A comparison of six adhesive tapes as tape lifts for efficient trace DNA recovery without the transfer of PCR inhibitors.

Tape-lifting is a non-destructive method employed in the laboratory to recover and collect trace DNA evidence from crime scene exhibits with porous surfaces. The success of tape-lifting is a balance between capturing the biological material and compatibility with downstream DNA extraction processes to ensure efficient release of the tape-lifted material during DNA extraction. In this study, six commercially available low-, regular- and high-tack adhesive tapes were evaluated. The low-tack S183 tape and the highly adhesive S-Hold tape were compared for DNA recovery efficiency from different materials commonly encountered in casework. All tape-lifts were processed using PrepFiler Express™ BTA and AutoMate Express™ Forensic DNA extraction systems, DNA samples quantitated by Quantifiler TRIO, amplified using Powerplex® 21 and VeriFiler™ PLUS (VFP), and analysed on a 3500xl genetic analyser to evaluate the quality of the resultant STR profiles obtained. The more adhesive S-Hold tape recovered comparable or more DNA than the low-tack S183 tape from the majority of materials tested. However, STR profiles obtained from S183 tape-lifts were of markedly higher quality compared to S-Hold tape-lifts. This was most evident for towel, denim and printed chiffon, where S-Hold samples exhibited severe PCR inhibition, with VFP internal quality markers confirming the presence of inhibitors. The findings suggest that strong adhesion is not necessarily beneficial for tape-lifting, as the low tack S183 tape was able to efficiently recover cellular material from the surface of porous substrates commonly encountered in casework, while avoiding the co-transfer of PCR-inhibitory substances from the sampled material.

Developmental validation of an efficient differential separation method incorporating the i-sep® DL spin column with high sperm DNA recovery for the processing of sexual assault samples.

The differential separation method is key to recovering a DNA profile of the sperm donor from sexual assault samples. However, low numbers of spermatozoa from the perpetrator are often swamped by the victim's epithelial cells or lost during the separation process, with the separation process labor-intensive, time-consuming, and operator-dependent. The self-sealing filter of the i-sep® DL spin column allows direct lysis of the substrate throughout the differential separation process while preventing intact sperm cells from passing through, maximizing DNA recovery, and separation of non-sperm and sperm cells present. This study investigated the efficacy of a modified differential separation method, which incorporated the i-sep® DL spin column in comparison with the conventional pellet-based differential separation method. Using semen dilution series and mock post-coital samples, the sensitivity, reproducibility, repeatability, and efficiency of sperm DNA recovery of the pellet-based differential to the i-sep® method were evaluated side by side. The i-sep® differential method was more sensitive in capturing sperm fraction DNA, with informative semen donor alleles detected from high dilutions of semen inputs where the pellet method has been unsuccessful. The i-sep® differential method reduces manual handling, generating repeatable, and reproducible results between operators. Re-extraction of samples previously processed by the pellet or i-sep® differential method showed that the pellet method failed to recover 15-88% of sperm fraction DNA, while the i-sep® differential method was able to recover >99% in the initial extraction. The i-sep® method is robust for processing sexual assault samples, overcoming the challenges of sperm DNA losses encountered by pellet-based methods.

Evaluation of DNA Extraction Methods for Processing Fingerprint Powder-Coated Forensic Evidence.

In unison, fingerprinting and DNA analysis have played a pivotal role in forensic investigations. Fingerprint powders that are available on the market can come in a range of colors and with specific properties. This study evaluated the efficiency of DNA extraction from samples coated with 3 brands of fingerprint powders: Lightning, Sirchie, and SupraNano, covering a range of colors and properties. A total of 23 fingerprint powders were tested using the Chelex, Promega DNA IQ™, and Applied Biosystems™ PrepFiler™ DNA extraction protocols. The DNA IQ™ and PrepFiler™ methods extracted higher yields of DNA in comparison to Chelex, which also accounted for better quality of PowerPlex x00AE; 21 DNA profiles recovered. There were no signs of degradation or inhibition in the quantification data, indicating that samples returning low DNA yield was due to interference during DNA extraction and not PCR inhibition. DNA profiles were recovered from the majority of fingerprint powders with only a single powder, Sirchie Magnetic Silver, failing to produce a profile using any of the methods tested. A link was observed between the DNA extraction chemistry, fingerprint powder property, that is, nonmagnetic, magnetic and aqueous, and the brand of fingerprint powder. Overall, the DNA IQ™ method was favorable for nonmagnetic fingerprint powders, while magnetic fingerprint powders produced more DNA profiles when extracted with the PrepFiler™ chemistry. This study highlights the importance of screening DNA extraction chemistries for the type of fingerprint powder used, as there is not a single DNA extraction method that suits all fingerprint powder brands and properties.

ERK/MAPK regulation of the androgen responsiveness of breast cancer cells.

The androgen receptor (AR) is the most widely expressed steroid hormone receptor in human breast cancers and androgens including 5alpha-dihydrotestosterone are potent inhibitors of breast cancer cell proliferation. The extracellular signal-regulated mitogen activated protein kinase (ERK/MAPK) pathway is hyperactivated in a proportion of breast tumors and can interact with steroid hormone receptor signaling by altering receptor phosphorylation, turnover, ligand, and cofactor interactions. To examine the effects of ERK/ MAPK hyperactivity on AR levels, MCF-7 cells were stably transfected with a plasmid encoding a constitutively active MEK1 protein to create MCF-7-DeltaMEK1 cells. Treatment of MCF-7-DeltaMEK1 with androgens caused a transient increase in AR protein levels, similar to that observed in untransfected MCF-7 cells treated with androgens. Androgens also inhibited the proliferation of MCF-7-DeltaMEK1 cells by 50-60% following 8 days of treatment in association with increased accumulation of cells in the G1 phase of the cell cycle. These results indicate that although ERK/MAPK hyperactivation in breast cancer cells is associated with reduced estrogen receptor (ERalpha) levels and antiestrogen resistance, AR levels are maintained and breast cancer cells remain susceptible to the growth inhibitory effects of androgens.