Tracey Dawson Cruz

STR profiles from DNA samples with "undetected" or low quantifiler results.

Abstract:  Screening methods capable of identifying DNA samples that will not yield short tandem repeat (STR) profiles are desired. In the past, quantitation methods have not been sensitive enough for this purpose. In this study, low level DNA samples were used to assess whether Quantifiler™ has a minimum quantitation value below which STR profiles would consistently fail to be detected. Buccal swabs were obtained and the DNA extracted, quantified, and serially diluted to concentrations ranging from 0.002 to 0.250 ng/μL. Samples were analyzed once with Quantifiler™, followed by Profiler Plus™ amplification and capillary electrophoresis analysis. An absolute minimum value below which STR results were unobtainable could not be defined. From the 96 low level samples tested, STR loci (including one full profile) were successfully amplified and detected from 27% of the samples “undetected” by Quantifiler™. However, no STR alleles were detected in 73% of these “undetected” samples, indicating that Quantifiler™ data may be useful for predicting STR typing success.

Characterization of human DNA in environmental samples

Environmental samples from indoor surfaces can be confounded by dust, which is composed largely of human skin cells and has been documented to contain roughly tens of micrograms of total DNA per gram of dust. This study complements previous published work by providing estimates of the quantity of amplifiable human DNA found in environmental samples from a typical indoor environment, categorized by the intensity of human traffic and visible quantity of dust. Dust was collected by surface swabbing standard 576 cm(2) areas in eight locations, and evaluated for total DNA quantity, presence of human DNA (mitochondrial and nuclear loci using conventional PCR), quantity of human nuclear DNA using quantitative PCR, and STR analysis. The total DNA content of 36 dust samples ranged from 9 to 28 ng/cm(2), and contained 0.2-1.1 pg/cm(2) of human DNA. Overall, human DNA was detected in 97% of 36 dust samples and 61% of samples yielded allele distributions of varying degrees of complexity when subjected to STR analysis. The implications of this study are twofold. First, the presence of dust in evidence can be a significant contamination source in forensic investigations because the human DNA component is of sufficient quality and quantity to produce allele calls in STR analysis. This can be effectively managed by implementing stringent protocols for collection and analysis of potential biological samples. A second implication is the use of dust as a source of evidence for identification of inhabitants within a defined location. In the latter case, a number of additional studies would be necessary to identify relevant pretreatments for environmental dust samples and to develop the necessary deconvolution techniques to separate the composite genotypes obtained.

Qiagen's Investigator™ Quantiplex Kit as a Predictor of STR Amplification Success from Low‐Yield DNA Samples,

Qiagens Investigator™ Quantiplex kit, a total human DNA quantitation kit, has a 200‐base pair internal control, fast cycling time, and scorpion molecules containing a covalently linked primer, probe, fluorophore, and quencher. The Investigator™ Quantiplex kit was evaluated to investigate a value under which complete short tandem repeat (STR) failure was consistently obtained. Buccal swabs were extracted using the Qiagen QIAamp® DNA Blood Mini Kit, quantified with the Investigator™ Quantiplex kit using a tested half‐volume reaction, amplified with the ABI AmpFlSTR® Identifiler kit, separated on the 3100Avant Genetic Analyzer, and data analyzed with GeneMapper® ID v.3.2. While undetected samples were unlikely to produce sufficient data for statistical calculations or CODIS upload (2.00 alleles and 0.82 complete loci on average), data may be useful for exclusionary purposes. Thus, the Investigator™ Quantiplex kit may be useful for predicting STR success. These findings are comparable with previously reported data from the Quantifiler™ Human kit.

Exploring the utility of genetic markers for predicting biological age

DNA evidence can be analyzed for genetic markers to determine phenotypes such as hair and eye color, ancestry, and even age estimation. Currently, telomere length is the only genetic biomarker that has been correlated to cell replication and replicative cell senescence--both strong indicators of tissue aging in humans. Unfortunately, while many studies have found a strong correlation between telomere length and age, many data sets show extreme variability, technical assay malfunction, inadequate evaluation of other variables that can impact telomere, altogether conflicting results, or insignificant correlations due to low sample size. Other, non-telomere based methods are problematic, as they often have only the ability to identify newborns or are only viable for specific tissue or cell types, and for most, the effects of outside variables have not been fully evaluated. Thus, telomeres remain the most promising biomarker for age estimation; mechanisms for telomere repeat attrition over time have been well documented. Unfortunately, assays currently used determine mean telomere length of a sample, are not precise or reproducible. New techniques should be robust enough to determine age across a broad spectrum of age ranges, and the effect of other variables (gender, race, disease, etc.), must be explored.

