An efficient and rapid method for cDNA cloning from difficult templates using codon optimization and SOE-PCR: with human RANK and TIMP2 gene as examples

Abstract

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.