[Construction of middle fragment deletion mutant with improved gene splicing by overlap extension].
OBJECTIVE
To 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.


METHODS
In 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.


RESULTS
A mutant with the 325th to 373rd amino acid codons deleted was constructed successfully.


CONCLUSIONS
The improved SOE is a very effective and reliable method to construct middle fragment deletion mutant. It is worthy to be popularized.
Li
Chen-hua
CH
National Laboratory of Medical Genetics, Central South University, Changsha, Hunan, PR China.
Fang
Hai-yan
HY
Deng
Xiao-yun
XY
Xia
Kun
K
Zheng
Duo
D
Xia
Jia-hui
JH
D019870:1-Phosphatidylinositol 4-Kinase / Q000235:genetics*; D001483:Base Sequence; D005822:Genetic Vectors / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods; D015202:Protein Engineering / Q000379:methods; D011994:Recombinant Proteins / Q000235:genetics; D017384:Sequence Deletion
D011994:Recombinant Proteins; D019870:1-Phosphatidylinositol 4-Kinase