标记染色体；它来源于Y染色体。同样，17p+的另一个片段被追踪到源自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.