SEGMENT-DIRECTED MUTAGENESIS - CONSTRUCTION INVITRO OF POINT MUTATIONS LIMITED TO A SMALL PREDETERMINED REGION OF A CIRCULAR DNA MOLECULE
SEGMENT-DIRECTED MUTAGENESIS - CONSTRUCTION INVITRO OF POINT MUTATIONS LIMITED TO A SMALL PREDETERMINED REGION OF A CIRCULAR DNA MOLECULE
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DOI：
10.1073/pnas.77.9.5375
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发表时间：
1980-01-01
期刊：
影响因子：
--
通讯作者：
BOTSTEIN, D
中科院分区：
文献类型：
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作者：
SHORTLE, D;KOSHLAND, D;BOTSTEIN, D
用一种有效地诱变闭合环状双链DNA分子预定片段的通用方法，在小质粒pBR322携带的β-内酰胺酶(Bla)基因的两个特定区域构建突变。片段定向突变的原理是利用单链同源DNA片段在DNA片段定义的片段内通过两步反应引导环状双链DNA的缺口。首先，大肠杆菌recA蛋白被用来催化同源单链DNA的同化，在环状DNA中产生一个置换环(D-loop)。然后，用少量的单链特异的S1核酸酶来切割移位的DNA。片段导向的缺口被转化为小缺口，然后专门用亚硫酸氢钠进行诱变。用bla基因中心的一小段(128碱基对)限制性内切酶片段进行诱变，发现7.5%的回收质粒为bla突变体，其中49/51的突变体位于一个与限制性片段的端点接近的缺失区间。当使用另一个覆盖基因开头的短片段时，也得到了类似的结果；这些突变中的许多都映射到编码信号序列的区域，该序列被认为与β-内酰胺酶的分泌有关。
A general method for efficiently mutagenizing a predetermined segment of a closed circular duplex DNA molecule was used to construct mutations in 2 specific regions of the .beta.-lactamase (bla) gene carried by the small plasmid pBR322. The principle of segment-directed mutagenesis is the use of a single-stranded homologous DNA fragment to direct the nicking of circular duplex DNA within a segment defined by the DNA fragment in a 2-step reaction. First, Escherichia coli recA protein is used to catalyze assimilation of the homologous single-stranded DNA, producing a displacement loop (D-loop) in the circular DNA. Then a small amount of the single-strand-specific S1 nuclease is used to nick the displaced DNA. The segment-directed nicks are converted to small gaps, which are then mutagenized specifically with sodium bisulfite. A short (128-base pair) restriction endonuclease fragment from the center of the bla gene was used to direct mutagenesis with the result that 7.5% of the recovered plasmids were bla- mutants and 49/51 of these mutants, mapped genetically, were found to lie in a deletion interval whose endpoints approximate those of the restriction fragment. Similar results were obtained when another short fragment covering the beginning of the gene was used; many of these mutations map in the region coding the signal sequence thought to be involved in secretion of .beta.-lactamase.