Generation of eight adjacent mutations in a single step using a site-directed mutagenesis kit.

Studies that characterize transcriptional regulatory elements involve the site-directed mutagenesis methodology to generate deletions, insertions and point mutations. Commercial PCR-based system kits are widely used to introduce up to four base changes in a target sequence. Sequential mutagenesis experiments allow more than four bases to be altered. In this work, we show that the QuikChange site-directed mutagenesis kit developed by Stratagene, with an optimum primer design, can change eight adjacent bases. This approach allows us to study the effect of DNA sequence changes on functionality of specific sequences from gene target regions, including promoters, exons and introns. As a result of this methodology, a faster and cheaper way of introducing this number of mutations is achieved in a single step with only one pair of primers, thus reducing the possibility of potential random mutation in the rest of the target sequence.