Ron M. Fourney

Fingerprint Enhancement Revisited and the Effects of Blood Enhancement Chemicals on Subsequent Profiler Plus ™ Fluorescent Short Tandem Repeat DNA Analysis of Fresh and Aged Bloody Fingerprints

This study was aimed at determining the effect of seven blood enhancement reagents on the subsequent Profiler Plus fluorescent STR DNA analysis of fresh or aged bloody fingerprints deposited on various porous and nonporous surfaces. Amido Black, Crowles Double Stain. 1,8-diazafluoren-9-one (DFO), Hungarian Red, leucomalachite green, luminol and ninhydrin were tested on linoleum, glass, metal, wood (pine, painted white), clothing (85% polyester/15% cotton, 65% polyester/35% cotton, and blue denim) and paper (Scott 2-ply and Xerox-grade). Preliminary experiments were designed to determine the optimal blood dilutions to use to ensure a DNA typing result following chemical enhancement. A 1:200 blood dilution deposited on linoleum and enhanced with Crowles Double Stain generated enough DNA for one to two rounds of Profiler Plus PCR amplification. A comparative study of the DNA yields before and after treatment indicated that the quantity of DNA recovered from bloody fingerprints following enhancement was reduced by a factor of 2 to 12. Such a reduction in the DNA yields could potentially compromise DNA typing analysis in the case of small stains. The blood enhancement chemicals selected were also evaluated for their capability to reveal bloodmarks on the various porous and nonporous surfaces chosen in this study. Luminol. Amido Black and Crowles Double Stain showed the highest sensitivity of all seven chemicals tested and revealed highly diluted (1:200) bloody fingerprints. Both luminol and Amido Black produced excellent results on both porous and nonporous surfaces, but Crowles Double Stain failed to produce any results on porous substrates. Hungarian Red, DFO, leucomalachite green and ninhydrin showed lower sensitivities. Enhancement of bloodmarks using any of the chemicals selected, and short-term exposure to these same chemicals (i.e., less than 54 days), had no adverse effects on the PCR amplification of the nine STR systems surveyed (D3S 1358, HumvWA, HumFGA, D8S1179, D21S11, D18S51, D5S818, D13S317, D7S820) or of the gender determination marker Amelogenin. The intensity of the fluorescent signals was very similar and the allele size measurements remained constant and identical to those of untreated bloody fingerprints. No additional background fluorescence was noted. Continuous exposure (for 54 days) to two of the seven enhancement chemicals selected (i.e., Crowles Double Stain and Hungarian Red) slightly reduced the amplification efficiency of the longer STR loci in profiles of fresh and 7 to 14-day-old bloodprints. This suggests that long-term exposure to these chemicals possibly affects the integrity of the DNA molecules. This study indicates that significant evidence can be obtained from fresh or aged bloody fingerprints applied to a variety of absorbent and nonabsorbent surfaces which are exposed to different enhancement chemicals for short or long periods of time. It also reaffirms that PCR STR DNA typing procedures are robust and provide excellent results when used in concert with fluorescence-based detection assays after fingerprint identification has taken place.

Enhanced Kinship Analysis and STR-based DNA Typing for Human Identification in Mass Fatality Incidents: The Swissair Flight 111 Disaster

A bioinformatic tool was developed to assist with the victim identification initiative that followed the Swissair Flight 111 disaster. Making use of short tandem repeat (STR) DNA typing data generated with AmpFlSTR Profiler Plus (PP) and AmpFlSTR COfiler(CO) kits, the software systematically compared each available STR genotype with every other genotype. The matching algorithm was based on the search for: (i) direct matches to genotypes derived from personal effects; and (ii) potential kinship associations between victims and next-of-kin, as measured by allele sharing at individual loci. The software greatly assisted parentage analysis by enabling kinship evaluation in situations where complete parentage trios were unavailable and, in some situations, with distantly related relatives. Exclusion of fortuitous kinship associations (FKA) was made possible through the recovery at the disaster site of at least one remains for every sought-after victim, and was incorporated into the kinship software. The data from the 13 combined STR loci produced 6 and 23 times fewer FKAs when compared with PP alone and AmpFlSTR Profiler (PR) alone, respectively. Identification leads or confirmations of identification were obtained for 218 victims for which DNA reference samples (personal effects and kin) had been submitted. Confirmation of an inferred kinship association was sought through frequency and likelihood calculations, as well as corroborative data from other identification modalities. The use of a simple, yet powerful, automated genotype comparison approach and the use of megaplexes with high power of discrimination (PD) values extended considerably the identification capabilities in the case of the Swissair disaster. The DNA typing identification modality proved to be a valuable component of the large arsenal of identification tools deployed in the aftermath of this disaster.

Systematic analysis of stutter percentages and allele peak height and peak area ratios at heterozygous STR loci for forensic casework and database samples.

