Woolford J L, Rosbash M
Nucleic Acids Res. 1979 Jun 11;6(7):2483-97. doi: 10.1093/nar/6.7.2483.
A method is presented for the purification of mRNAs and the identification of structural gene sequences in recombinant DNA molecules. RNA is hybridized to double-stranded linear DNA such that R-loops are formed between most DNAs and their complementary RNA sequences. These R-loops are purified from unhybridized RNAs by gel filtration chromatography in the presence of a high concentration of salt. The complementary RNAs are released from the R-loops by heating, and are assayed by gel electrophoresis or cell free translation to determine their purity and to identify the proteins for which they code. We have demonstrated that recombinant DNAs containing sequences for abundant or moderately abundant mRNAs of Saccharomyces cerevisiae can be identified by this means.
本文介绍了一种纯化mRNA以及鉴定重组DNA分子中结构基因序列的方法。使RNA与双链线性DNA杂交，从而在大多数DNA与其互补RNA序列之间形成R环。在高浓度盐存在的情况下，通过凝胶过滤色谱法从未杂交的RNA中纯化这些R环。通过加热从R环中释放互补RNA，并通过凝胶电泳或无细胞翻译进行检测，以确定其纯度并鉴定它们所编码的蛋白质。我们已经证明，通过这种方法可以鉴定出含有酿酒酵母丰富或中等丰富mRNA序列的重组DNA。
