Construction of middle fragment deletion mutant with improved gene splicing by overlap extension.
- Author:
Chen-hua LI
1
;
Hai-yan FANG
;
Xiao-yun DENG
;
Kun XIA
;
Duo ZHENG
;
Jia-hui XIA
Author Information
- Publication Type:Journal Article
- MeSH: 1-Phosphatidylinositol 4-Kinase; genetics; Base Sequence; Genetic Vectors; genetics; Molecular Sequence Data; Mutagenesis, Site-Directed; Mutation; genetics; Polymerase Chain Reaction; methods; Protein Engineering; methods; Recombinant Proteins; genetics; Sequence Deletion
- From: Chinese Journal of Medical Genetics 2004;21(1):52-55
- CountryChina
- Language:Chinese
-
Abstract:
OBJECTIVETo construct a phosphatidylinositol 4-kinase beta (PI4K-beta) mutant with the 325th to 373rd amino acid codons deleted, and try to develop a simple method for constructing middle fragment deletion mutant.
METHODSIn line with the mechanism of gene splicing by overlap extension(SOE), an additional PCR was used to get the PI4K-beta mutant in which the 325th to 373rd amino acid codons were deleted. Then the mutated gene was cloned into pCMV-Tag4A mammalian expression vector.
RESULTSA mutant with the 325th to 373rd amino acid codons deleted was constructed successfully.
CONCLUSIONThe improved SOE is a very effective and reliable method to construct middle fragment deletion mutant. It is worthy to be popularized.
