人肝癌SMMC-7721细胞中POLD1基因CDS突变的检测

Mutational analysis of the protein-coding sequence of the POLD1 gene in human hepato-cellular carcinoma cell line SMMC-7721

目的检测人肝癌细胞株SMMC-7721中POLD1基因CDS的突变情况,以期从DNA复制最关键酶的编码基因POLD1的突变为干预细胞恶性增殖提供新的思路。方法设计人POLD1基因CDS特异性引物,以人肝癌细胞株SMMC-7721总RNA为模板进行RT-PCR扩增,将扩增产物纯化后加入"腺嘌呤"并与T载体连接,将连接产物进行测序,与数据库中的序列进行比对。结果成功扩增了人肝癌细胞中POLD1基因的CDS片段。SMMC-7721细胞中POLD1基因的CDS存在11个碱基替换,1个碱基缺失。结论人肝癌细胞SMMC-7721中POLD1基因的CDS发生了突变,进而导致其编码的氨基酸序列发生改变。

Objective To analyze mutations in the protein-coding region of the POLD1 gene in the human hepatocarcinoma cell line SMMC-7721 ;this gene encodes an enzyme critical for DNA replication. Methods Total RNA was isolated from SMMC-7721 cultures,and the POLD1 gene coding region was amplified by RT-PCR,purified and A-tailed, and then cloned into a T-vector. Mutations were detected by sequencing. Results The protein-coding region of the POLD1 gene was successfully amplified and found to contain 12 mutations, 11 of which were nucleotide changes and 1 was a nucleotide deletion. Conclusions SMMC-7721 en-codes a mutated POLD1 gene, and the mutations lead to changes in the protein sequence.