Two-dimensional S1 nuclease heteroduplex mapping: detection of rearrangements in bacterial genomes.
Two-dimensional S1 nuclease heteroduplex mapping: detection of rearrangements in bacterial genomes.
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二维 S1 核酸酶异源双链图谱：检测细菌基因组中的重排。
DOI：
10.1073/pnas.81.9.2723
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发表时间：
1984
影响因子：
11.1
通讯作者：
Inouye,M
中科院分区：
文献类型：
--
作者：
Yee,T;Inouye,M
建立了一种用于细菌全染色体基因重排和重复序列检测的二维S1核酸酶异源双链作图方法。为了检测两种变异细菌菌株之间的DNA重排，将来自两种菌株的总染色体DNA制备物用识别四个碱基的限制性内切酶消化，混合在一起，变性，复性，并在第一维聚丙烯酰胺平板凝胶上分离。切下凝胶条并浸泡在含有S1核酸酶的缓冲液中，S1核酸酶扩散到条带中并使DNA片段在单链区域发生变性。然后将消化的DNA在垂直于第一维度的第二维上进行电泳。被S1核酸酶消化的DNA异源双链体在明亮的未分辨的同源双链体条带下分辨为不同的斑点。本报告介绍了测试的模型系统，由两个几乎同源的大肠杆菌菌株，和应用这种方法在检测与相变化粘球菌黄的DNA重排。
A method of two-dimensional S1 nuclease heteroduplex mapping was developed to detect gene rearrangements and repeated sequences in total bacterial chromosomes. To detect DNA rearrangements between two variant bacterial strains, total chromosomal DNA preparations from the two strains are digested with four-base-recognizing restriction enzymes, mixed together, denatured, renatured, and separated on first-dimension polyacrylamide slab gels. Gel strips are cut out and soaked in a buffer containing S1 nuclease, which diffuses into the strips and digests the DNA fragments at single-stranded regions. The digested DNA is then electrophoresed in a second dimension perpendicular to the first dimension. DNA heteroduplexes that were digested by the S1 nuclease are resolved as distinct spots below a bright unresolved band of homoduplex. This report describes testing of this method on a model system consisting of two nearly isogeneic strains of Escherichia coli, and the application of this method in detecting DNA rearrangements associated with phase variation in Myxococcus xanthus.