Quantification of 3′OH DNA Breaks by Random Oligonucleotide-Primed Synthesis (ROPS) Assay

Abstract

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 μg 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 10⁵ bp.