The Latest DNA-Related Technical Reports

Species origin in biological materials, mitochondrial DNA analysis, and identification of biological stains are included in the latest batch of final technical reports available from NIJ via the National Criminal Justice Reference Service.

See below for additional details. These reports are the results of NIJ-funded projects but were not published by the U.S. Department of Justice.

Title: A Rapid, Efficient, and Effective Assay to Determine Species Origin in Biological Materials (pdf, 61 pages)

Authors: Jared M. Latiolais

Abstract: The primary research objective was to develop a single nucleotide polymorphism (SNP) panel to identify animal species from forensic samples of unknown biological origin by using mitochondrial DNA (mtDNA)-based markers. The NIJ-funded research collected mtDNA samples from a small number of common species potentially present at North American crime scenes (e.g., bear, cat, cow, deer/elk, pig), including some that were not previously stored in animal gene data banks. Samples of mtDNA were collected for 90 species in 10 taxa, sequenced, and compiled in a database of three mtDNA genes.

The technology laboratory was able to identify taxa-specific SNPs that could be exploited in assay form; each SNP was tested using the ABI SNaPshot Multiplex kit, which is readily adapted to thermal cycler and capillary electrophoresis instrumentation present in many forensic laboratories. Proof of concept was demonstrated for using degenerate polymerase chain reaction and multiplexed, single-base extension to identify unknown animals in forensic samples. Although the taxa-specific SNPs work appropriately and robustly, liberal thermal cycling parameters for “universal” primers appear to contribute to an elevated downstream signal-to-noise ratio and baseline anomalies.

Title: Assessing Deep Sequencing Technology for Human Forensic Mitochondrial DNA Analysis (pdf, 90 pages)

Author: Mark R. Wilson, Ph.D.

Abstract: Human mitochondrial DNA (mtDNA) analysis, in a forensic setting, is currently limited in breadth (the quantity of sequence data obtained) and depth (the ability to detect minor variants arising from mutations but present at very low levels). The goal of this research was to generate information on the whole mtDNA genome sequence from limited DNA samples, which would greatly expand the potential uses of this marker system. The researchers evaluated two new methods of DNA sequence analysis to obtain massively parallel mtDNA sequence information (deep sequencing) from hair, buccal, and blood samples. This has revealed that subtle mixtures are present in forensic DNA samples that are not currently detected by current forensic mtDNA analysis.

The researchers found that a combination of enhanced DNA extraction, whole-genome amplification of the extracts, multiplexed polymerase chain reactions around the mtDNA genome, and direct sequencing of the DNA samples on the Illumina MiSeq instrument resulted in mtDNA sequence information from hair shafts that matched information found in blood and buccal extracts from the same donors. A direct sample preparation step, using the Illumina Nextera-XT kit, easily collected ample template mtDNA from the enhanced extraction and amplification steps to perform the other applications. This allows the direct processing of any double-stranded DNA, including amplicons, for deep sequencing in a much simpler and cost-effective manner.

Title: Validation of Highly-Specific Protein Markers for the Identification of Biological Stains (pdf, 54 pages)

Author: Phillip B. Danielson, Ph.D.

Abstract: Although DNA analysis of an evidentiary swab may reveal the presence of a DNA profile consistent with an alleged victim, the DNA profile cannot indicate whether the DNA came from saliva, vaginal fluid, urine, or a host of other sources. The ability to confidently associate a DNA extract with a specific tissue source or to accurately characterize mixed stains, however, can provide criminal investigators with critical information. This research aimed to (1) recruit volunteers and collect samples of forensically relevant body fluids (peripheral and menstrual blood, vaginal fluid, semen, urine, and saliva), (2) use a high-sensitivity mass spectrometry technology (Q-TOF) to combine six panels of highly specific protein biomarkers into a single multiplex assay to evaluate the target stain specificity on samples of each of the six body fluids from 50 research participants, and (3) validate the specificity of the candidate biomarkers and the Q-TOF multiplex assay in 37 forensic casework-type samples.

Single- and mixed-source samples recovered from various substrates or exposed to environmental contaminants/insults were also assessed; the fluids were accurately detected with one or more of the high-specificity biomarkers. This information will help to facilitate the commercial production of such assays, including development of a commercial mass spectrometry approach based on the multiplex assay described in this research.

Title: Microchip Analyzer for Forensic Short Tandem Repeat Typing of Single Cells (pdf, 39 pages)

Authors: Tao Geng, Richard A. Mathies

Abstract: Short tandem repeat (STR) typing at the single-cell level is a promising tool for human forensic identification. For the majority of crimes, the biological evidence is composed of mixtures of cells, and hence a mix of DNA genotypes, at relatively low concentrations from multiple individuals. This can lead to subsequent challenges in interpreting the results of STR analysis, especially if the number of contributors exceeds two. A more difficult situation is when the perpetrator cells are much rarer than the victim cells, resulting in preferential amplification of the victim DNA and the inability to detect the perpetrator genotype. The currently available methods are inefficient, have low throughput, a high possibility of sample cross-contamination, and a lack of universality.

In this report, the authors describe a novel single-cell STR typing method with high sensitivity, fidelity, and throughput that combines microfluidic droplet generation with a single-cell, multiplex emulsion polymerase chain reaction (PCR). Individual cells are isolated within microdroplets, which subsequently function as miniaturized reactors for PCR amplification, producing high-quality STR profiles from single cells at high throughput. This method can be applied to the analysis of forensic evidence samples involving low-abundance materials and multiple suspects, thereby solving the classic mixture analysis problem.

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