A Rapid Method for Creating Gene Mutations and Fusions Using PCR-Ligation-PCR Mutagenesis

Here we describe a novel PCR method for site-specific mutagenesis. Unlike splice overlap extension (SOE) PCR, this technique does not require primers with overlapping complementary sequences, but still maintains sequence specificity at the recombination junction. Like the PCR process itself, the principle is only inherently obvious after it has been explained. Remarkably, this simple technique can be applied universally to generate gene fusions, deletions, insertions and point mutations. The method is practical, cost effective and reproducible, and it therefore offers many advantages over existing techniques and commercially available mutagenesis kits.