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背景与目的:研究小鼠淋巴瘤L5178Y3.2.7c-tk+/-细胞tk基因的自发突变频率和分子突变。材料与方法:进行4次平行试验,计算自发突变频率。选择tk基因自发突变子(tk-/-),提取基因组DNA,经等位基因特异性PCR扩增,杂合性缺失(Lossofheterozygosity,LOH)分析和测序等技术,分析其分子突变谱。结果:L5178Y细胞tk基因的平均自发突变频率为145×10-6,tk基因自发突变LOH平均发生率为79.8%。由LOH分析的试验结果显示,自发突变的LOH在大集落(Largeclone,LC)与小集落(Smallclone,SC)之间差异无统计学意义。序列分析证实,自发tk基因点突变主要为碱基置换。结论:tk基因的自发突变是以功能性等位基因缺失为主的分子突变,其中点突变以碱基置换为主。
BACKGROUND & AIM: To investigate the frequency of spontaneous mutation and molecular mutation of tk gene in mouse lymphoma L5178Y3.2.7c-tk +/- cells. Materials and Methods: Four parallel experiments were performed to calculate the frequency of spontaneous mutations. Select the tk gene spontaneous mutant (tk - / -), extracted genomic DNA, allele-specific PCR amplification, loss of heterozygosity (LOH) analysis and sequencing techniques, analysis of its molecular mutation spectrum. Results: The average spontaneous mutation frequency of tk gene in L5178Y cells was 145 × 10-6, and the average incidence of spontaneous mutation of tk gene was 79.8%. The results of LOH analysis showed that LOH with spontaneous mutation was not significantly different between Largeclone (LC) and Smallclone (SC). Sequence analysis confirmed that spontaneous tk gene point mutations are mainly base substitutions. CONCLUSION: The spontaneous mutation of tk gene is a molecular mutation dominated by functional allele deletion, of which point mutation is dominated by base substitution.