首页> 美国卫生研究院文献>Nucleic Acids Research >Production of DNA bifilarly substituted with bromodeoxyuridine in the first round of synthesis: branch migration during isolation of cellular DNA.
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Production of DNA bifilarly substituted with bromodeoxyuridine in the first round of synthesis: branch migration during isolation of cellular DNA.

机译:在合成的第一轮中用溴脱氧尿苷双取代的DNA的生产:分离细胞DNA期间的分支迁移。

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摘要

Incubation of human lymphoid cells with bromodeoxyuridine (BrdUrd) for short periods produces three classes of DNA containing analog: DNAHL (hybrid DNA, density approximately equal to 1.75 g/cm3), DNAint (intermediate density DNA, density approximately equal to 1.71 g/cm3), and DNAHH (DNA with both strands containing analog, density approximately equal to 1.80 g/cm3). Preparations of DNAint yield DNAHH after extensive shearing and/or treatment with single strand specific endonuclease. Cross-linking of pulse-labeled (BrdUrd + 3HdT) DNA in cells by treatment with trioxsalen and near UV light before lysis prevents the appearance of DNAHH.Cross-linking after lysis has little effect. A large fraction of DNAHH is obtained after incubation of cells with caffeine. Extraction of DNA at high salt concentration or cross-linking with trioxsalen and near UV light drastically reduced the amount of DNAHH obtained from caffeine-treated cells. We conclude that most DNAHH arises from in vitro branch migration in isolated DNA growing points.
机译:将人淋巴细胞与溴脱氧尿嘧啶核苷(BrdUrd)短时间孵育会产生三类包含类似物的DNA:DNAHL(杂交DNA,密度约等于1.75 g / cm3),DNAint(中等密度DNA,密度约1.71 g / cm3) )和DNAHH(两条链都含有类似物的DNA,密度大约等于1.80 g / cm3)。 DNAint的制备在广泛剪切和/或用单链特异性核酸内切酶处理后可产生DNAHH。裂解前用三氧沙林和近紫外光处理细胞中的脉冲标记(BrdUrd + 3HdT)DNA交联可防止DNAHH的出现,裂解后的交联作用很小。在将咖啡因与细胞一起孵育后,将获得大部分的DNAHH。在高盐浓度下提取DNA或与三氧沙林交联并在近紫外光下大大减少了从咖啡因处理过的细胞中获得的DNAHH的量。我们得出的结论是,大多数DNAHH来自分离的DNA生长点中的体外分支迁移。

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