Construction of middle fragment deletion mutant with improved gene splicing by overlap extension / 中华医学遗传学杂志

ABSTRACT

<p><b>OBJECTIVE</b>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.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>A mutant with the 325th to 373rd amino acid codons deleted was constructed successfully.</p><p><b>CONCLUSION</b>The improved SOE is a very effective and reliable method to construct middle fragment deletion mutant. It is worthy to be popularized.</p>