Molecular characterisation of the nucleic acids recovered from aged forensic samples

Abstract

The molecular composition of the genetic substrate recovered from seven aged forensic samples has been extensively investigated. A simple enzymatic test based on DNAseI incubation of the extracts showed that the UV-fluorescent material from the forensic specimens is composed of nucleic acids, with the DNA fraction representing at least 90% of the total amount. Since spectrophotometric determinations of the extracts showed unreliable results due to anomalous OD 260 /OD 280 ratios, quantification of the nuclease-sensitive genetic material was performed by a slightly modified agarose plate method. The first quantitative data on exogenous contamination in aged forensic samples are provided by slot-blot hybridisation of the extracts to human, bacterial and fungal probes. Only limited amounts of human and contaminant DNA were detected in the samples. The molecular integrity of the primary structure of these aged DNA samples was analysed by reversed-phase HPLC/MS. The data show a good correlation between the degree of chemical damage and the ability to hybridise to molecular probes. The ability to achieve specific genetic profiles was assessed by multiplex PCR amplification of STR loci. Our data show that accurate determination of the molecular composition of the DNA recovered from forensic samples can be extremely useful for a reliable evaluation of the PCR typing results.

Estimating the integrity of aged DNA samples by CE

“…Samples f ‐PI1 and f ‐PI3, previously analysed by HPLC/MS 33, showed a good correspondence in terms of uDNAb (at that time, the values were 0.930 for f ‐PI1 and 0.907 for f ‐PI3). This observation should be considered as a further demonstration of the present procedure accuracy as two different techniques like HPLC/MS and CE/UV provided consistent data on the same samples analysed in different laboratories and times.…”

“…Six out of them showed a substantial reduction of the MW below 400 bp (see Table 2). In order to exclude that the observed fluorescence could arise from unknown contaminants, aliquots of each sample were incubated in presence of the endonuclease DNase I, as already described 33. This enzyme completely digested the UV‐fluorescent material recovered from eight samples while a consistent reduction of the signal was observed for five samples (see Table 2).…”

“…In the case of aged/forensic samples, exogenous (bacterial/fungal) contamination can be a relevant source of UV‐absorbing substances. However, it has been already shown by hybridisation techniques 33 that such contamination provides only a partial explanation for the bulks of genetic material. In our samples, even though contamination could not be excluded at all, both fragmentation and DNA chemical decay provide more reasonable explanations for no or low sensitivity to the Real‐Time PCR quantitative assay.…”

“…DNA was recovered from all samples using a standard organic extraction protocol based on phenol/chloroform purification and ethanol precipitation 33. All these DNA samples were further purified through Ultracel 100 K Amicon Ultra columns (Millipore, MA; USA).…”

“…DNA can be hydrolysed either by enzymatic or heat‐acid procedures 34. Aged DNA samples can be scarcely sensitive to the enzymatic activity of the DN aseI 33, 35, therefore a heat‐acid protocol has to be adopted for these samples. Although this protocol itself causes a further, albeit low, DNA damage 34, all the experiments were performed in formic acid (90%, v/v) at 170°C for 30 min as lower temperatures (140, 110, and 80°C) gave unsatisfactory yields of hydrolysis especially concerning the pyrimidimic compounds 36, 37.…”