title: 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.
authors.affiliation: Department of Medical Genetics, College of Basic Medicine, Third Military Medical University, Chongqing 400038, China.
authors.forename: Gang
authors.initials: G
authors.lastname: Huang
authors.forename: Qianjun
authors.initials: Q
authors.lastname: Wen
authors.forename: Qiangguo
authors.initials: Q
authors.lastname: Gao
authors.forename: Fang
authors.initials: F
authors.lastname: Zhang
authors.forename: Yun
authors.initials: Y
authors.lastname: Bai
mesh_terms: D000595:Amino Acid Sequence; D001482:Base Composition; D001483:Base Sequence; D002277:Carcinoma; D045744:Cell Line, Tumor; D003001:Cloning, Molecular / Q000379:methods*; D003062:Codon; D003110:Colonic Neoplasms; D018076:DNA, Complementary / Q000235:genetics* / Q000378:metabolism; D005091:Exons / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D053246:Receptor Activator of Nuclear Factor-kappa B / Q000235:genetics* / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D019716:Tissue Inhibitor of Metalloproteinase-2 / Q000235:genetics* / Q000378:metabolism
chemical_list: D003062:Codon; D018076:DNA, Complementary; D053246:Receptor Activator of Nuclear Factor-kappa B; C560823:TIMP2 protein, human; C505686:TNFRSF11A protein, human; D019716:Tissue Inhibitor of Metalloproteinase-2