Slilaty S N, Fung M, Shen S H, Lebel S
Biotechnology Research Institute, National Research Council of Canada, Montreal, Quebec.
Anal Biochem. 1990 Feb 15;185(1):194-200. doi: 10.1016/0003-2697(90)90279-i.
An approach for generating structures capable of directing full-length complementary-strand synthesis for double-stranded plasmid DNA is described. The structures are formed following heat denaturation and cooling of linearized plasmid DNA molecules in the presence of what is referred to as a "closing" oligonucleotide. Consisting of a sequence complementary to the free ends of one of the two plasmid strands, the closing oligonucleotide functions as an agent for recircularization of a DNA strand and generation of a primer-circular template structure suitable for polymerase-dependent full-length complementary-strand synthesis and ligation into a covalently closed heteroduplex molecule. When combined with a mutagenic oligonucleotide and uracil-substituted DNA templates, this approach allows site-directed mutagenesis to be performed directly on double-stranded DNA with a mutant formation efficiency of about 50%, a level amenable to rapid screening by DNA sequencing.
本文描述了一种生成能够指导双链质粒DNA全长互补链合成的结构的方法。这些结构是在所谓的“封闭”寡核苷酸存在下，线性化质粒DNA分子热变性并冷却后形成的。封闭寡核苷酸由与两条质粒链之一的自由末端互补的序列组成，其作用是使DNA链重新环化，并生成适合于依赖聚合酶的全长互补链合成并连接成共价闭合异源双链分子的引物-环状模板结构。当与诱变寡核苷酸和尿嘧啶取代的DNA模板结合时，这种方法允许直接在双链DNA上进行定点诱变，突变形成效率约为50%，这一水平便于通过DNA测序进行快速筛选。
