Thomason Lynn C, Sawitzke James A, Li Xintian, Costantino Nina, Court Donald L
Basic Science Program, GRCBL-Molecular Control & Genetics Section, Frederick National Laboratory for Cancer Research, Leidos Biomedical Research, Inc, Frederick, Maryland.
Molecular Control and Genetics Section, Gene Regulation and Chromosome Biology, National Cancer Institute at Frederick, National Institutes of Health, Frederick, Maryland.
Curr Protoc Mol Biol. 2014 Apr 14;106:1.16.1-1.16.39. doi: 10.1002/0471142727.mb0116s106.
The bacterial chromosome and bacterial plasmids can be engineered in vivo by homologous recombination using PCR products and synthetic oligonucleotides as substrates. This is possible because bacteriophage-encoded recombination proteins efficiently recombine sequences with homologies as short as 35 to 50 bases. Recombineering allows DNA sequences to be inserted or deleted without regard to location of restriction sites. This unit first describes preparation of electrocompetent cells expressing the recombineering functions and their transformation with dsDNA or ssDNA. It then presents support protocols that describe several two-step selection/counter-selection methods of making genetic alterations without leaving any unwanted changes in the targeted DNA, and a method for retrieving onto a plasmid a genetic marker (cloning by retrieval) from the Escherichia coli chromosome or a co-electroporated DNA fragment. Additional protocols describe methods to screen for unselected mutations, removal of the defective prophage from recombineering strains, and other useful techniques.
利用PCR产物和合成寡核苷酸作为底物，通过同源重组可在体内对细菌染色体和细菌质粒进行工程改造。这之所以可行，是因为噬菌体编码的重组蛋白能有效地重组同源性低至35至50个碱基的序列。重组工程允许在不考虑限制酶切位点位置的情况下插入或删除DNA序列。本单元首先描述表达重组工程功能的电感受态细胞的制备及其用双链DNA或单链DNA进行转化。然后介绍了一些支持方案，这些方案描述了几种两步选择/反选择方法，用于在不使目标DNA留下任何不需要的变化的情况下进行基因改造，以及一种从大肠杆菌染色体或共电穿孔的DNA片段中将遗传标记物回收到质粒上的方法（通过回收进行克隆）。其他方案描述了筛选未选择突变的方法、从重组工程菌株中去除缺陷原噬菌体的方法以及其他有用技术。
