title: PCR-mediated generation of a gene disruption construct without the use of DNA ligase and plasmid vectors.
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.
authors.affiliation: Institute of Biological Sciences, University of Tsukuba, Tsukuba, Tennodai 1-1-1, Ibaraki 305-8572, Japan.
authors.forename: Hidekazu
authors.initials: H
authors.lastname: Kuwayama
authors.forename: Shinji
authors.initials: S
authors.lastname: Obara
authors.forename: Takahiro
authors.initials: T
authors.lastname: Morio
authors.forename: Mariko
authors.initials: M
authors.lastname: Katoh
authors.forename: Hideko
authors.initials: H
authors.lastname: Urushihara
authors.forename: Yoshimasa
authors.initials: Y
authors.lastname: Tanaka
mesh_terms: D000483:Alleles; D000818:Animals; D002460:Cell Line; D011088:DNA Ligases / Q000378:metabolism; D017931:DNA Primers / Q000235:genetics; D004023:Dictyostelium / Q000235:genetics*; D018390:Gene Targeting / Q000191:economics / Q000379:methods*; D017125:Genes, Protozoan / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics; D016254:Mutagenesis, Insertional / Q000235:genetics / Q000379:methods; D016297:Mutagenesis, Site-Directed / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000191:economics / Q000379:methods*; D011995:Recombination, Genetic / Q000235:genetics; D012689:Sequence Homology, Nucleic Acid; D013997:Time Factors; D014170:Transformation, Genetic
chemical_list: D017931:DNA Primers; D011088:DNA Ligases