Basnakian A G, James S J
National Center for Toxicological Research, FDA, Division of Nutritional Toxicology, Jefferson, Arkansas 72079, USA.
DNA Cell Biol. 1996 Mar;15(3):255-62. doi: 10.1089/dna.1996.15.255.
A simple and precise assay is presented for quantification of the relative number of 3'OH ends (breaks) present in DNA molecules. The assay is based on the ability of the Klenow fragment polymerase to initiate random oligonucleotide-primed synthesis from the reannealed 3'OH ends of single-stranded (ss) DNA. After a denaturation-reassociation step, the ssDNA serves as its own primer by randomly reassociating itself or to other ssDNA molecules. Under strictly defined reaction conditions (time, temperature, concentration of precursors) the incorporation of [32P]dNTP into newly synthesized DNA will be proportional to the initial number of 3'OH ends (breaks). The assay is specific for the detection of 3'OH ends and requires only 0.25 micrograms of DNA for analysis. It has application for the detection of the relative number of breaks per DNA molecule generated in vitro by endonucleases or in vivo during normal processes of DNA repair and also for the detection of DNA strand breaks from genotoxic DNA damaging agents. Although specific for 3'OH DNA ends, the assay can be adapted to measure 3'P (5'OH) DNA ends or breaks induced by oxidative DNA damaging agents by pretreatment of the DNA with alkaline phosphatase or Escherichia coli exonuclease III. The assay is capable of quantifying first several breaks per 10(5) bp.
本文介绍了一种简单而精确的分析方法，用于定量DNA分子中存在的3'OH末端（断裂）的相对数量。该分析方法基于Klenow片段聚合酶从单链（ss）DNA重新退火的3'OH末端起始随机寡核苷酸引发合成的能力。经过变性-复性步骤后，ssDNA通过自身随机重新退火或与其他ssDNA分子重新退火，作为自身的引物。在严格定义的反应条件（时间、温度、前体浓度）下，[32P]dNTP掺入新合成DNA的量将与初始3'OH末端（断裂）的数量成正比。该分析方法对3'OH末端的检测具有特异性，分析仅需0.25微克DNA。它可用于检测核酸内切酶在体外产生的或在DNA修复正常过程中体内产生的每个DNA分子的断裂相对数量，也可用于检测遗传毒性DNA损伤剂导致的DNA链断裂。尽管该分析方法对3'OH DNA末端具有特异性，但通过用碱性磷酸酶或大肠杆菌核酸外切酶III对DNA进行预处理，可将其改编用于测量由氧化性DNA损伤剂诱导的3'P（5'OH）DNA末端或断裂。该分析方法能够定量每10（5）bp中的前几个断裂。
