General method for quantifying base adducts in specific mammalian genes.
General method for quantifying base adducts in specific mammalian genes.
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定量特定哺乳动物基因中碱基加合物的通用方法。
DOI：
10.1073/pnas.85.11.3723
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发表时间：
1988
影响因子：
11.1
通讯作者：
Sancar,A
中科院分区：
文献类型：
--
作者：
Thomas,DC;Morton,AG;Bohr,VA;Sancar,A
已经开发出一种通用方法来测量哺乳动物基因组特定序列中 DNA 加合物的形成和去除。加合的基因组 DNA 用适当的限制性酶消化，用大肠杆菌 UvrABC 核酸切除酶（ABC 核酸外切酶）处理，进行碱性凝胶电泳，并通过 Southern 杂交探测特定序列。 ABC 核酸外切酶切割含有大加合物的 DNA，从而降低 Southern 杂交中全长片段的强度，与探测序列中存在的加合物数量成比例。该方法与 Bohr 等人开发的方法类似。 [Bohr, V. A., Smith, C. A., Okumoto, D. S. & Hanawalt, P. C. (1985) Cell 40, 359-369] 使用 T4 核酸内切酶 V 定量嘧啶二聚体。然而，由于 ABC 核酸外切酶的底物范围广泛，我们的方法可用于定量特定基因组序列中的多种 DNA 加合物。
A general method has been developed to measure the formation and removal of DNA adducts in defined sequences of mammalian genomes. Adducted genomic DNA is digested with an appropriate restriction enzyme, treated with Escherichia coli UvrABC excision nuclease (ABC excinuclease), subjected to alkaline gel electrophoresis, and probed for specific sequences by Southern hybridization. The ABC excinuclease incises DNA containing bulky adducts and thus reduces the intensity of the full-length fragments in Southern hybridization in proportion to the number of adducts present in the probed sequence. This method is similar to that developed by Bohr et al. [Bohr, V. A., Smith, C. A., Okumoto, D. S. & Hanawalt, P. C. (1985) Cell 40, 359-369] for quantifying pyrimidine dimers by using T4 endonuclease V. Because of the wide substrate range of ABC exinuclease, however, our method can be used to quantify a large variety of DNA adducts in specific genomic sequences.