Hobden A N, Read M J, Dykes C W, Harford S
Anal Biochem. 1985 Jan;144(1):75-8. doi: 10.1016/0003-2697(85)90085-5.
A solution hybridization procedure for the rapid identification of M13 clones carrying a particular sequence is described. The method, which employs a radiolabeled oligonucleotide probe, can discriminate between sequences which differ by only a single base, and can therefore be used for the identification of mutant sequences created by oligonucleotide-directed mutagenesis. Samples of phage-containing supernatant from cultures of M13-infected Escherichia coli are incubated with radiolabeled probe in the presence of sodium dodecyl sulfate. The mixtures are then subjected to agarose gel electrophoresis to separate hybrid molecules from unbound probe and hybridization is detected by autoradiography. This solution hybridization procedure is quicker and more convenient than membrane hybridization and has the added advantage that more than one probe can be used on a given gel.
本文描述了一种用于快速鉴定携带特定序列的M13克隆的溶液杂交程序。该方法采用放射性标记的寡核苷酸探针，能够区分仅相差一个碱基的序列，因此可用于鉴定由寡核苷酸定向诱变产生的突变序列。将来自M13感染的大肠杆菌培养物的含噬菌体上清液样品与放射性标记的探针在十二烷基硫酸钠存在下孵育。然后将混合物进行琼脂糖凝胶电泳，以从未结合的探针中分离杂交分子，并通过放射自显影检测杂交情况。这种溶液杂交程序比膜杂交更快、更方便，并且具有在给定凝胶上可使用不止一种探针的额外优势。
