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

Zhonghua Yi Xue Yi Chuan Xue Za Zhi. 2004 Feb;21(1):52-5.
[Article in Chinese]

Abstract

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.

Conclusion: The improved SOE is a very effective and reliable method to construct middle fragment deletion mutant. It is worthy to be popularized.

Publication types

  • English Abstract
  • Research Support, Non-U.S. Gov't

MeSH terms

  • 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*

Substances

  • Recombinant Proteins
  • 1-Phosphatidylinositol 4-Kinase