A comprehensive evaluation of Cell Elution Method (CEM) for forensic DNA analysis from smoked cigarettes.

Cigarette stubs are commonly encountered trace DNA samples at crime scenes. Standard laboratory practice typically involves direct lysis of the stub for DNA extraction, leading to the co-extraction of DNA-degrading and inhibiting constituents from smoke and tobacco. This process can result in lower-quality DNA profiles. There has been limited focus on developing specific sample processing techniques that minimize these degrading agents and inhibitors before DNA extraction, which could significantly enhance the quality of DNA profiles. This study evaluates a previously established Cell Elution Method (CEM) against the conventional Direct Lysis Method (DLM) for DNA extraction from cigarette stubs. DNA quantity, quality, and subsequent STR profiles were assessed in 80 smoked cigarette stubs, comprising both flavoured and unflavoured types. While CEM exhibited comparable DNA yield from both flavoured (0.17 ng) and unflavoured (0.19 ng) cigarettes, DLM showed significant variability in average DNA yield for unflavoured (0.05 ng) and for flavoured (0.25 ng) cigarettes. Notably, CEM-treated samples demonstrated lower Degradation Index (DI) values compared to DLM-treated ones for both the types of cigarettes. Consequently, STR profiling success rates were higher with CEM, with 95 % of flavoured and 55 % of unflavoured samples yielding informative profiles, compared to 80 % and 0 %, respectively, for DLM. In unflavoured stubs, Amelogenin marker amplification was achieved in 35 % of CEM-treated samples, significantly outperforming the 5 % success rate with DLM. Additionally, CEM resulted in higher average allele recovery rates for both flavoured (58.98 %) and unflavoured (33.41 %) samples compared to DLM. These findings indicate that CEM outperforms DLM in producing higher-quality DNA profiles from cigarette stubs. Thus, CEM can be a choice of method for processing cigarette stub prior to DNA extraction.

Forensic evaluation of mitochondrial DNA heteroplasmy in Gujarat population, India.

BACKGROUND: Owing to its high copy number and its small size, mtDNA analysis is the most reliable choice when biological materials from crime scenes are degraded or have mixed STR profiles. AIM: To examine the occurrence of heteroplasmy along with its frequency and pattern in both HV1 and HV2 regions of the mtDNA among unrelated individuals from India. SUBJECTS AND METHODS: Mitochondrial DNA control region [hypervariable region one (HV1) and hypervariable region two (HV2)] were analysed in blood and buccal tissues of 104 unrelated individuals from the Indian state of Gujarat. RESULTS: A high frequency of point heteroplasmy (PH) and length heteroplasmy (LH) was revealed. PH was detected in 7.69% of the population, with a higher frequency observed in blood than in buccal samples. However, there were no statistically significant differences in PH between the two tissues (Chi-square = 0.552, p ≥ 0.05). A total of six PH positions were detected: three at HV1, and another three at HV2. The studied population showed 46.15% LH in the HV1 and HV2 regions of both tissues. The LH positions observed in the Gujarat population were the same as those previously reported at HV1 np16184-16193 and HV2 np303-315. CONCLUSIONS: Our findings suggest that differences in the pattern of heteroplasmy found in different tissues can complicate the forensic analysis, on the other hand, the probability of a match between the questioned and reference samples increases when the heteroplasmy is identical in both tissues. Variability of PH among persons and even within tissues recommends analysing multiple tissue samples before drawing a conclusion in forensic mtDNA analyses.

Comparative evaluation of different human dental tissues and alveolar bone for DNA quantity and quality for forensic investigation.

In this study, the efficacy of dental tissues (cementum, dentine and pulp) and alveolar bone as a potential source of DNA was tested in terms of the quality and quantity using nuclear and mitochondrial markers for forensic investigation.This study found dentine as the best source of DNA with only 5.36% imbalanced (PHR<0.7) heterozygous loci. Pulp showed the highest quantity of DNA but exhibited 22.3% imbalanced (PHR<0.7) heterozygous loci. Cementum with highest (46.67%) heterozygote imbalance proved to be the last choice as a source of DNA. Alveolar bone exhibited the second-highest total yield of DNA/mg of tissue. All Global Filer™ STR loci were amplified in 70% samples of fresh alveolar bone whereas for 30% samples, only partial profile was generated along with successful sex determination. All the dental tissues and alveolar bone samples amplified non STR markers (D-loop, Cytochrome Oxidase I, SRY, AMEL). Of the alveolar bones from archival samples, one sample exhibited full STR profile whereas other alveolar bone samples gave partial profiles. This study substantiates alveolar bone as an alternate source of nuclear and mitochondrial DNA.

A systematic evaluation of 'Bidi - a hand-rolled cigarette' as a forensic DNA evidence.

Bidis are small handmade cigarettes consisting of ~0.2 g of tobacco flakes rolled in a dried leaf of 'Tendu' (Diospyros melanoxylon) or Piliostigma racemosum and tied with a thin thread. They have gained worldwide popularity among smokers and are often collected as forensic DNA evidence from crime scenes and processed similarly to cigarette butts. However, bidi's composition and manufacturing process differs distinctly from the cigarette, and hence the optimal processing for DNA analysis should not be assumed to be similar to cigarette butts. In the present study, the methodical evaluation of the bidi for DNA analysis is reported and an additional process of cell elution from bidi stubs prior to DNA extraction is systematically compared with the direct lysis of bidi stubs which is identical to the standard practice in forensic labs for cigarette butts. In terms of cell recovery from bidi stubs, the SDS based Cell Elution Buffer (CEB) proved to be better than Tween20 based CEB. The three components (cell-elute, supernatant, and processed stub) obtained after the cell elution process of smoked bidi stubs showed varying amounts of DNA. The cell-elute and processed stub exhibited high quality DNA, resulting in 90-100% of the samples giving a full STR profile. On the contrary, the directly lysed stubs yielded high quantity but low quality of DNA, resulting in only 40% of the samples with full STR profiles. The cell elution process enables three components namely cell-elute, supernatant and processed stub from the same bidi to be used for forensic DNA analysis, although the cell-elute proved to be the best source of DNA for STR profiling. The current study demonstrates that the additional cell elution process proved to be an essential step prior to DNA extraction procedure for bidi stubs.