Lin H C, Lei S P, Wilcox G
Anal Biochem. 1985 May 15;147(1):114-9. doi: 10.1016/0003-2697(85)90016-8.
A modification of Hong's systematic DNA sequencing strategy is described. The original procedure has been simplified and transfectant yield increased. After DNase I limited cleavage in the presence of Mn2+, the single-cut linear DNA does not have to be separated from supercoiled or open circular DNA on an agarose gel. After ligation, the DNA is digested with a second restriction endonuclease for which a unique cleavage site resides between the insert and the first restriction endonuclease cutting site. The original intact DNA is linearized whereas the deleted subclone is not. The background is decreased to an undetectable level. This DNA sequencing strategy was tested on a 1.4-kb DNA fragment containing the araC regulatory gene from Erwinia carotovora. A set of subclones sufficient to sequence the fragment on both strands was produced in 2 days and the yield was at least 60-fold higher than in the original protocol.
本文描述了对洪氏系统DNA测序策略的一种改进。原始程序已被简化，转染子产量有所提高。在Mn2+存在的情况下进行DNase I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，用第二种限制性内切酶消化DNA，其独特的切割位点位于插入片段和第一个限制性内切酶切割位点之间。原始完整DNA被线性化，而缺失的亚克隆则不会。背景降低到不可检测的水平。该DNA测序策略在一个包含胡萝卜软腐欧文氏菌araC调控基因的1.4kb DNA片段上进行了测试。在两天内产生了一组足以对该片段进行双链测序的亚克隆，产量比原始方案至少高60倍。
