Correction of a mutation in a synthetic gene by DREAM technique, a site-directed mutagenesis

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 amount of silent mutations and accordingly carries various restriction enzyme sites.One 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 flanking fragments of the site to be mutated,with only one fragment carrying the mutation.The amplified fragments were then joined by restriction enzyme cut and ligation,resulting in the required mutation.Results:With this strategy,a two-base deletion in a synthetic gene(namely soluble human tissue factor,sTF)was successfully recovered,which restored the reading frame.Conclusion:This is an easy-to-use technique for site-directed mutagenesis which is efficient and can be easily adopted in any molecular biology research setting.This strategy reduces the possibility of unexpected mutations resulted from overlapping PCR and synthesis of long primers,and could serve as an option of site-directed mutagenesis.This technique is named as designed restriction enzyme assisted mutagenesis or DREAM.