Lee Y M, Lee S C
Institute of Biological Chemistry, School of Medicine, National Taiwan University, Taiwan, Republic of China.
Anal Biochem. 1988 Dec;175(2):521-4. doi: 10.1016/0003-2697(88)90577-5.
A modification of Lin's systematic DNA sequencing strategy is described. A method based on the religation of compatible cohesive ends generated by Sau3AI and BamHI was developed. The original procedure has been simplified and the yield of transfectant has been greatly improved. After complete digestion with BamHI and limited cleavage with Sau3AI, the single-cut linear DNA does not have to be separated from the supercoil or the open circular DNA on an agarose gel. After ligation, the DNA is digested with the restriction enzyme between the cloning site and BamHI site again. The original intact DNA is linearized, whereas the deleted subclone is not. Therefore the background is decreased to an undetectable level. This DNA sequencing strategy was tested on a 1.4-kb cDNA fragment containing the haptoglobin-related sequences. It is not necessary to purify large amounts of RF DNA (500 ng is enough) to get enough subclones. A set of subclones was produced in 1 day and the yield of plaques was about sixfold higher than that published.
本文描述了对林氏系统DNA测序策略的一种改进。开发了一种基于Sau3AI和BamHI产生的兼容粘性末端重新连接的方法。原始程序得到了简化，转染子的产量也有了很大提高。用BamHI完全消化并经Sau3AI有限切割后，单切线性DNA无需在琼脂糖凝胶上与超螺旋或开环DNA分离。连接后，DNA再次用限制酶在克隆位点和BamHI位点之间消化。原始完整DNA被线性化，而缺失的亚克隆则不会。因此背景降低到无法检测的水平。这种DNA测序策略在一个包含触珠蛋白相关序列的1.4 kb cDNA片段上进行了测试。无需纯化大量的RF DNA（500 ng就足够）来获得足够的亚克隆。一天内就产生了一组亚克隆，噬菌斑的产量比已发表的高出约六倍。
