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| Correction of a mutation in a synthetic gene by DREAM technique, a site-directed mutagenesis |
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Abstract Objective: To develop a simple and efficient way to perform site-directed mutagenesis. Methods: DNA sequence to be mutated was reversely translated into degenerate codons, which contains large sum of silent mutations and accordingly carries various restriction enzyme sites. A silent mutant sequence with appropriate restriction enzyme was selected as the template. Two outwards primers containing the restriction enzyme were synthesized, with only one harboring the aimed mutation. Then two polymerase chain reactions were carried out with the above primers to amplify the fragments at both sides of the site to be mutated, with only one fragment carrying the site. The amplified fragments were then joined by restriction enzyme cut and ligation, resulting in the wanted mutation. Results: A two-base deletion in a synthetic gene (namely soluble human tissue factor) was successfully corrected in this way. Conclusion: This is an easy-to-use technique for site-directed mutagenesis which can be easily adopted in any molecular biology research settings. This strategy has several variations, including a universal design which uses one of the restriction enzymes cutting outside the recognition site, such as BsaI and SapI. In this universal design, the reverse translation and search for restriction enzyme are omitted. This technique is designated as DREAM: Designed Restriction Enzyme Assisted Mutagenesis.
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Received: 31 July 2006
Published: 10 May 2010
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