Oliner J D, Kinzler K W, Vogelstein B
Johns Hopkins Oncology Center, Baltimore, MD 21231.
Nucleic Acids Res. 1993 Nov 11;21(22):5192-7. doi: 10.1093/nar/21.22.5192.
This report describes an efficient method to clone PCR products exploiting endogenous Escherichia coli enzymatic activities. PCR products are engineered to contain terminal sequences identical to sequences at the two ends of a linearized vector. PCR products and vector DNA are then simply co-transfected into E. coli strain JC8679, obviating the requirement for enzymatic treatment of the PCR product or in vitro ligation. The high rate of homologous recombination in this strain results in efficient incorporation of the insert into the vector, a process we refer to as in vivo cloning (IVC).
本报告描述了一种利用大肠杆菌内源性酶活性克隆PCR产物的有效方法。对PCR产物进行工程改造，使其包含与线性化载体两端序列相同的末端序列。然后将PCR产物和载体DNA简单地共转染到大肠杆菌菌株JC8679中，无需对PCR产物进行酶处理或体外连接。该菌株中同源重组的高发生率导致插入片段高效整合到载体中，我们将这一过程称为体内克隆（IVC）。
