Development of an effective method of human DNA extraction from soil as forensic evidence.

In forensic investigations, it is crucial to detect a suspect's DNA from the available evidence. In an outdoor crime scene, the evidence may be mixed with the soil. However, soil is speculated to inhibit DNA extraction from forensic science samples. In the field of soil microbiology, it is necessary to extract DNA directly from soil to analyse its microbial composition. In this study, we investigated whether skim milk used in procedure of DNA extraction from soil samples could be applied to forensic science to enhance human DNA extraction efficiency from soil mixed with forensic evidence, such as blood, buccal cells, and skin cells. The use of additive reagents, skim milk and bovine serum albumin (BSA), are known to blocking reagent. In the selection of additive reagents experiment, about blood sample, using the skim milk and BSA were found to increase the DNA yield. Therefore, we observed extracted DNA yield from blood, buccal cells, and skin cells when skim milk and BSA were used as additive reagents. The DNA recovery rate was high across all samples upon addition of skim milk. However, in STR analysis, a non-specific peak was detected in the extracted DNA in the presence of skim milk, which was not detected in the presence of BSA, indicating its suitability in forensic analysis. Our study suggests that addition of BSA can efficiently aid the extraction of DNA from forensic evidence mixed with soil.

Evaluation of bovine thymic function by measurement of signal joint T-cell receptor excision circles.

A signal joint T-cell receptor excision circle (sjTREC) is a circular DNA produced by T-cell receptor α gene rearrangement in the thymus. Measurements of sjTREC values have been used to evaluate thymic function. We recently established a quantitative PCR (QPCR) assay of bovine sjTREC. In the present study, we used this QPCR assay to measure the sjTREC value in bovine peripheral blood mononuclear cells and we then evaluated the relationships between sjTREC values and peripheral blood T-cell number, growth stage, gender, and meteorological season. The sjTREC value was highest at the neonatal stage, and its value subsequently decreased with age. On the other hand, the peripheral T-cell number increased with age. The sjTREC value in calves up to 50-days old was significantly higher for males than for females, suggesting that thymic function might differ by gender. In addition, the sjTREC value and the peripheral T-cell number were significantly higher in calves in the summer season than in calves in the winter season. These data suggest that bovine thymic function is highly variable and varies according to the growth stage, gender, and environmental factors such as air temperature or the UV index.

Detection and quantification of bovine signal joint T-cell receptor excision circles.

A signal joint (sj) T-cell receptor excision circle (TREC) is produced by T-cell receptor (TCR) gene rearrangements during αß T-cell maturation in the thymus. sjTREC have been studied as a marker of thymic function in several spices. We designed specific primers for δrec-ψJα sj region to identify the location of the bovine sjTREC region and determined the nucleotide sequence of the PCR product. The obtained sequences were subjected to a BLAST search, which identified a matching region. This matching region contained TCR δ genes and was identified on bovine chromosome 10. We also confirmed the polymorphism of the sj region by sequencing of 10 PCR products, and observed irregular insertion of bases in the δrec-ψJα recombination signal sequence. We then developed a quantitative PCR (QPCR) assay for evaluation of sjTRECs level in order to evaluate bovine thymic function for application in the veterinary clinic. This QPCR assay specifically amplified the sj region of bovine sjTREC and could detected 10(1)-10(7) copy numbers of sjTRECs. Using this assay we found that the number of sjTRECs in peripheral blood mononuclear cells was less than 10% that of the thymus.