Parker R C, Watson R M, Vinograd J
Proc Natl Acad Sci U S A. 1977 Mar;74(3):851-5. doi: 10.1073/pnas.74.3.851.
The cleavage of DNA by restriction endonucleases can be limited by the addition of ethidium bromide. When closed circular DNA is used as a substrate, DNA with one-site cleavages of one or both strands can be made by adding appropriate amounts of dye. The singly cleaved DNA is a complete set of full-length permuted linear molecules. Fractionation of the products of a digestion of the permuted linears with a single-hitting restriction endonuclease by gel electrophoresis yields a series of bands that can be used to determine relative molecular weights of the DNA fragments in the gel without the introduction of standards. It is possible to determine the relative molecular weight of a fragment to within +/-2.5%. These molecular weights immediately allow the determination of the HindIII and Hpa I maps of simian virus 40. The HindIII map of bacteriophage PM2 was determined by this method with one ambiguity that was resolved by using traditional techniques.
通过添加溴化乙锭可以限制限制性内切核酸酶对DNA的切割。当使用闭环DNA作为底物时，通过添加适量的染料可以制备单链或双链有一个切割位点的DNA。单链切割的DNA是一组完整的全长重排线性分子。用单切割限制性内切核酸酶消化重排线性分子的产物，通过凝胶电泳进行分离，可得到一系列条带，无需引入标准品即可用于确定凝胶中DNA片段的相对分子量。可以将片段的相对分子量测定在±2.5%以内。这些分子量可立即用于确定猿猴病毒40的HindIII和Hpa I图谱。噬菌体PM2的HindIII图谱就是用这种方法测定的，其中有一个模糊之处通过传统技术得以解决。
