Quantitative Analysis of DNA Interstrand Crosslink Induced by Chloroethylnitrosoureas with Real-Time Fluorometric Assay

Chloroethylnitrosoureas (CENUs) were an important family of anticancer agents in the clinical treatment of human malignancies. Several lines of evidences indicated that their cytotoxicity was related to the interstrand crosslink of DNA. In this study, a method for the determination of DNA crosslink was established using real-time fluorescence quantitative PCR. The DNA crosslink ratio induced by two CENUs, Semustine (Me-CCNU) and Carmustine (BCNU) were quantitatively analyzed and compared. The results showed that BCNU and Me-CCNU exhibit different crosslink trend of double strand DNA. Evagreen, a fluorescent dye, was used as the probe in the assay. This study provided direct evidence that real-time fluorescence quantitative PCR (qPCR) was a more convenient, efficient and stable method to detect DNA crosslink.