摘要
重叠PCR技术是以聚合酶链式反应为基础,将多条DNA片段进行融合;PCR-酶切连接主要应用于基因片段连接及载体构建,这两种方法在基因工程与分子生物学领域已得到广泛应用。以多毛番茄抗冷转录因子LhCBF1基因、EGFP基因及RD29A启动子为例,分别采用一步法重叠PCR、二步法重叠PCR及PCR-酶切连接技术,优化PCR体系及反应条件,成功构建LhCBF1-EGFP及RD29A::EGFP融合基因。研究为重叠PCR及PCR-酶切连接技术的改进及构建融合基因方法的选择提供了有益参考。
Overlap extension PCR（OE-PCR） is frequently used for gene fragments fusion based on polymerase chain reaction. PCR, restriction enzyme digestion and ligation are primarily applied to the construction of recombinant vectors. These two techniques are in widespread application in the fields of gene engineering and molecular biology. LhCBF1 gene （L.hirsutum CRT-binding factor 1）, EGFP gene and promoter RD29A sequence were taken as example and successfully obtained LhCBF1-EGFP and RD29A：：EGFP fusion genes by using one step and two step OE-PCR, PCR-digestion-ligation method and optimized PCR protocol and system. This study provides significant theoretical basis for OE-PCR, PCR-digestion-ligation and fusion gene construction techniques.
出处
《东北农业大学学报》
CAS
CSCD
北大核心
2012年第1期155-160,共6页
Journal of Northeast Agricultural University
基金
黑龙江省教育厅海外学人项目
黑龙江省科技厅科技攻关项目(GA06B103-1)
东北农业大学科技创新团队项目
