Construction of fused recombinant vector of EGFR gene containing G719S and T790M by overlap PCR
10.13602/j.cnki.jcls.2018.02.17
- VernacularTitle:重叠PCR法构建宫颈癌相关的EGFR基因G719S和T790M融合重组载体
- Author:
Weilei DONG
1
;
Huahua XIANG
;
Hua PENG
;
Yongqing GONG
;
Jing ZHOU
;
Hongquan ZHANG
;
Zifen GUO
Author Information
1. 南华大学附属第一医院妇产科
- Keywords:
EGFR gene;
vector construction;
cervical cancer;
overlap PCR
- From:
Chinese Journal of Clinical Laboratory Science
2018;36(2):139-141,147
- CountryChina
- Language:Chinese
-
Abstract:
Objective To construct the recombinant pMD19-exon18-exon20 plasmid containing locus G719S and T790M of EGFR gene associated with cervical cancer,which may provide a template for preparing the mutant recombinant vector of EGFR gene.Methods Using the healthy human genome DNA as templates,the segments of exon 18 and exon 20 of EGFR gene were amplified by two pairs of specific primers which were designed based on the sequences of overlapping and complementary area.The amplified segments were linked by overlap PCR.The products of linked exon18-exon20 were further inserted into the vector pMD19-T.The constructed recombi nant pMD19-exon18-exon20 plasmid was finally transformed into competent cells E.coli DH5α and then identified by PCR with bacterial solution and genome sequencing.Results The amplified fragments of exon18 and exon20 were clearly appeared at 778 bp and 596 bp and the fused product of exon18-exon20 was showed at 1 374 bp on agarose gel electrophoresis.The recombinant plasmid of fusion EGFR gene was consistent with the expected results via bacterial PCR assay and DNA sequencing.Conclusion We successfully fused the segments of exon18 with exon20 and constructed the recombinant expression vector of EGFR gene by using overlap PCR method.
