David R. Foran

Polymerase Resistance to Polymerase Chain Reaction Inhibitors in Bone

Abstract:u2002 Amplification of DNA from aged or degraded skeletal remains can be a challenging task, in part due to naturally occurring inhibitors of the polymerase chain reaction. PCR inhibitors may act by inactivating a polymerase itself, or compete with or bind other reaction components, although various polymerases may be differentially susceptible to such insult. In this study, ten thermostable polymerases from six bacterial species were examined for their ability to amplify DNA in the presence of bone‐derived or individual PCR inhibitors. Two polymerases, one from Thermus aquaticus and one from Thermus thermophilus, showed lower susceptibility to inhibition from bone, while polymerases from Thermus flavus were highly susceptible. Addition of bovine serum albumin improved the activity of most of the enzymes. Taken together, the results indicate that thermostable DNA polymerases have different susceptibility to bone‐derived PCR inhibitors, and that those most often used in forensic laboratories may not be optimal when working with DNA from skeletal remains.

Aging Blow Fly Eggs Using Gene Expression : A Feasibility Study

Abstract:u2002 Forensic entomology can aid death investigations by using predictable developmental changes to estimate the ages of flies associated with a body. In developmental stages that do not increase in size however, including the egg and pupa, it can be difficult to objectively refine an age estimate beyond the limits of the stage duration. Gene transcript levels, changing throughout development, represent a potential data source useful for objectively identify smaller units of developmental time. The expression of three genes (bcd, sll, cs) was profiled throughout the maturation of blow fly eggs to determine the feasibility of predicting age, identifying significant linear trends in expression during their development. Models estimating egg age made predictions within 2u2003h of true age when all expression data were available, while the presence/absence of cs transcripts identified two age classes, together indicating that gene expression can be used to more precisely predict blow fly age.

The utility of whole genome amplification for typing compromised forensic samples.

ABSTRACT: Biological evidence has become invaluable in the crime laboratory; however, it may exist in limited quantity and/or quality. Given this, the ability to amplify total DNA obtained from evidence, in an unbiased manner, would be highly advantageous. Methods for whole genome amplification (WGA) have the potential to fulfill this role, resulting in a virtually unlimited supply of DNA. In the research presented, two WGA methods, improved primer extension preamplification and multiple displacement amplification (MDA), were tested using commercial kits. Control DNA, artificially degraded DNA, and DNA from fresh blood, aged blood, hair shafts, and aged bones underwent WGA, followed by short tandem repeat and mitochondrial DNA analysis. The methods did amplify DNA, but performed poorly on forensically relevant samples; the maximum amplicon size was reduced, and MDA often resulted in extraneous bands following polymerase chain reaction. Taken together, WGA appears to be of limited forensic utility unless the samples are of a very high quality.

Spatial and Temporal Influences on Bacterial Profiling of Forensic Soil Samples

Abstract:u2002 Bacterial content may be helpful in differentiating forensic soil samples; however, the effectiveness of bacterial profiling depends on several factors, including uniqueness among different habitat types, the level of heterogeneity within a habitat, and changes in bacterial communities over time. To examine these, soils from five diverse habitats were tested over a 1u2003year period using terminal restriction fragment length polymorphism (TRFLP) analysis. Soil samples were collected at central locations monthly, and 10 feet in cardinal directions quarterly. Similarity indices were found to be least related among habitats, while the greatest bacterial similarities existed among collection locations within a habitat. Temporally, however, bacterial content varied considerably, and there was substantial overlap in similarity indices among habitats during different parts of the year. Taken together, the results indicate that while bacterial DNA profiling may be useful for forensic soil analysis, certain variables, particularly time, must be considered.

Bacterial profiling of soil using genus-specific markers and multidimensional scaling

Abstract:u2002 Forensic identification of soil based on microbial DNA fingerprinting has met with mixed success, with research efforts rarely considering temporal variability or local heterogeneity in soil’s microbial makeup. In the research presented, the nitrogen fixing bacteria rhizobia were specifically examined. Soils were collected monthly from five habitats for 1u2003year, and quarterly in each cardinal direction from the main collection site. When all habitats were compared simultaneously using Terminal Restriction Fragment Length Polymorphism analysis of the rhizobial recA gene and multidimensional scaling, only two were differentiated over a year’s time, however pairwise comparisons allowed four of five soils to be effectively differentiated. Adding in 10‐foot distant soils as “questioned” samples correctly grouped them in 40–70% of cases, depending on restriction enzyme used. The results indicate that the technique has potential for forensic soil identification, although extensive anthropogenic manipulation of a soil makes such identification much more tentative.

Simplified field preservation of tissues for subsequent DNA analyses.

