Birnboim H C, Jevcak J J
Cancer Res. 1981 May;41(5):1889-92.
DNA strand breaks can be detected with great sensitivity by exposing crude cell lysates to alkaline solutions and monitoring the rate of strand unwinding. As little as one strand break per chromosome can be detected. Previous methods for measuring strand unwinding have required physical separation of single- from double-stranded molecules. We now describe conditions under which unwinding can be monitored directly using a fluorescent dye, thus greatly simplifying the analysis. Breaks due to irradiation of blood samples by 60Co gamma-rays at doses as low as 0.05 to 0.1 gray (5 to 10 rads) were detectable. Rapid rejoining of strand breaks during in vitro incubation at 37 degrees could readily be observed following a dose of one gray. Since the procedure is very rapid and cells can be analyzed directly without the requirement for culturing or radiolabeling, the procedure could be useful in cancer chemotherapy if in vivo damage is to be monitored or for testing the in vitro sensitivity of cells to drugs.
通过将粗制细胞裂解物暴露于碱性溶液中并监测链解旋速率，可极其灵敏地检测到DNA链断裂。每条染色体上低至一个链断裂都能被检测到。以往测量链解旋的方法需要将单链分子与双链分子进行物理分离。我们现在描述了一种条件，在此条件下可使用荧光染料直接监测解旋，从而极大地简化了分析过程。低至0.05至0.1戈瑞（5至10拉德）的60Coγ射线照射血样所导致的断裂均可被检测到。在1戈瑞剂量照射后，很容易观察到在37摄氏度体外孵育期间链断裂的快速重新连接。由于该程序非常快速，且无需培养或放射性标记即可直接分析细胞，因此如果要监测体内损伤或测试细胞对药物的体外敏感性，该程序在癌症化疗中可能会很有用。
