Rapid screening of a human genomic library in yeast artificial chromosomes for single-copy sequences.
Rapid screening of a human genomic library in yeast artificial chromosomes for single-copy sequences.
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DOI：
10.1073/pnas.86.15.5898
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发表时间：
1989-08
影响因子：
11.1
通讯作者：
C. Traver;S. Klapholz;R. Hyman;R. W. Davis
中科院分区：
文献类型：
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作者：
C. Traver;S. Klapholz;R. Hyman;R. W. Davis
从原代成纤维细胞DNA中产生了酿酒酵母中的酵母人工染色体（YAC）文库，该文库由30，000个克隆组成，平均插入大小为0.1兆碱基对的人DNA。对YAC载体进行了修饰，以使大肠杆菌质粒中的人DNA插入片段的两端能够回收，并赋予哺乳动物细胞G418抗性。利用酵母原生质球在海藻酸钙薄层中再生的能力，建立了一种快速的酵母菌落杂交方法。该方法允许直接复制平板接种并将菌落从初级转化平板处理至硝酸纤维素过滤器。已经建立了酵母菌落杂交条件，以在人基因组DNA的YAC文库中鉴定与具有独特单拷贝序列的人DNA探针具有同源性的人工染色体。通过与DNA探针杂交，鉴定了具有来自人Xq28区域的0.1兆碱基对插入物的人工染色体，所述DNA探针检测因子VIII基因3'端附近的独特序列。
A yeast artificial chromosome (YAC) library in Saccharomyces cerevisiae consisting of 30,000 clones with an average insert size of 0.1 megabase pair of human DNA has been generated from primary fibroblast DNA. A YAC vector was modified to enable the recovery of both ends of a human DNA insert in plasmids in Escherichia coli and to confer G418 resistance to mammalian cells. A rapid method for yeast colony hybridization was used that exploits the ability of yeast spheroplasts to regenerate in a thin layer of calcium alginate. This method permits direct replica plating and processing of colonies from the primary transformation plate to nitrocellulose filters. Yeast colony hybridization conditions have been established to identify, within a YAC library of human genomic DNA, artificial chromosomes with homology to human DNA probes of unique single-copy sequence. An artificial chromosome with a 0.1-megabase-pair insert from the human Xq28 region has been identified by hybridization to a DNA probe that detects a unique sequence near the 3' end of the factor VIII gene.