同源区段长度对 In-Fusion 技术连接效率的影响

摘要

目的在目的基因原载体（DNA 模板）与克隆载体相同的条件下，探讨了 PCR 扩增片段（含目的基因）的末端与载体间同源区段长度对 In-Fusion 连接效率的影响，并建立以In-Fusion 技术为基础的高通量克隆方法。方法以糖基水解酶 Lxyl-p1-2（2.4 kb）基因突变库的构建为例，设计引物使目的基因的两端分别与线性化载体末端含有15~200 bp 重叠序列，并通过易错 PCR 方法获得含目的基因的 PCR 扩增片段，运用 In-Fusion 技术将 PCR 扩增片段定向克隆至表达载体 pPIC3.5K 中。结果对于长度大约为2.4 kb 的较大片段 DNA 的克隆， PCR 扩增片段的末端与载体间最适同源区段长度约为100 bp，此时连接效率最高，其阳性重组率高达90%左右，是常规酶切连接法的3倍。与后者相比，该技术将克隆周期由3~4 d 缩短为1~2 d。结论优化的同源区段长度可以显著提高 In-Fusion 技术对于2.4 kb 目的基因与载体的连接效率，该方法尤其适用于定向进化过程中基因突变库的构建。%Objective To explore the effect of length of homologous sequence between the ends of PCR amplicon (harboring the targeted gene) and the vector on the In-Fusion recombination efficiency, when the template vector is the same as the cloning vector, and to establish an efficient high-throughput cloning method based on In-Fusion technique. Methods With construction of the mutant library of the glycoside hydrolase Lxyl-p1-2 (2.4 kb) as an example, different primers were designed on the basis of the various positions of the vector. The PCR fragments (each harboring the targeted gene) were obtained by error-prone PCR. Each fragment contained two over-lapping sequences (15 - 200 bp in length) at the two ends with each of the corresponding ends of the linearized vector. These PCR amplicons were directionally cloned into the vector pPIC3.5K by In-Fusion technique. Results For the cloning of the 2.4 kb DNA fragments by the In-Fusion method, the highest DNA ligation efficacy was obtained when the over-lapping sequence between the PCR fragments and the linearized vector at any end was 100 bp in length, with the highest recombination rate of around 90%. The recombination rate was three times higher than that of the conventional restriction enzyme cloning method. Moreover, the cloning period was drastically shortened from 3 - 4 d of the conventional method to 1 - 2 d of the present method. Conclusion The optimized length of homologous sequence obtained in this paper may significantly increase the ligation efficacy between the 2.4 kb targeted gene and the vector when the In-Fusion technique was applied. The method is particularly suitable to construct the mutant library in the study of directed evolution.