Abstract:u2002 Successful DNA‐based identification of mass disaster victims depends on acquiring tissues that are not highly degraded. In this study, multiple protocols for field preservation of tissues for later DNA analysis were tested. Skin and muscle samples were collected from decaying pig carcasses. Tissues were preserved using cold storage, desiccation, or room temperature storage in preservative solutions for up to 6u2003months. DNA quality was assessed through amplification of successively larger segments of nuclear DNA. Solution‐based storage, including a DMSO/NaCl/EDTA mixture, alcohols, and RNAlater preserved DNA of the highest quality, refrigeration was intermediate, and desiccation was least effective. Tissue type and extent of decomposition significantly affected stored DNA quality. Overall, the results indicate that any tissue preservation attempt is far superior to delaying or forgoing preservation efforts, and that simple, inexpensive methods can be highly effective in preserving DNA, thus should be initiated as quickly as possible.

The influence of swabbing solutions on DNA recovery from touch samples.

There has been minimal research into how to best obtain DNA from touch samples. Many forensic laboratories simply moisten a swab with water and use it for collecting cells/DNA from evidentiary samples. However, this and other methods have not been objectively studied in order to maximize DNA yields. In this study, fingerprints were collected using swabs moistened with water or laboratory or commercially available detergents, including sodium dodecyl sulfate (SDS), Triton X‐100, Tween 20, Formula 409®, and Simple Green®. Prints were swabbed, DNA isolated using an organic extraction, yields quantified, and relative yields compared. In all cases, the detergent‐based swabbing solutions outperformed water, with SDS and Triton X‐100 producing significant increases in yield. Short tandem repeat profiles were consistent with the individuals that placed them. Subsequent analysis of SDS concentrations for collecting touch DNA demonstrated an increase in DNA yield with increasing SDS concentration, with an optimal concentration of approximately 2%.

The Recovery and Analysis of Mitochondrial DNA from Exploded Pipe Bombs

Abstract:u2002 Improvised explosive devices (IEDs) represent one of the most common modes of arbitrarily injuring or killing human beings. Because of the heat generated by, and destruction to, an IED postconflagration, most methods for identifying who assembled the device are ineffective. In the research presented, steel pipe bombs were mock‐assembled by volunteers, and the bombs detonated under controlled conditions. The resultant shrapnel was collected and swabbed for residual cellular material. Mitochondrial DNA profiles were generated and compared blind to the pool of individuals who assembled the bombs. Assemblers were correctly identified 50% of the time, while another 19% could be placed into a group of three individuals with shared haplotypes. Only one bomb was assigned incorrectly. In some instances a contaminating profile (mixture) was also observed. Taken together, the results speak to the extreme sensitivity the methods have for identifying those who assemble IEDs, along with precautions needed when collecting and processing such evidence.

Next‐Generation Sequencing of the Bacterial 16S rRNA Gene for Forensic Soil Comparison: A Feasibility Study

Soil has the potential to be valuable forensic evidence linking a person or item to a crime scene; however, there is no established soil individualization technique. In this study, the utility of soil bacterial profiling via next‐generation sequencing of the 16S rRNA gene was examined for associating soils with their place of origin. Soil samples were collected from ten diverse and nine similar habitats over time, and within three habitats at various horizontal and vertical distances. Bacterial profiles were analyzed using four methods: abundance charts and nonmetric multidimensional scaling provided simplification and visualization of the massive datasets, potentially aiding in expert testimony, while analysis of similarities and k‐nearest neighbor offered objective statistical comparisons. The vast majority of soil bacterial profiles (95.4%) were classified to their location of origin, highlighting the potential of bacterial profiling via next‐generation sequencing for the forensic analysis of soil samples.

Assessing the Utility of Soil DNA Extraction Kits for Increasing DNA Yields and Eliminating PCR Inhibitors from Buried Skeletal Remains

DNA identification of human remains is often necessary when decedents are skeletonized; however, poor DNA recovery and polymerase chain reaction (PCR) inhibition are frequently encountered, a situation exacerbated by burial. In this research, the utility of integrating soil DNA isolation kits into buried skeletal DNA analysis was evaluated and compared to a standard human DNA extraction kit and organic extraction. The soil kits successfully extracted skeletal DNA at quantities similar to standard methods, although the two kits tested, which differ mechanistically, were not equivalent. Further, the PCR inhibitors calcium and humic acid were effectively removed using the soil kits, whereas collagen was less so. Finally, concordant control region sequences were obtained from human skeletal remains using all four methods. Based on these comparisons, soil DNA isolation kits, which quickened the extraction process, proved to be a viable extraction technique for skeletal remains that resulted in positive identification of a decedent.