Kao F T, Yu J W
Eleanor Roosevelt Institute for Cancer Research, Denver, CO 80206.
Proc Natl Acad Sci U S A. 1991 Mar 1;88(5):1844-8. doi: 10.1073/pnas.88.5.1844.
A procedure has been described for microdissection and microcloning of human chromosomal DNA sequences in which universal amplification of the dissected fragments by Mbo I linker adaptor and polymerase chain reaction is used. A very large library comprising 700,000 recombinant plasmid microclones from 30 dissected chromosomes of human chromosome 21 was constructed. Colony hybridization showed that 42% of the clones contained repetitive sequences and 58% contained single or low-copy sequences. The insert sizes generated by complete Mbo I cleavage ranged from 50 to 1100 base pairs with a mean of 416 base pairs. Southern blot analysis of microclones from the library confirmed their human origin and chromosome 21 specificity. Some of these clones have also been regionally mapped to specific sites of chromosome 21 by using a regional mapping panel of cell hybrids. This chromosome microtechnology can generate large numbers of microclones with unique sequences from defined chromosomal regions and can be used for processes such as (i) isolating corresponding yeast artificial chromosome clones with large inserts, (ii) screening various cDNA libraries for isolating expressed sequences, and (iii) constructing region-specific libraries of the entire human genome. The studies described here demonstrate the power of this technology for high-resolution genome analysis and explicate their use in an efficient search for disease-associated genes localized to specific chromosomal regions.
本文描述了一种用于人类染色体DNA序列显微切割和微克隆的方法，该方法采用Mbo I连接子衔接头和聚合酶链反应对切割片段进行通用扩增。构建了一个非常大的文库，包含来自人类21号染色体30个切割染色体的700,000个重组质粒微克隆。菌落杂交显示，42%的克隆含有重复序列，58%的克隆含有单拷贝或低拷贝序列。完全Mbo I切割产生的插入片段大小范围为50至1100个碱基对，平均为416个碱基对。对文库中的微克隆进行Southern印迹分析，证实了它们的人类来源和21号染色体特异性。其中一些克隆还通过使用细胞杂种区域定位板被区域定位到21号染色体的特定位点。这种染色体微技术可以从特定染色体区域产生大量具有独特序列的微克隆，可用于以下过程：(i)分离具有大插入片段的相应酵母人工染色体克隆；(ii)筛选各种cDNA文库以分离表达序列；(iii)构建整个人类基因组的区域特异性文库。本文所述的研究证明了该技术在高分辨率基因组分析中的强大功能，并阐述了其在有效寻找定位于特定染色体区域的疾病相关基因中的应用。
