Principles of 3-step and 1-step PCR. A) Single gene deletion using a 3-step PCR. The first step involves amplifying independently the upstream and downstream regions of the target gene and the kanamycin resistance cassette. Then the three fragments obtained are mixed together and a third amplification performed to augment the yield of the product. B) Single gene deletion using a 1-step PCR. The first step is performed using pUC4K (Genbank X06404) as template and a mixture of long and short primers. The long primers (F100-kan/R100-kan) contain at their 5' extremity 100-nt homology to the flanking regions of the target gene and at their 3' extremity 22–24 nt homology to the kanamycin resistance cassette. The short primers Fup/Rdown are homologous to the 5' extremities of the long primers. C) Deletion of large chromosomal region using a 3-step PCR. The first step requires the amplification of MAR2xT7 transposon with the upstream and downstream region of genes of interest. Two transposon mutants are used as template to generate the up and down regions. Then the two fragments are mixed together to generate the desired PCR product and a third step performed to augment the yield of the product.
Lesic and Rahme BMC Molecular Biology 2008 9:20 doi:10.1186/1471-2199-9-20