Comparison of different proteases and direct cell lysis methods used for the recovery of exogenous DNA from fingernail evidence
Izzo, Caitlin Rose
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Fingernail samples are analyzed in forensic casework to determine the source of the nail and/or to recover a foreign profile from beneath the nail. When extracting from a fingernail sample, it is possible to recover deoxyribonucleic acid (DNA) of the nail donor from within the nail and from the surface of the nail; similarly, foreign DNA may also be present on and recovered from the nail surface. When attempting to recover the latter, fingernail samples present particular problems. Often, the foreign component is masked by the greater mass of donor DNA present within and on the nail sample. This masking effect is exacerbated by the use of proteinase K (PK) in DNA extractions, as PK, with an average of 200 cut sites per keratin molecule, is capable of breaking open the keratin matrix of the nail and exposing the nail DNA intercalated in the matrix. Directly extracting nail clippings, in contrast to swabbing or scraping, would further introduce nail DNA when using proteinase K. The present study explores whether utilizing other proteases (ZyGEM, Acrosolv, and Factor Xa) with fewer cut sites than PK or direct lysis methods (IGEPAL® CA-630 and MAWI iSWABTM-ID) would minimize recovery of nail DNA from within the nail and thus mitigate the masking effect often seen with fingernail samples. The endogenous DNA extraction efficiency of each suggested method was compared with the manufacturer’s standard QIAGEN QIAamp® DNA Investigator extraction protocol for hand-washed and/or laboratory-cleaned nails. The extraction results from the hand-washed nails demonstrate variability both within samples from the same donor and between donors. In contrast to previously published literature, a comparison of the results between the hand-washed and cleaned nails suggests that much of the endogenous DNA recovered from fingernail samples is derived from DNA on the surface rather than from within the nail. QIAamp® extraction with the inclusion of dithiothreitol (DTT) recovered significantly more DNA (0.845 ± 0.651 nanograms of DNA per milligram of nail [ng DNA/mg nail]; p = 0.0045) than the same protocol without DTT (0.278 ± 0.253 ng DNA/mg nail). IGEPAL® recovered the least endogenous DNA (0.005 ± 0.012 ng DNA/mg nail) from the nail. The ZyGEM extraction recovered the second lowest amount (0.163 ± 0.161 ng DNA/mg nail) and both the Acrosolv (0.546 ± 0.607 ng DNA/mg nail) and MAWI’s iSWABTM-ID (0.681 ± 0.780 ng DNA/mg nail) methods recovered more DNA than the QIAamp® protocol without DTT. An assessment of the electropherograms resulting from cleaned fingernails across all extraction methods for one donor showed that both IGEPAL® and MAWI failed to recover a complete profile, whereas the remaining methods were able to recover complete profiles of the nail donor. An assessment of donor variability found variations in terms of endogenous nail DNA recovery. Fingernails were also spiked with blood, saliva, or semen to assess the recovery of foreign DNA. The extractions of the spiked nail samples demonstrate variability across all samples, owing, to some degree, to inconsistencies of sample preparation. IGEPAL®’s inability to recover complete foreign profiles suggests that the method may not be viable for extraction of fingernail samples. Conversely, the ZyGEM, Acrosolv, and MAWI extraction methods demonstrate potential as alternative extraction methods for fingernail samples and would benefit from additional experimentation.