Department of Medical Genetics, College of Basic Medicine, Third Military Medical University, Chongqing 400038, China.
Biotechnol Lett. 2011 Oct;33(10):1939-47. doi: 10.1007/s10529-011-0656-y. Epub 2011 Jun 10.
As gene cloning from difficult templates with regionalized high GC content is a long recognized problem, we have developed a novel and reliable method to clone such genes. Firstly, the high GC content region of the target cDNA was synthesized directly after codon optimization and the remaining cDNA fragment without high GC content was generated by routine RT-PCR. Then the entire redesigned coding sequence of the target gene was obtained by fusing the above available two cDNA fragments with SOE-PCR (splicing by overlapping extension-PCR). We have cloned the human RANK gene (ten exons; CDS 1851 bp) using this strategy. The redesigned cDNA was transfected into an eukaryotic expression system (A459 cells) to verify its expression. RT-PCR and western blotting confirmed this. To validate our method, we also successfully cloned human TIMP2 gene (five exons; CDS 660 bp) also having a regionalized high GC content. Our strategy for combining codon optimization and SOE-PCR to clone difficult genes is thus feasible and potentially universally applicable.
由于从具有区域高 GC 含量的困难模板中克隆基因是一个长期存在的问题，我们开发了一种新颖而可靠的方法来克隆此类基因。首先，在密码子优化后直接合成目标 cDNA 的高 GC 含量区域，然后通过常规 RT-PCR 生成没有高 GC 含量的剩余 cDNA 片段。然后，通过 SOE-PCR（重叠延伸-PCR 拼接）融合上述两种可用的 cDNA 片段，获得目标基因的整个重新设计的编码序列。我们使用这种策略克隆了人类 RANK 基因（十个外显子；CDS 1851bp）。重新设计的 cDNA 被转染到真核表达系统（A459 细胞）中以验证其表达。RT-PCR 和 Western blotting 证实了这一点。为了验证我们的方法，我们还成功克隆了具有区域高 GC 含量的人类 TIMP2 基因（五个外显子；CDS 660bp）。因此，我们结合密码子优化和 SOE-PCR 克隆困难基因的策略是可行的，并且具有潜在的普遍适用性。
