A novel PCR-mediated method for one-tube generation of a gene disruption construct

Abstract

A novel PCR-based method is reported for generating a gene disruption construct which requires no purification of PCR fragments and enables the whole procedure to be completed in one tube very rapidly. The procedure starts with PCR amplification of both the 5′ and 3′ regions of a particular gene in one tube. Then, exonuclease I is added to the tube to remove the residual primers. After heat inactivation of the enzyme, a marker cassette DNA fragment is added and fusion PCR is performed to build up a gene disruption construct. The gene disruption construct is subsequently amplified with the outermost primers in the amount necessary for transformation. In order to distinguish the gene disruption construct from the remaining intact gene allele, the outermost primers are designed to have GC-rich tag sequences that anneal at a higher temperature, ensuring the specific amplification of the gene disruption construct.