Rapid, Efficient, and Effective Assay to Determine Species Origin in Biological Materials.

摘要

This report details the NIJ-funded research performed by The Bode Technology Group, Inc. (Bode) over a two year period from July 2008 to August 2010. The primary objective of the research was to develop a single nucleotide polymorphism (SNP) panel for the discrimination of species identity from forensic samples of unknown biological origin; using Mitochondrial DNA (mtDNA) based markers. Bodes approach was to develop proof of concept for the assay by focusing on a small number of ubiquitous species potentially present at North American crime scenes. Using sequence data pulled from NCBI GenBank as well as over 100,000 base pairs of sequence generated on-site, we were able to create gene alignments across all included taxa and species for three mtDNA genes including cytochrome oxidase I (COI), NADH dehydrogenase subunit 4 (ND4), and Cytochrome b (Cytb). The development of canonical SNPs confirmatory for species identity was performed using a neighbor joining phylogenetic algorithm with 10,000 subsequent bootstrap replicates. Potential SNPs were identified by consistency index (CI) values of 1.0 and screened by eye for exclusivity. The resultant SNPs candidates were categorized by gene of origin, species indication, and placement within individual gene sequence. Our intent is for this assay to be used by forensic scientists encountering an unknown biological specimen as an initial screening tool. Using the ABI SNaPshotTM Multiplex kits as the base chemistry for our assay requires multiplexing of both initial polymerase chain reaction (PCR) amplification primers as well as single base extension (SBE) primers. An SBE primer was developed for each of the SNPs for both the forward and reverse strand conformations. Polymerase chain reaction (PCR) primers were then created using the consensus by plurality sequence generated from the alignments to identify conserved areas across all samples. The reactions for each PCR primer were optimized to work across the widest range of species possible then multiplexed into one amplification reaction. This initial reaction provides an ample volume of amplified sample to be used as starting material for SBE reactions, thus it is possible to perform several SBE mini-multiplex reactions from one PCR reaction. The mini multiplexes are plagued by inconsistent representation of species identifying peaks at low template DNA concentrations as well as heightened background noise and the presence of random spurious peaks. A number of methods were attempted to decrease background signal, however they seem to be generated through the use of liberal PCR conditions during the PCR reaction and thus unavoidable. Therefore although SBE primers were spaced so as to enable multiplexing into one single assay; we have tested only the use of three separate mini-multiplexes. To date we have demonstrated the proof of concept for the use of degenerate PCR and multiplexed SBE for the identification of unknown animal samples utilizing three mini multiplexes. However, the continuing presence of random background noise and baseline anomalies deters us from suggesting this method for use as anything other than a research tool. The initial results indicate a presumptive assay with a good deal of promise which needs further development before being ready for widespread use.