CONSTRUCTION OF SMALL-INSERT GENOMIC DNA LIBRARIES HIGHLY ENRICHED FOR MICROSATELLITE REPEAT SEQUENCES
CONSTRUCTION OF SMALL-INSERT GENOMIC DNA LIBRARIES HIGHLY ENRICHED FOR MICROSATELLITE REPEAT SEQUENCES
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DOI：
10.1073/pnas.89.8.3419
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发表时间：
1992-04-15
影响因子：
11.1
通讯作者：
DUYK, G
中科院分区：
文献类型：
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作者：
OSTRANDER, EA;JONG, PM;DUYK, G
我们描述了一种高效的方法，用于构建富含高度多态性的简单重复序列的小插入基因组文库。用这种方法，产生了其中40-50%的成员含有（CA）n重复的文库，代表了几乎等于常规小插入物基因组DNA文库的50倍富集。简而言之，在噬菌粒载体中构建平均插入物大小小于500个碱基对的基因组文库。该文库在大肠杆菌的dut ung菌株中的扩增允许文库作为闭合环状单链DNA回收，其中尿嘧啶经常代替胸腺嘧啶掺入。该DNA用作第二链DNA合成的模板，在高温下通过热稳定DNA聚合酶用（CA）n或（TG）n寡核苷酸引发。将该混合物转化到野生型E.大肠杆菌菌株导致引物延伸产物的回收，这是对含尿嘧啶的单链DNA分子进行强遗传选择的结果。以这种方式，回收了高度富集靶向的含微卫星克隆的文库。这种方法是广泛适用的，并且可以用于产生标记选择的文库，所述文库携带来自cDNA、全基因组、单染色体或更受限制的感兴趣的染色体区域的任何简单序列重复。
We describe an efficient method for the construction of small-insert genomic libraries enriched for highly polymorphic, simple sequence repeats. With this approach, libraries in which 40-50% of the members contain (CA)n repeats are produced, representing an almost-equal-to 50-fold enrichment over conventional small-insert genomic DNA libraries. Briefly, a genomic library with an average insert size of less than 500 base pairs was constructed in a phagemid vector. Amplification of this library in a dut ung strain of Escherichia coli allowed the recovery of the library as closed circular single-stranded DNA with uracil frequently incorporated in place of thymine. This DNA was used as a template for second-strand DNA synthesis, primed with (CA)n or (TG)n oligonucleotides, at elevated temperatures by a thermostable DNA polymerase. Transformation of this mixture into wild-type E. coli strains resulted in the recovery of primer-extended products as a consequence of the strong genetic selection against single-stranded uracil-containing DNA molecules. In this manner, a library highly enriched for the targeted microsatellite-containing clones was recovered. This approach is widely applicable and can be used to generate marker-selected libraries bearing any simple sequence repeat from cDNAs, whole genomes, single chromosomes, or more restricted chromosomal regions of interest.