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.

The molecular characterization of a depurinated trial DNA sample can be a model to understand the reliability of the results in forensic genetics

“…The protocol described here follows our previous studies based on DNA depurination under mild acidic conditions and represents an evolution toward its simplification. Depurination was performed in water at 70°C for 1, 2.5, 5, 7.5, and 10 h. Reagents, chemicals, and even the DNA sample here used (sample TSPV2, deriving from 500 mL of fresh blood, in water at concentration of 75 ng/μL) are the same as described elsewhere, as well as the methods employed to characterize the molecular features of the damaged DNA samples .…”

“…The first sample was the “trial sample” (TS) that was prepared from eight aliquots of sample TSPV2 incubated at 70°C for 5 h. The pooled aliquots were adjusted to the final volume of 2.4 mL with water and then carefully mixed. This solution was then subdivided into 40 aliquots of 60 μL each.…”

“…To verify the outcome of the depurination protocol, the amount of released bases was assessed, in each of the eight samples, by MEKC as described previously . This test showed that the mean percentage release of the purines was 5.8 ± 0.5 and 4.6 ± 0.3 for guanine and adenine, respectively (as calculated by the formula A fX /A totX , where A fX is the amount of a given free base X and A totX is the amount of the same base bound to the DNA before the treatment) .…”

“…To check the main molecular features of the TS and QSS, four randomly selected aliquots of each stock were analyzed as described elsewhere in two different working sessions, the first one immediately after the sample preparation and the second one after a 2‐month freezing storage.…”

Capillary electrophoresis analysis of DNA primary structure: Toward a quality control test for the reliability of the STR-typing from forensic specimens

“…An experimental protocol was set up to partially degrade a trial DNA sample by a rapid and reliable protocol in order to employ the treated samples for further assessments [3]. CE analysis of these samples showed smaller peaks both for G and A compared to the control sample TSPV2.…”