应用基因组步移克隆小鼠Doc-1R基因组序列

Cloning of the mouse Doc-1R gene by genomic walking

目的 获得小鼠 Doc- 1R基因组序列。方法 根据小鼠 Doc- 1R基因的 c DNA序列设计、合成特异引物 ,应用基因组步移策略对小鼠基因组步移文库进行扩增。以含有特殊接头 (adaptor)的小鼠基因组步移文库作为模板 ,用巢式 PCR法扩增目的片段。结果 应用此方法得到约 1.5 kb的目的片段 ,经测序分析证实 Doc- 1R基因克隆成功。此基因含有 4个外显子、3个内含子 ,外显子与内含子接头符合 GT- AG法则。结论 基因组步移方法简单、可靠、有效 。

Objective: To obtain the genomic sequences of the mouse Doc-1R gene. Methods: Gene-specific primers were designed and synthesized based on the cDNA sequences of the mouse Doc-1R gene. With the use of genomic walking strategy, the mouse genomic walking library was amplified by the polymerase chain reaction(PCR). Mouse genomic library constructed with a special adaptor was utilized as a template to amplify the desired fragment by nested PCR. Results: A desired fragment of 1.5 kb was obtained. Sequence analysis of the desired fragment confirmed that the genomic cloning of the Doc-1R gene was successful. This gene contains four exons and three introns. All of the splice donor/acceptor site sequences are in accordance with the consensus 'GT-AG' rule. Conclusion: The genomic walking strategy is simple, efficient and reliable; it is an ideal method of cloning genomic fragments.