QUANTIFICATION OF 3'OH DNA BREAKS BY RANDOM OLIGONUCLEOTIDE-PRIMED SYNTHESIS (ROPS) ASSAY

A simple and precise assay is presented for quantification of the rela tive 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 [P-32]dNTP into newly synthesized DNA will be proportional to the init ial number of 3'OH ends (breaks). The assay is specific for the detect ion of 3'OH ends and requires only 0.25 mu g of DNA for analysis, It h as application for the detection of the relative number of breaks per DNA molecule generated in vitro by endonucleases or in vivo during nor mal processes of DNA repair and also for the detection of DNA strand b reaks from genotoxic DNA damaging agents. Although specific for 3'OH D NA ends, the assay can be adapted to measure 3'P (5'OH) DNA ends or br eaks induced by oxidative DNA damaging agents by pretreatment of the D NA with alkaline phosphatase or Escherichia coil exonuclease In. The a ssay is capable of quantifying first several breaks per 10(5) bp.