DNA transfer by examination tools--a risk for forensic casework?

The introduction of profiling systems with increased sensitivity has led to a concurrent increase in the risk of detecting contaminating DNA in forensic casework. To evaluate the contamination risk of tools used during exhibit examination we have assessed the occurrence and level of DNA transferred between mock casework exhibits, comprised of cotton or glass substrates, and high-risk vectors (scissors, forceps, and gloves). The subsequent impact of such transfer in the profiling of a target sample was also investigated. Dried blood or touch DNA, deposited on the primary substrate, was transferred via the vector to the secondary substrate, which was either DNA-free or contained a target sample (dried blood or touch DNA). Pairwise combinations of both heavy and light contact were applied by each vector in order to simulate various levels of contamination. The transfer of dried blood to DNA-free cotton was observed for all vectors and transfer scenarios, with transfer substantially lower when glass was the substrate. Overall touch DNA transferred less efficiently, with significantly lower transfer rates than blood when transferred to DNA-free cotton; the greatest transfer of touch DNA occurred between cotton and glass substrates. In the presence of a target sample, the detectability of transferred DNA decreased due to the presence of background DNA. Transfer had no impact on the detectability of the target profile, however, in casework scenarios where the suspect profiles are not known, profile interpretation becomes complicated by the addition of contaminating alleles and the probative value of the evidence may be affected. The results of this study reiterate the need for examiners to adhere to stringent laboratory cleaning protocols, particularly in the interest of contamination minimisation, and to reduce the handling of items to prevent intra-item transfer.

Mutational interference mapping experiment (MIME) for studying RNA structure and function

RNA regulates many biological processes; however, identifying functional RNA sequences and structures is complex and time-consuming. We introduce a method, mutational interference mapping experiment (MIME), to identify, at single-nucleotide resolution, the primary sequence and secondary structures of an RNA molecule that are crucial for its function. MIME is based on random mutagenesis of the RNA target followed by functional selection and next-generation sequencing. Our analytical approach allows the recovery of quantitative binding parameters and permits the identification of base-pairing partners directly from the sequencing data. We used this method to map the binding site of the human immunodeficiency virus-1 (HIV-1) Pr55(Gag) protein on the viral genomic RNA in vitro, and showed that, by analyzing permitted base-pairing patterns, we could model RNA structure motifs that are crucial for protein binding.