Differentiation of apurinic/apyrimidinic sites and single-strand breaks in DNA by formamide- and alkaline-sucrose gradient sedimentation.
The excision repair of DNA damaged by physical or chemical agents may produce either apurinic/apyrimidinic (AP) sites or single-strand breaks (SSB) in the DNA. Alkaline-sucrose gradient sedimentation and alkaline elution, techniques generally used for the study of DNA repair which depend upon high pH to denature the DNA, cannot differentiate between these possibilities. A simple method for the quantitative measurement of SSB in DNA which leaves any AP sites intact is presented. This method relies upon the separation by size of the fragments resulting from the denaturation of the DNA under neutral conditions by sedimentation through gradients of sucrose in formamide. By combining the use of both formamide- and alkaline-sucrose sedimentation methods, we can quantify both AP sites and SSB in DNA.
D001080:Apurinic Acid / Q000032:analysis*; D002499:Centrifugation, Density Gradient; D055598:Chemical Phenomena; D002621:Chemistry; D004260:DNA Repair; D004277:DNA, Single-Stranded / Q000032:analysis*; D004279:DNA, Viral / Q000032:analysis; D005559:Formamides; D006863:Hydrogen-Ion Concentration; D009691:Nucleic Acid Denaturation; D011119:Polynucleotides / Q000032:analysis*; D013056:Spectrophotometry, Ultraviolet; D013395:Sucrose; D013604:T-Phages / Q000235:genetics
D004277:DNA, Single-Stranded; D004279:DNA, Viral; D005559:Formamides; D011119:Polynucleotides; C001790:apyrimidinic acid; D001080:Apurinic Acid; C031066:formamide; D013395:Sucrose