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.

Evolutionary linkage between eukaryotic cytokinesis and chloroplast division by dynamin proteins

“…Wild-type D. discoideum (AX2) and the mutants derived from AX2 were cultured as previously described (33,34). The dlpA, dlpB, and dlpC genes were disrupted by insertion of the blasticidin S-resistant gene via homologous recombination (33).…”

“…The dlpA, dlpB, and dlpC genes were disrupted by insertion of the blasticidin S-resistant gene via homologous recombination (33). Spores were germinated by heat shock as described previously (19), and DNase-treated total RNA was used for RT-PCR analyses.…”

Engineering super mycovirus donor strains of chestnut blight fungus by systematic disruption of multilocus vic genes

“…Disruption of vic genes was performed by homologous recombination in C. parasitica strain DK80 using PCRgenerated disruption fragments based on the strategy of Kuwayama et al (40) with modifications as described by Zhang et al (25) to include flanking loxP sites as described previously (23) for the single disruptions of the individual vic1a-2 and vic3b-1 genes. The gene disruption fragments containing loxPflanked SMGs were excised by providing the Cre recombinase in trans via anastomosis with a compatible C. parasitica Cre-producing strain as described by Zhang et al (25).…”

d‐threo‐tetrahydrobiopterin is synthesized via 1′‐oxo‐2′‐d‐hydroxypropyl‐tetrahydropterin inDictyostelium discoideumAx2

“…To select transformant HL5 medium was supplemented with 10 lg/ml blasticidin S [15]. Transformation was performed with S phase synchronized method [16]. Cells were synchronized in S phase by incubation at 9.6°C for 14 h followed by incubation at 22°C for 2.5 h at a density of 1.0 · 10 6 cells/ml in HL5 medium.…”