E F Huffine | PubMed Health

PubMed2001

Improved MtDNA sequence analysis of forensic remains using a "mini-primer set" amplification strategy.

Mitochondrial DNA (mtDNA) analysis of highly degraded skeletal remains is often used for forensic identification due largely to the high genome copy number per cell. Literature from the "ancient DNA" field has shown that highly degraded samples contain populations of intact DNA molecules that are severely restricted in size (1-4). Hand et al. have demonstrated the targeting and preferential amplification of authentic human DNA sequences with small amplicon products of 150 bp or less (1,2). Given this understanding of ancient DNA preservation and amplification, we report an improved approach to forensic mtDNA analysis of hypervariable regions 1 and 2 (HV1/HV2) in highly degraded specimens. This "mini-primer set" (MPS) amplification strategy consists of four overlapping products that span each of the HV regions and range from 126 to 170 bp, with an average size of 141 bp. For this study, 11 extracts representing a range of sample quality were prepared from nonprobative forensic specimens. We demonstrate a significant increase in MPS amplification success when compared to testing methods using approximately 250 bp amplicons. Further, 16 of 17 independent amplifications previously "unreported" due to mixed sequences provided potentially reportable sequence data from a single, authentic template with MPS testing.

Bone and Bones↗

PubMed2000

Formation of an interfering substance, 3,4-dimethyl-5-phenyl-1,3-oxazolidine, during a pseudoephedrine urinalysis.

During fatal aviation accident investigations, biosamples from the victims are submitted to the FAA Civil Aeromedical Institute (CAMI) for drug analysis. In the process of one such analysis by CAMI, an unknown substance was found in a urine sample. Simultaneous screening by thin layer chromatography (TLC) and gas chromatography/FID (GC/FID) suggested the presence of pseudoephedrine. A subsequent routine confirmation analysis of a separate urine aliquot by GC Fourier transform infrared (GC/FTIR) and GC mass spectrometry (GC/MS) indicated that the retention times of the unknown substance matched with those of pseudoephedrine. However, its infrared and mass spectra were different--the -OH and -NH groups were missing, a C-O-C group was present, and the molar mass was 12 atomic mass units (amu) more than that of pseudoephedrine. A subsequent literature search suggested that ephedrine-like amines react with aldehydes to form oxazolidines. Therefore, the 12-amu increase could be accounted for by condensation of pseudoephedrine with formaldehyde. Since this aldehyde is present in various grades of methanol and ethyl acetate, and these solvents were used during the solid-phase extraction, 3,4-dimethyl-5-phenyl-1,3-oxazolidine was synthesized by using (+)-pseudoephedrine HCl and formaldehyde. The analytical findings of the synthesized compound were consistent with those of the unknown interfering substance, confirming that it was the oxazolidine. Aldehyde contaminants in solvents or specimens can transform drugs of interest and may result in misidentification of a compound originally present in specimens. Therefore, chemicals used in analyses should be of the highest available purity, and a multi-analytical approach should be adopted to maintain a high degree of quality assurance.

Accidents, Aviation↗