Abstract
BackgroundMultiple approaches for the site-directed mutagenesis (SDM) have been developed. However, only several of them are designed for simultaneous introduction of multiple nucleotide alterations, and these are time consuming. In addition, many of the existing multiple SDM methods have technical limitations associated with type and number of mutations that can be introduced, or are technically demanding and require special chemical reagents.ResultsIn this study we developed a quick and efficient strategy for introduction of multiple complex mutations in a target DNA without intermediate subcloning by using a combination of connecting SDM and suppression PCR. The procedure consists of sequential rounds, with each individual round including PCR amplification of target DNA with two non-overlapping pairs of oligonucleotides. The desired mutation is incorporated at the 5' end of one or both internal oligonucleotides. DNA fragments obtained during amplification are mixed and ligated. The resulting DNA mixture is amplified with external oligonucleotides that act as suppression adapters. Suppression PCR limits amplification to DNA molecules representing full length target DNA, while amplification of other types of molecules formed during ligation is suppressed. To create additional mutations, an aliquot of the ligation mixture is then used directly for the next round of mutagenesis employing internal oligonucleotides specific for another region of target DNA.ConclusionA wide variety of complex multiple mutations can be generated in a short period of time. The procedure is rapid, highly efficient and does not require special chemical reagents. Thus, MALS represents a powerful alternative to the existing methods for multiple SDM.
Highlights
Multiple approaches for the site-directed mutagenesis (SDM) have been developed
MALS procedure (Mutagenesis by Amplification, Ligation and Suppression PCR) allows the generation of all types of mutations depending on the design of the internal oligonucleotides used (Figure 1)
To demonstrate the efficiency of the method we sequentially introduced four different nucleotide alterations in a model 4 kb DNA sequence originating from the genomic DNA of phage λ (Figure 2)
Summary
Multiple approaches for the site-directed mutagenesis (SDM) have been developed. only several of them are designed for simultaneous introduction of multiple nucleotide alterations, and these are time consuming. A popular approach employs several pairs of mutagenic primers for sequential rounds of mutagenesis This procedure is robust, it is time-consuming because it (page number not for citation purposes). BMC Biotechnology 2009, 9:83 http://www.biomedcentral.com/1472-6750/9/83 requires a subcloning procedure between rounds of mutagenesis [3] Another strategy utilizes combining mutagenic oligonucleotides in the same reaction [4,5,6,7]. NonPCR based template amplification in combination with ligation of growing DNA strands reduces the rate of spontaneous errors Despite the advantages, this procedure has limitations, such as restrictions in the number, sizes and types of mutations that can be introduced simultaneously [2]. The need for resolution of these products greatly reduces the overall efficiency of the method
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