To assist the interpretation of STR DNA typing results from forensic casework samples containing mixtures, the range of heterozygous allele peak height and peak area ratios (HR) and stutter percentages (stutter %) for the loci comprised in the AmpFlSTR Profiler Plus (PP) kit were assessed on 468 database and 275 casework single source samples. Stutter % medians were similar for database and casework samples, ranging from 2% to 7%. The upper limit of the stutter value range was 16%, calculated as median +3 SD, although lower locus-specific values could be used. HR medians were 93 +/- 6.5% for database samples, 88 +/- 12% for casework samples. For casework samples, the maximum signal imbalance noted was 52%, calculated as median -3 SD. No significant difference was observed between peak height and peak area calculated values. This study shows the importance of selecting the proper reference database for the establishment of HR threshold values.

Automated processing of forensic casework samples using robotic workstations equipped with nondisposable tips: contamination prevention.

Abstract:  An automated process has been developed for the analysis of forensic casework samples using TECAN Genesis RSP 150/8 or Freedom EVO liquid handling workstations equipped exclusively with nondisposable tips. Robot tip cleaning routines have been incorporated strategically within the DNA extraction process as well as at the end of each session. Alternative options were examined for cleaning the tips and different strategies were employed to verify cross‐contamination. A 2% sodium hypochlorite wash (1/5th dilution of the 10.8% commercial bleach stock) proved to be the best overall approach for preventing cross‐contamination of samples processed using our automated protocol. The bleach wash steps do not adversely impact the short tandem repeat (STR) profiles developed from DNA extracted robotically and allow for major cost savings through the implementation of fixed tips. We have demonstrated that robotic workstations equipped with fixed pipette tips can be used with confidence with properly designed tip washing routines to process casework samples using an adapted magnetic bead extraction protocol.

Validation of a DNA IQ™-based extraction method for TECAN robotic liquid handling workstations for processing casework

A semi-automated DNA extraction process for casework samples based on the Promega DNA IQ™ system was optimized and validated on TECAN Genesis 150/8 and Freedom EVO robotic liquid handling stations configured with fixed tips and a TECAN TE-Shake™ unit. The use of an orbital shaker during the extraction process promoted efficiency with respect to DNA capture, magnetic bead/DNA complex washes and DNA elution. Validation studies determined the reliability and limitations of this shaker-based process. Reproducibility with regards to DNA yields for the tested robotic workstations proved to be excellent and not significantly different than that offered by the manual phenol/chloroform extraction. DNA extraction of animal:human blood mixtures contaminated with soil demonstrated that a human profile was detectable even in the presence of abundant animal blood. For exhibits containing small amounts of biological material, concordance studies confirmed that DNA yields for this shaker-based extraction process are equivalent or greater to those observed with phenol/chloroform extraction as well as our original validated automated magnetic bead percolation-based extraction process. Our data further supports the increasing use of robotics for the processing of casework samples.

Assessing a novel room temperature DNA storage medium for forensic biological samples.

The ability to properly collect, analyze and preserve biological stains is important to preserving the integrity of forensic evidence. Stabilization of intact biological evidence in cells and the DNA extracts from them is particularly important since testing is generally not performed immediately following collection. Furthermore, retesting of stored DNA samples may be needed in casework for replicate testing, confirmation of results, and to accommodate future testing with new technologies. A novel room temperature DNA storage medium, SampleMatrix™ (SM; Biomatrica, Inc., San Diego, CA), was evaluated for stabilizing and protecting samples. Human genomic DNA samples at varying amounts (0.0625-200 ng) were stored dry in SM for 1 day to 1 year under varying conditions that included a typical ambient laboratory environment and also through successive freeze-thaw cycles (3 cycles). In addition, spiking of 1-4 × SM into samples prior to analysis was performed to determine any inhibitory effects of SM. Quantification of recovered DNA following storage was determined by quantitative PCR or by agarose gel electrophoresis, and evaluation of quantitative peak height results from multiplex short tandem repeat (STR) analyses were performed to assess the efficacy of SM for preserving DNA. Results indicate no substantial differences between the quality of samples stored frozen in liquid and those samples maintained dry at ambient temperatures protected in SM. For long-term storage and the storage of low concentration samples, SM provided a significant advantage over freezer storage through higher DNA recovery. No detectable inhibition of amplification was observed at the recommended SM concentration and complete profiles were obtained from genomic DNA samples even in the presence of higher than recommended concentrations of the SM storage medium. The ability to stabilize and protect DNA from degradation at ambient temperatures for extended time periods could have tremendous impact in simplifying and improving sample storage conditions and requirements. The current work focuses on forensics analysis; however this technology is applicable to all endeavors requiring storage of DNA.

