Tachibana Akira, Tohiguchi Kazuo, Ueno Takayuki, Setogawa Yuichi, Harada Ayako, Tanabe Toshizumi
Department of Bioengineering, Graduate School of Engineering, Osaka City University, Sugimoto 3-3-138, Sumiyoshi-ku, Osaka 558-8585, Japan.
J Biosci Bioeng. 2009 Jun;107(6):668-9. doi: 10.1016/j.jbiosc.2009.01.019.
Ligation-independent cloning (LIC) is a useful method for efficient directional cloning of a PCR product. LIC requires a specially designed vector containing a long stretch of sequence that is missing any one of the four nucleotides. When the linearized vector is treated with T4 DNA polymerase, in the presence of the absent base, long single-stranded overhangs are generated that are suitable for cloning. In this study, long and efficient sticky ends for LIC were produced by sequential T4 DNA polymerase treatments at non-specific sequences on a commercially available vector. All restriction enzyme sites become available in the current LIC.
不依赖连接酶的克隆（LIC）是一种用于高效定向克隆PCR产物的有用方法。LIC需要一种经过特殊设计的载体，该载体包含一段长序列，其中缺少四种核苷酸中的任何一种。当线性化载体用T4 DNA聚合酶处理时，在缺少的碱基存在下，会产生适合克隆的长单链突出端。在本研究中，通过在市售载体上的非特异性序列上依次用T4 DNA聚合酶处理，产生了用于LIC的长而有效的粘性末端。在当前的LIC中，所有限制酶位点都可用。
