Work

PCR-mediated generation of a gene disruption construct without the use of DNA ligase and plasmid vectors

Year: 2002

Type: article

Abstract: We introduce a PCR-based procedure for generating a gene disruption construct. This method depends on DNA fragment fusion by the PCR technique and requires only two steps of PCR to obtain a sufficient amount of the gene disruption construct for one transformation experiment. The first step involves three separate PCR syntheses of a selectable marker cassette and the 5'- and 3'-regions of a target gene. Of the four primers used in amplification of the 5'- and 3'-regions of the target gene, two primers placed proximal to the site of the marker cassette are designed to have sequence tags complementary to the 5'- or 3'-side of the marker cassette. The two primers used in PCR synthesis of the marker cassette are complementary to the tagged primers. By fusion PCR, the 5' and 3' PCR products are linked to the marker cassette via the regions of tagged primers that overlap. A sufficient amount of the disruption construct can be directly amplified with the outermost primers. This method is simple, rapid and relatively inexpensive. In addition, there is the freedom of attaching long flanking regions to any selectable marker cassette.

Source: Nucleic Acids Research

DOI: 10.1093/nar/30.2.e2

Authors: H. Kuwayama, Shinji Obara, Takahiro Morio, Mariko Katoh, Hideko Urushihara, Yoshimasa Tanaka

Institution: University of Tsukuba

Language: English

Topic: CRISPR and Genetic Engineering

Subfield: Molecular Biology

Field: Biochemistry, Genetics and Molecular Biology

Domain: Life Sciences

Open Access status: bronze

Funders: University of California, San Diego, Japan Society for the Promotion of Science

Award: Genome analysis of the cellular slime molds to understand the genetic system controlling cell differentiation