LPS-stimulated tumor necrosis factor-alpha and interleukin-6 mRNA and cytokine responses following acute psychological stress

The purpose of this study was to examine the effect of acute psychological stress on LPS-stimulated TNF-α and IL-6 mRNA expression. Twenty-one healthy male subjects participated in 20 min of acute stress. Blood samples for norepinephrine and LPS-stimulated TNF-α and IL-6 cytokines and mRNA were drawn prior to, immediately after and 1-h after stress. Stress-induced increases in anxiety scores, cortisol, plasma norepinephrine, and heart rate demonstrated that the experimental protocol elicited an acute stress response. LPS-stimulated TNF-α mRNA decreased significantly immediately post-stress and partially recovered at 1h post-stress, whereas LPS-stimulated IL-6 mRNA exhibited a significant change across time, with an increase immediately after stress and a decrease 1h after stress. Trends in LPS-stimulated TNF-α and IL-6 cytokine concentrations followed the patterns of mRNA expression. A negative correlation of body mass index (BMI) and percent change of LPS-stimulated TNF-α mRNA was observed immediately post-stress, and BMI positively correlated with percent change of LPS-stimulated IL-6 cytokine levels immediately following stress. These findings demonstrated that acute psychological stress affects LPS-stimulated IL-6 and TNF-α gene expression. These results also indicate that BMI may impact the effects of psychological stress on cytokine responses to immune challenge. Further examination of the effects of stress on synthesis of other cellular cytokines and investigation of the association of BMI and stress responses will provide a more clear representation of the cytokine responses to acute psychological stress. In addition, studies examining the influence of gender on the response of immune cell subsets to acute stress and the possible mediating effect of BMI are warranted.

dcDegenerate oligonucleotide primed-PCR for multilocus, genome-wide analysis from limited quantities of DNA.

This study modified the degenerate oligonucleotide primed-polymerase chain reaction (DOP-PCR)-based whole genome amplification method for improvement of downstream genome-wide analysis of low copy number DNA samples (≤0.100 ng). Experiments involved altering the degeneracy of the DOP primer, nonspecific cycle number, and adding proofreading polymerases. Increasing the degeneracy of the primer and the number of cycles that use a low annealing temperature should improve the nonspecific amplification of the DOP-PCR reaction. The addition of proofreading enzymes should allow for longer amplification products, increasing the genome coverage of the reaction. Low-input DNA quantities were examined for the primer and the cycle number studies using standard DOP-PCR parameters. The optimized DOP-PCR technique was then implemented for the polymerase study. All DOP-PCR products were amplified by using a multiplex microsatellite amplification kit to evaluate products from multiple chromosomes, followed by separation and detection by capillary electrophoresis. The 10 N primer, 12 nonspecific cycles, and the addition of the DeepVent proofreading enzyme all significantly increased the number of short tandem repeat alleles successfully amplified. All modifications also lowered the rate of allele drop-in, or sporadic additional allele occurrence, when compared with DOP-PCR results published earlier. Further, an average of >0.50 intralocus heterozygote peak ratios were observed for most DNA input quantities examined. These results show that modifications of the traditional DOP-PCR reaction (dcDOP-PCR) to include the use of a more degenerate primer (10 N), 12 nonspecific cycles, and a proofreading enzyme allows for a more complete, balanced chromosome amplification from limited and/or compromised clinical and biological samples.

