Rapid in vitro splicing of coding sequences from genomic DNA by isothermal recombination reaction-based PCR
Chen, Wenxuan | Zeng, Dongchang | Shen, Rongxin | Ma, Xingliang | Zhang, Qunyu | Chen, Letian | Liu, Yao-Guang | Zhu, Qinlong
Cloning of coding sequence (CDS) is an important step for gene function research. Here, we reported a simple and efficient strategy for assembling multiple-exon into an intron-free CDS from genomic DNA (gDNA) by an isothermal recombination reaction-based PCR (IRR-PCR) method. As an example, a 2067-bp full-length CDS of the anther-specific expression gene OsABCG15, which is composed of seven exons and six introns, was generated by IRR-PCR using genomic DNA of rice leaf as the template. Actually, this approach can be wildly applied to any DNA sequences assembly to achieve CDS cloning, gene fusion and multiple site-directed mutagenesis in functional genomics studies in vitro.
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