用DREAM技术纠正人工合成基因中的突变

目的：介绍一种简便、有效的定点突变技术。方法：根据突变位点附近的DNA序列推导出氨基酸序列，再以此氨基酸序列进行逆翻译，这样在不改变氨基酸序列的前提下可以得到数目巨大的隐性突变体（silent mutants），这些突变体中包含大量的限制性内切酶位点，选择合适的酶切位点设计引物用PCR技术扩增两侧DNA片段，然后以相应酶切融合这两个片段即可完成定点突变。结果：用该方法成功地在人工合成的含有缺失的可溶性组织因子基因的472位插入C，T两个碱基，校正了阅读框架，获得了预期的目的基因。结论：该方法简便、有效, 避免了多轮PCR和合成长引物导致突变的可能性，这种改进的PCR 定点诱变技术我们称之为“设计限制酶辅助突变”（Designed Restriction Enzyme Assisted Mutagenesis, DREAM）。此技术简单方便, 诱变的成功率高, 适于实验室常规应用。

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.