小鼠Mcm6基因电子克隆的验证解析.docVIP

小鼠Mcm6基因电子克隆的验证 PAGE 小鼠Mcm6基因电子克隆的验证 摘 要 小鼠Mcm6（Mus musculus minichromosome maintenance deficient 6）基因是通过电子克隆获得的基因。以liver,Mus.musculus为关键词检索得到UniGene 数据库，选择其中一个EST序列 HYPERLINK /UniGene/seq.cgi?ORG=MmSID=8442862 D86726.1（种子序列）作为电子探针，获得该序列的电子延伸产物EST重叠群。再与基因组草图进行序列校正，获得了全长为3191bp的cDNA序列。该基因定位于小鼠1号染色体和8号染色体上，将该基因命名为Mcm6。但这种根据“基因组草图搜索法”获得的全长cDNA序列，最后尚需要通过实验来验证。本研究的目的就是通过实验来验证电子克隆基因Mcm6是否为真正具有生物学功能的基因。 本实验从新鲜的小鼠肝脏组织中提取总RNA，经逆转录合成cDNA。根据Mcm6的电子克隆全长cDNA序列，在同一阅读框架终止密码子附近及其上游设计1条引物,在阅读框架起始密码子附近及其下游设计1条引物。再以cDNA为模板，利用上述引物扩增目的基因片段，琼脂糖凝胶电泳检测PCR产物，扩增产物大小为379bp。通过比较根据基因组草图搜索法预测的和实验扩增的目的片段大小，即可判断我们是否获得该基因的全长cDNA 序列。 结果证实成功克隆该基因，因而验证了Mcm6基因电子克隆的正确性。本实验为该基因功能的进一步研究奠定了基础。 关键词：小鼠，Mcm6，电子克隆,逆转录，PCR扩增。 Identification of sillcon cloning gene Mcm6 Abstract Mcm6（Mus musculus minichromosome maintenance deficient 6）gene is obtained by sillcon cloning. First of all, they obtain a UniGene cluster which roots in Mouse’s brain by use the key word Mus musculus ,brain to searches UniGene database,then with one EST sequence HYPERLINK /UniGene/seq.cgi?ORG=MmSID=8442862 D86726.1(Seed sequence) as an electronic probe, access to the electronic extension of the EST product sequence contig. With the draft genome sequence correction, access to the full-length cDNA sequences of 3191bp. The gene located in mouse chromosome 1 and chromosome 8. They named it Mcm6 gene. However, this full-length cDNA sequences under the draft genome search also needs to verify through experiments. The purpose of this study is to verify whether Mcm6 is a Biological function of genes through experiments. In this experiment we extract total RNA from fresh Mus musculus liver tissue,and synthesis cDNA by Reverse Transcription. According to the sillcon cloning sequence of full-length cDNA of Mcm6, design a primer near the stop codon and its upstream in the same reading frame, design another primer in the reading frame near the initiation codon and its downstream. We made the cDNA of mouse liver tissue of the Mus musculus a