不依赖基因序列和连接反应克隆(SLIC)方法的改进

Improvement of the sequence and ligation-independent cloning(SLIC)method

SLIC(Sequence and Ligation Independent Cloning)是一种不依赖于基因序列和连接反应的高效基因克隆新方法.为了进一步提高该方法的重组效率,将T4连接酶缓冲液改换成退火缓冲液,并使用相应的退火程序,可使重组效率提高至少10倍.此外,参加重组的若干DNA片段在同一反应体系中用T4 DNA聚合酶处理,再使用相同的方法退火,其重组效率仍然可以提高至少10倍.因此,利用本研究所建立的SLIC改进方法,不仅可以提高重组效率,而且还可以用于多DNA片段的高效重组,减少研究者的时间和节约实验成本.

SLIC （sequence and ligation-independent cloning） is a new efficient cloning method which can assemble multiple DNA fragments with complementary ends precisely in vitro. In the present study, the potential secondary structure in the complementary overhangs were analyzed and that may obstacle the recombination, a modified annealing buffer and the programmed annealing steps were designed to replace the original T4 ligation buffer. The experimental data show that the modified SLIC can increase the recombination rate at least 10-fold higher than that of previous method. Additionally, when several fragments involving in homologous recombination were treated with T4 DNA polymerase in one tube and subsequently conducted the programmed annealing steps in annealing buffer, a similar high recombination rate could be observed as well. The study demonstrated that the modified SLIC method can not only increase the recombination rate, but also be used for efficient recombination of multiple DNA fragments.