基于PIPE现象的质粒克隆位点序列设计与功能评价

Sequence Design and Functional Evaluation of Plasmid Cloning Site Based on PIPE Phenomenon

分子克隆是现代生物学研究的核心技术之一,是基因工程、蛋白质工程中的重要手段。为提高分子克隆实验的操作效率,本研究设计并合成基于聚合酶引物不完全延伸(polymerase incomplete primer extension,PIPE)现象的质粒克隆位点序列。并以此为基础统一相关引物的设计方案,避免传统酶切———连接法中需针对不同载体MCS序列设计不同引物的缺点。该方案利用13 bp定长接头序列,在同一体系中使用2对引物、2种线性化模板同时扩增载体和插入片段,通过20个循环,在1次PCR过程中即合成可供转化使用的带缺口质粒产物。在NEB Q5酶系统中,利用此法将3种荧光素酶序列插入pET-15b及pET-21b(+)载体,均获得成功。且利用商品化感受态细胞(转化效率>5×108 cfu/μg)转化后所获得转化子数量均在300个以上,其中含插入片段的阳性克隆比例可达85%以上。基于本方案的设计及作用原理,可将其应用于10 kb以内载体和插入片段的快速重组。且具有通用性强、耗时少、阳性克隆得率高和成本低等优点,是传统DNA重组方法的有益补充,可作为各实验室的常规分子克隆手段之一。

Molecular cloning is one of the core technologies in modern biological research and an important means in genetic engineering and protein engineering.In order to improve the efficiency of molecular cloning experiments,we have designed and synthesized a plasmid cloning site sequence based on“polymerase incomplete primer extension(PIPE)”phenomenon.Thus,to unify the sequence of related primers,which makes designing unique primers for different MCS sequences as in traditional digestion-ligation method no longer required.This novel cloning site utilizes two pairs of primers with a 13 bp fixed-length linker,to simultaneously amplify vectors and insertions from two linearized templates in the same PCR system,then generates plasmid products with nicks for transformation in a single PCR reaction with 20 cycles.With NEB Q5 DNA polymerase applied,three luciferase sequences were successfully inserted into the pET-15b and pET-21b(+)vectors by this method.Moreover,the number of clones obtained by using commercial competent cells(>5×108 cfu/μg)was more than 300,and the ratio of positive clones containing desire insertion was over 85%.According to the principle of this protocol,it is suitable for rapid recombination of vectors and insertions less than 10 kb.The method is highly versatile,time-saving,robust and economic with high positive clone yield.Which makes it a useful supplement to the traditional DNA recombination method,and can be used as one of the routine molecular cloning protocols in most laboratory.