用DREAM技术纠正人工合成基因中的突变

Correction of a Mutation in a Synthetic Gene by DREAM Technique,a Site-directed Mutagenesis

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(silent mutations),这些突变中包含大量的限制性内切酶位点,选择合适的酶切位点,自酶切位点向两侧合成引物(其中仅一个引物含有突变),用这些引物扩增两侧DNA片段(其中仅一个片段含有突变),然后将这两个片段以相应的内切酶切割后融合即可完成定点突变。结果:用该方法成功地纠正了人工合成的可溶性组织因子基因中两个碱基的缺失,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,突变成功率高,适合于在一般分子生物学实验室使用;该方法能避免了因多轮PCR和合成长引物导致突变的可能性,可作为一种定点突变的替代方法。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)。

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 carried out with the above primers to amplify fragment carrying the mutation. The amplified the the aimed mutation. Then two polymerase chain reactions were flanking fragments of the site to be mutated, with only one 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.