5-溴脱氧尿嘧啶核苷标记酵母基因组DNA的方法

A Method of Labeling Genome DNA of Saccharomyces cerevisiae with BrdU

胸腺嘧啶类似物5-溴脱氧尿嘧啶核苷(BrdU)标记技术是一种研究DNA复制、修复等生命过程的有效手段。由于酿酒酵母(Saccharomyces cerevisiae)中缺少胸腺嘧啶核苷酸补救途径,胞外BrdU不能有效的渗入到基因组中,使该技术在酿酒酵母中的应用受到极大制约。通过在基因组中引入单纯疱疹病毒胞苷激酶(HSV-TK)和人类平衡核苷转运蛋白(hENT1)基因,工作建立了BrdU标记酵母基因组DNA的方法。在生长对数中期加入0.2mg/ml BrdU,离体检测法检测发现,标记3h的荧光信号较1h、5h时强;胞内检测法结果显示,标记3h时55.3%的基因组DNA中能够渗入BrdU。该工作为酿酒酵母DNA复制、修复等方面提供了直接有效的研究方法。

DNA labeling with thymidine analogue bromodeoxyuridine（BrdU） is a powerful tool for analyzing DNA replication and repair.But the technique was limited to use for Saccharomyces cerevisiae due to lack of thymidine salvage pathway which promoted BrdU binding to DNA.Two genes,herpes simplex virus thymidine kinase（HSV-TK） and human equilibrative nucleoside transporter 1（hENT1）,were inserted to the yeast genomic DNA by using an integration plasmid p405.The results showed that when the concentration of BrdU was 0.2mg/ml,it can be labeled to S.cerevisiae genomic DNA during the mid-logarithmic period.The fluorescence signal intensity at 3h was stronger than those at 1h or 5h in vitro.And data from detecting in vivo showed that 55.3% of the S.cerevisiae genomic DNA can be integrated with BrdU.These indicated that a new method with BrdU for labeling to S.cerevisiae genomic DNA was established.The protocol can be used easily for analysis of genomic DNA replication and repair for S.cerevisiae.