Sensitive and Specific Quantification of Human Genomic Deoxyribonucleic Acid (DNA) in Forensic Science Specimens: Casework Examples

We describe the forensic science application of a method for quantification of human genomic deoxyribonucleic acid (DNA). The two cases cited in this report involve DNA samples extracted from skin tissue and bloodstained clothing recovered from different crime scenes. High-molecular-weight DNA was recovered from both specimens, and the concentrations of these DNAs were estimated to be approximately 0.5 microgram/microL by ethidium bromide/agarose gel electrophoresis. Using the human-specific DNA probe p17H8 (locus D17Z1) to quantify the amount of human genomic DNA in these samples, it is shown that less than 1% of the DNA isolated from the skin tissue is of human origin and that the DNA isolated from the bloodstained clothing is effectively devoid of human DNA sequences. These case examples illustrate the need to quantify not only the total amount of DNA recovered from forensic casework material, but also the proportion of the DNA that is of human origin.

Validation of highly polymorphic fluorescent multiplex short tandem repeat systems using two generations of DNA sequencers.

Validation studies are a crucial requirement before implementation of new genetic typing systems for clinical diagnostics or forensic identity. Two different fluorescence-based multiplex DNA profiling systems composed of amelogenin, HumD21S11 and HumFGA (referred to as multiplex 1A), and HumD3S1358, HumD21S11 and HumFGA (multiplex 1B) have been evaluated for use in forensic identification using the Applied Biosystems Model 373A and Prism 377 DNA Sequencers, respectively. Experiments were aimed at defining the limit of target DNA required for reliable profiling, the level of degradation that would still permit amplification of the short tandem repeat (STR) loci examined, and the robustness of each locus in the multiplexes after samples were exposed to environmental insults. In addition, the specificity of the multiplexes was demonstrated using nonhuman DNAs. Forensically relevant samples such as cigarette butts, chewing gum, fingernails and envelope flaps were processed using both an organic extraction procedure and a QIAamp protocol. DNAs and resultant multiplex STR profiles were compared. The validation of the triplex STR systems was extended to include over 140 nonprobative casework specimens and was followed with a close monitoring of initial casework (over 300 exhibits). Our results document the robustness of these multiplex STR profiling systems which, when combined with other multiplex systems, could provide a power of discrimination of approximately 0.9999.

Optimized manual and automated recovery of amplifiable DNA from tissues preserved in buffered formalin and alcohol-based fixative

Archival tissue preserved in fixative constitutes an invaluable resource for histological examination, molecular diagnostic procedures and for DNA typing analysis in forensic investigations. However, available material is often limited in size and quantity. Moreover, recovery of DNA is often severely compromised by the presence of covalent DNA-protein cross-links generated by formalin, the most prevalent fixative. We describe the evaluation of buffer formulations, sample lysis regimens and DNA recovery strategies and define optimized manual and automated procedures for the extraction of high quality DNA suitable for molecular diagnostics and genotyping. Using a 3-step enzymatic digestion protocol carried out in the absence of dithiothreitol, we demonstrate that DNA can be efficiently released from cells or tissues preserved in buffered formalin or the alcohol-based fixative GenoFix. This preparatory procedure can then be integrated to traditional phenol/chloroform extraction, a modified manual DNA IQ or automated DNA IQ/Te-Shake-based extraction in order to recover DNA for downstream applications. Quantitative recovery of high quality DNA was best achieved from specimens archived in GenoFix and extracted using magnetic bead capture.

AmpFlSTR Profiler Plus and AmpFlSTR COfiler analysis of tissues stored in GenoFix, a new tissue preservation solution for mass disaster DNA identification.

A preliminary study was conducted to assess the capability of a new alcohol-based tissue fixative, GenoFix, to preserve DNA from biopsy tissues stored at room temperature and/or -20 degrees C in a freezer, for subsequent short tandem repeat (STR) DNA typing analysis. Fresh human smooth muscle samples were stored at room temperature in GenoFix for one month and up to one year and seven months before being processed using the megaplex STR systems, AmpFlSTR Profiler Plus and AmpFlSTR COfiler. Alternatively, muscle tissues in GenoFix were placed at -20 degrees C in a freezer for up to 3 1/2 years following two to three months in the fixative at room temperature. DNA analysis was also carried out on tissues stored in GenoFix for one month at room temperature and subsequently paraffin-embedded and stored at room temperature for four years. The AmpFlSTR Profiler Plus and AmpFlSTR COfiler STR profiles produced, using DNA extracted from all fixed tissue samples, were of very good quality. The fluorescent signals were well balanced across the nine STR loci or six loci comprised in the megaplexes surveyed and profiles showed no differences with those observed for the control blood of the respective donor patients. Continuous exposure to GenoFix at room temperature (up to one year and seven months) did not compromise the STR typing analysis of the fixed tissues. No adverse effects were noted on the STR typeability of tissues fixed with GenoFix and stored at -20 degrees C in a freezer for up to 3 1/2 years. STR profiles generated from the paraffin-embedded tissues fixed in GenoFix were of excellent quality. This preliminary study suggests that GenoFix can be used to store tissue samples at room temperature for up to one year and seven months or at -20 degrees C in a freezer for longer storage (up to 3 1/2 years). This new and odorless tissue fixative promotes tissue and DNA preservation in a very effective manner and as such may prove useful in criminal investigations or mass disaster identifications carried out in remote locations and in which a small or large number of tissue samples are collected for further analyses.