Multiple site directed mutagenesis strategy based on total RNA and RT-PCR method.

Site-directed PCR-based mutagenesis methods are widely used to generate mutations. All published methods work on DNA clones carrying the target sequence. However, DNA clones are not always available. We have previously published a RT-PCR-based site-directed mutagenesis method starting from total RNA to overcome this problem. In this article, we report an improvement of our previous method to facilitate introduction of multiple mutations into a target sequence. We demonstrate the efficacy and feasibility of this strategy by mutation of the human beta-actin gene. BamHI restriction endonuclease cleavage sites were generated within the gene to assist screening. Using three mutagenic primers in a single RT-PCR reaction, seven different clones were produced carrying three single and four multiple mutations. An investigation of the effect of the cycle number and elongation time of the PCR reactions revealed that both have an influence on the ratio of clones carrying single and multiple mutations. An optimized protocol was established for efficient multiple site-directed mutagenesis.