Church G M, Gilbert W
Proc Natl Acad Sci U S A. 1984 Apr;81(7):1991-5. doi: 10.1073/pnas.81.7.1991.
Unique DNA sequences can be determined directly from mouse genomic DNA. A denaturing gel separates by size mixtures of unlabeled DNA fragments from complete restriction and partial chemical cleavages of the entire genome. These lanes of DNA are transferred and UV-crosslinked to nylon membranes. Hybridization with a short 32P-labeled single-stranded probe produces the image of a DNA sequence "ladder" extending from the 3' or 5' end of one restriction site in the genome. Numerous different sequences can be obtained from a single membrane by reprobing. Each band in these sequences represents 3 fg of DNA complementary to the probe. Sequence data from mouse immunoglobulin heavy chain genes from several cell types are presented. The genomic sequencing procedures are applicable to the analysis of genetic polymorphisms, DNA methylation at deoxycytidines, and nucleic acid-protein interactions at single nucleotide resolution.
独特的DNA序列可以直接从小鼠基因组DNA中确定。变性凝胶按大小分离来自整个基因组完全酶切和部分化学切割的未标记DNA片段混合物。这些DNA条带被转移并通过紫外线交联到尼龙膜上。与短的32P标记单链探针杂交产生从基因组中一个酶切位点的3'或5'端延伸的DNA序列“阶梯”图像。通过重新杂交可以从单个膜上获得许多不同的序列。这些序列中的每条带代表与探针互补的3 fg DNA。展示了来自几种细胞类型的小鼠免疫球蛋白重链基因的序列数据。基因组测序程序适用于遗传多态性分析、脱氧胞苷处的DNA甲基化以及单核苷酸分辨率下的核酸-蛋白质相互作用分析。
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