Separation of uncompromised whole blood mixtures for single source STR profiling using fluorescently-labeled human leukocyte antigen (HLA) probes and fluorescence activated cell sorting (FACS)

Analysis of biological mixtures is a significant problem for forensic laboratories, particularly when the mixture contains only one cell type. Contributions from multiple individuals to biologic evidence can complicate DNA profile interpretation and often lead to a reduction in the probative value of DNA evidence or worse, its total loss. To address this, we have utilized an analytical technique that exploits the intrinsic immunological variation among individuals to physically separate cells from different sources in a mixture prior to DNA profiling. Specifically, we applied a fluorescently labeled antibody probe to selectively bind to one contributor in a mixture through allele-specific interactions with human leukocyte antigen (HLA) proteins that are expressed on the surfaces of most nucleated cells. Once the contributors cells were bound to the probe, they were isolated from the mixture using fluorescence activated cell sorting (FACS)-a high throughput technique for separating cell populations based on their optical properties-and then subjected to STR analysis. We tested this approach on two-person and four-person whole blood mixtures where one contributor possessed an HLA allele (A*02) that was not shared by other contributors to the mixture. Results showed that hybridization of the mixture with a fluorescently-labeled antibody probe complimentary to the A*02 alleles protein product created a cell population with a distinct optical profile that could be easily differentiated from other cells in the mixture. After sorting the cells with FACS, genetic analysis showed that the STR profile of this cell population was consistent with that of the contributor who possessed the A*02 allele. Minor peaks from the A*02 negative contributor(s) were observed but could be easily distinguished from the profile generated from A*02 positive cells. Overall, this indicates that HLA antibody probes coupled to FACS may be an effective approach for generating STR profiles of individual contributors from forensic mixtures.

A Case of Amelogenin Y-null: A simple primer binding site mutation or unusual genetic anomaly?

A thirteen year old boy was murdered by a gunshot wound to the head. In order to confirm identity of the boy, samples were sent to the Instituto de Ciencias Forenses de Puerto Rico (PR-ICF) DNA laboratory. Autosomal DNA results exhibited only an X at the Amelogenin locus, whereas the autopsy results reported the child to be anatomically male. The sample was amplified with four separate YSTR marker systems. While a full Y-STR profile for the father of the boy was obtained, the boy only amplified at STR markers on the p arm of the Y chromosome. Theories that could account for this large absence of Y-STR results include an X-Y translocation or Yp isochromosome.

Specific melanin content in human hairs and mitochondrial DNA typing success.

This study investigated whether a difference exists in the ability to obtain quality mitochondrial DNA (mtDNA) sequence data from hair shafts due to specific melanin content differences. Eumelanin, the pigment in darker hairs, protects nuclear DNA in the skin by absorbing and scattering UV radiation. In contrast, sensitized pheomelanin, the predominate melanin in red hairs and some blond hairs, is unable to prevent DNA damage in skin upon exposure to UV radiation. It has been reported in the literature that darker hairs (predominate eumelanin content) have a higher mtDNA sequencing success rate than lighter colored hairs. However, others have reported to the contrary when different methodologies are used. In this study, 2-cm hair fragments were cut from dark brown, red, and gray white hairs and typed using standard casework mtDNA sequence analysis methods. All 30 hair fragments produced quality mtDNA sequence data on first attempt from the second half of hypervariable region 1. These results are likely due to the apparent shielding of mtDNA by the hard protein of the hair shaft fiber from radiation-induced damage, regardless of melanin type, after 10-months minimal solar exposure. Nonetheless, this study may serve as a guide for future quantitative studies that investigate hair mtDNA photodamage in circumstances of increased solar, chemical, environmental, or mechanical damage.

An Optimized DNA Analysis Workflow for the Sampling, Extraction, and Concentration of DNA obtained from Archived Latent Fingerprints

DNA profiles have been obtained from fingerprints, but there is limited knowledge regarding DNA analysis from archived latent fingerprints—touch DNA “sandwiched” between adhesive and paper. Thus, this study sought to comparatively analyze a variety of collection and analytical methods in an effort to seek an optimized workflow for this specific sample type. Untreated and treated archived latent fingerprints were utilized to compare different biological sampling techniques, swab diluents, DNA extraction systems, DNA concentration practices, and post‐amplification purification methods. Archived latent fingerprints disassembled and sampled via direct cutting, followed by DNA extracted using the QIAamp® DNA Investigator Kit, and concentration with Centri‐Sep™ columns increased the odds of obtaining an STR profile. Using the recommended DNA workflow, 9 of the 10 samples provided STR profiles, which included 7–100% of the expected STR alleles and two full profiles. Thus, with carefully selected procedures, archived latent fingerprints can be a viable DNA source for criminal investigations including cold/postconviction cases.