Looney J E, Han J H, Harding J D
Gene. 1984 Jan;27(1):67-73. doi: 10.1016/0378-1119(84)90239-7.
We describe a method for detecting specific DNA sequences cloned in M13 phage vectors, based on the procedure of Woo (in Wu, R., Methods in Enzymology, Vol. 68, Academic Press, New York, 1979, pp. 389-395). M13 plaques are adsorbed to a nitrocellulose filter that has been pre-saturated with bacteria. The filter is incubated on an agar plate to amplify the phage; the DNA is alkali-denatured and then hybridized with a radioactive RNA probe. Unlike standard procedures, this method detects and distinguishes M13 plaques containing phage particles which harbor either the coding or non-coding (RNA-like) DNA strand, when single-stranded RNA is used as probe. We have optimized this procedure with M13 clones containing mouse histidine tRNA gene sequences and have used it to determine the sequence of both strands of a mouse glycine tRNA gene.
我们描述了一种基于Woo的方法（见Wu, R.主编的《酶学方法》第68卷，学术出版社，纽约，1979年，第389 - 395页）检测克隆于M13噬菌体载体中的特定DNA序列的方法。将M13噬菌斑吸附到预先用细菌饱和的硝酸纤维素滤膜上。将滤膜在琼脂平板上孵育以扩增噬菌体；DNA经碱变性后与放射性RNA探针杂交。与标准程序不同的是，当使用单链RNA作为探针时，该方法可检测并区分含有携带编码或非编码（类RNA）DNA链的噬菌体颗粒的M13噬菌斑。我们用含有小鼠组氨酸tRNA基因序列的M13克隆优化了该程序，并将其用于确定小鼠甘氨酸tRNA基因两条链的序列。
