CONSTRUCTION OF LIBRARIES ENRICHED FOR SEQUENCE REPEATS AND JUMPING CLONES, AND HYBRIDIZATION SELECTION FOR REGION-SPECIFIC MARKERS
CONSTRUCTION OF LIBRARIES ENRICHED FOR SEQUENCE REPEATS AND JUMPING CLONES, AND HYBRIDIZATION SELECTION FOR REGION-SPECIFIC MARKERS
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DOI：
10.1073/pnas.91.1.88
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发表时间：
1994-01-04
影响因子：
11.1
通讯作者：
WEISSMAN, SM
中科院分区：
文献类型：
--
作者：
KANDPAL, RP;KANDPAL, G;WEISSMAN, SM
我们描述了一种简单而快速的方法，用于构建高度富集二聚体、三聚体和四聚体核苷酸重复基序的小插入基因组文库。该方法涉及使用通过PCR扩增小插入物超声处理的基因组噬菌体文库或通过单引物PCR扩增Mbo I消化和接头连接的基因组DNA回收的DNA插入物。将基因组DNA插入物热变性并与生物素化的寡核苷酸杂交。将生物素化的杂交体保留在Veclidin-抗生物素蛋白基质上并特异性洗脱。PCR扩增并克隆该cDNA。用这种方法富集（CA）n微卫星的文库中超过90%的克隆含有含有CA重复的插入片段的克隆。我们还使用该方案富集（CAG）n和（AGAT）n序列重复和Not I跳跃克隆。我们已经使用了丰富的文库与适应的cDNA选择方法，以丰富的重复基序编码的酵母人工染色体。
We describe a simple and rapid method for contructing small-insert genomic libraries highly enriched for dimeric, trimeric,and tetrameric nucleotide repeat motifs. The approach involves use of DNA inserts recovered by PCR amplification of a small-insert sonicated genomic phage library or by a single-primer PCR amplification of Mbo I-digested and adaptor-ligated genomic DNA. The genomic DNA inserts are heat denatured and hybridized to a biotinylated oligonucleotde. The biotinylated hybrids are retained on a Vectrex-avidin matrix and eluted specifically. The eluate is PCR amplified and cloned. More than 90% of the clones in a library enriched for (CA)n microsatellites with this approach contained clones with inserts containing CA repeats. We have also used this protocol for enrichment of (CAG)n and (AGAT)n sequence repeats and for Not I jumping clones. We have used the enriched libraries with an adaptation of the cDNA selection method to enrich for repeat motifs encoded in yeast artificial chromosomes.