一种利用IIS型限制性内切酶进行无缝堆垒的酶促组装方法  

A Method for Seamless Stack Enzymatic Assembly of DNA Molecules Using IIS Type Restriction Endonuclease

作　　者：唐焱锋 周天顺 刘玲 余东[2,3] 董皓 段美娟[4] 袁定阳 Tang Yanfeng;Zhou Tianshun;Liu Ling;Yu Dong;Dong Hao;Duan Meijuan;Yuan Dingyang(Long Ping Branch,Graduate School of Hunan University,Changsha,410125;Hunan Hybrid Rice Research Center and State Key Laboratory of Hy-brid Rice,Changsha,410125;Hunan Academy of Agricultural Sciences,Changsha,410125;College of agriculture,Hunan Agricultural University,Changsha,410125)

机构地区：[1]湖南大学研究生院隆平分院,长沙410125 [2]湖南杂交水稻研究中心,杂交水稻全国重点实验室,长沙410125 [3]湖南省农业科学院,长沙410125 [4]湖南农业大学农学院,长沙410125

出　　处：《分子植物育种》2024年第7期2407-2412,共6页Molecular Plant Breeding

基　　金：湖南杂交水稻研究中心科技创新项目(2018ZD02);湖南省科技人才托举工程项目中青年学者培养计划项目(2019TJ-Q08)共同资助。

摘　　要：酶切-酶连是分子组装常用的传统重组方法,但其应用受到组装效率低、限制性酶切位点少、易产生突变碱基及无法实现无缝克隆等因素制约。为克服以上限制因素,我们提出一种利用IIS型限制性内切酶进行无缝堆垒的酶促组装方法。该方法是一种利用IIS型限制性内切酶识别位点位于切割位点之外的特性,在线性化质粒载体和与目的片段同源重组过程中不会破坏识别位点,通过多轮酶促组装实现目的序列的无缝克隆的二元酶促组装方法。我们通过该方法对EAT1基因CDs序列组装验证了该方法的可行性与实用性。Enzyme digestion-enzyme linked is a traditional method for molecular assembly,however,it's appli-cation is restricted by low assembly efficiency,few restriction sites,easy to produce mutant sites and unable to achieve seamless cloning.In order to overcome the above limitations,we propose a Seamless Stack Enzymatic Assembly using IIS type restriction endonuclease.This method is a binary enzyme assembly method which uses IIS type restriction endonuclease recognition site outside the cutting site,and does not destroy the recognition site in the process of linearizing plasmid carrier and homologous recombination with the target fragment,and realizes the seamless cloning of the target sequence by multi round enzyme assembly.And we verified the feasibility and practicability of this method by assembling the CDs sequence of EAT1 gene.

关 键 词：无缝堆垒 酶促组装 IIS型限制性内切酶 

分 类 号：Q78[生物学—分子生物学] Q55