CE analysis and molecular characterisation of depurinated DNA samples

Abstract

A DNA sample was partially degraded by scalar heat-acid treatments to study the extent of apurinic-apyrimidinic (A-P) lesions produced along the molecule. A CE-UV method allowed us to measure the rate of depurination at pH 5.0 and 70°C which was calculated to be 5.41×10(-6) s(-1) for adenine and 6.27×10(-6) s(-1) for guanine. CE identified depurination on treated samples when it occurred with a loss of >4% of the basic moieties. The molecular features of the A-P enriched samples were investigated by using molecular assays (agarose gel electrophoresis, UV spectrophotometry and quantitative PCR) and the consistency of the results of the STR typing were compared with the degree of depurination of the PCR template. The treated DNA samples showed molecular features such as fragmentation, altered OD(260) /OD(280) ratios and decreased ability of the quantitative PCR to synthesise the human target, related to the severity of depurination. A satisfactory correlation between the degree of damage and the amount of residual PCR-sensitive target sequences was also demonstrated (r(2) =0.9717). The conventional and mini-STR typing of the samples showed that the genetic outcome was influenced by a depurination damage that exceeded 4% when locus drop-outs and artefactual PCR results were evident. As the success of STR typing depends on the integrity of the DNA recovered from the samples, the CE-UV, physical and molecular assays described here are proposed as a set of useful methods in the analysis of certain forensic and clinical samples, for a critical evaluation of the outcome of the genetic testing.