Tracey Cruz - Academia.edu (original) (raw)

Papers by Tracey Cruz

Journal of Laboratory Automation, Sep 11, 2020

Thermo Fisher Scientific released the Precision ID Ancestry Panel, a 165-single-nucleotide polymo... more Thermo Fisher Scientific released the Precision ID Ancestry Panel, a 165-single-nucleotide polymorphism (SNP) panel for ancestry prediction that was initially compatible with the manufacturer's massively parallel sequencer, the Ion Torrent Personal Genome Machine (PGM). The semiautomated workflow using the panel with the PGM involved several timeconsuming manual steps across three instruments, including making templating solutions and loading sequencing chips. In 2014, the manufacturer released the Ion Chef robot, followed by the Ion S5 massively parallel sequencer in late 2015. The robot performs the templating with reagent cartridges and loads the chips, thus creating a fully automated workflow across two instruments. The objective of the work reported here is to compare the performance of two massively parallel sequencing systems and ascertain if the change in the workflow produces different ancestry predictions. For performance comparison of the two systems, forensic-type samples (n = 16) were used to make libraries. Libraries were templated either with the Ion OneTouch 2 system (for the PGM) or on the Ion Chef robot (for the S5). Sequencing results indicated that the ion sphere particle performance metrics were similar for the two systems. The total coverages per SNP and SNP quality were both higher for the S5 system. Ancestry predictions were concordant for the mock forensic-type samples sequenced on both massively parallel sequencing systems. The results indicated that automating the workflow with the Ion Chef system reduced the labor involved and increased the sequencing quality.

Humana Press eBooks, 2006

... Table 2 Commercially Available Fluorescent STR Multiplexes Average power of Name Vendor discr... more ... Table 2 Commercially Available Fluorescent STR Multiplexes Average power of Name Vendor discriminationa STR loci included FFFL Promega Corporation 1 : 2700 F13A1, FES/FPS, F13B, LPL AmpF1STR® Blue Applied Biosystems 1 : 5000 D3S1358, VWA, FGA ...

Low copy number (LCN) DNA evidence (≤100pg) can become difficult to analyze with traditional STR ... more Low copy number (LCN) DNA evidence (≤100pg) can become difficult to analyze with traditional STR analysis due to allele drop-out and increased stochastic effects. To overcome these limitations, whole genome amplification (WGA) has been investigated. Degenerate oligonucleotide-primed PCR (DOP-PCR), one form of WGA, uses a partially degenerate primer and a low annealing temperature (30ºC) to generate non-specific DNA fragments from throughout the genome. Data from preliminary studies using the DOP-PCR pre-amplification technique showed small increases in STR allele amplification; however, stochastic issues affecting data interpretation were prevalent. Thus, a modified DOP-PCR technique has been developed, dcDOP-PCR. Experiments included varying the number of non-specific cycles in the initial phase of the DOP-PCR reaction, as well as altering the degeneracy of the DOP-PCR primer and adding proofreading polymerases to the reaction mixture. All samples were DOP-PCR amplified using 3, 4, 7, 9, 12, and 15 cycles during the initial non-specific amplification round as well as using both a 10N and 16N degenerate primer. Additionally, several proofreading enzyme combinations, including Taq:Pfu, Taq:Deep Vent, Taq:Tgo, Platinum Pfx, and ABI GeneAmp High Fidelity (TaqGold and an unknown proprietary enzyme(s)) were evaluated. Data generated under these experimental conditions were compared to data collected using the standard, previously described DOP-PCR approach which includes a 6N degenerate primer, 5 non-specific cycles, and Taq polymerase only. Serially-diluted, QIAamp-extracted DNA samples ranging from 0.25ng down to 7.8pg were evaluated for all initial studies. All DOP-PCR products were amplified with the AmpFlSTR ® Profiler Plus ® STR kit followed by separation and detection by CE (ABI 3100Avant). The 10N degenerate primer, 12 non-specific cycles, and the addition of DeepVent proofreading enzyme in the DOP-PCR reaction all significantly increased the number of alleles successfully amplified and detected. Further, these modifications, when combined, lowered the rate of sporadic additional allele occurrence (dropin), when compared to the previously published DOP-PCR results. Additionally, intra-locus heterozygote peak ratios were consistently ≥0.6 for most low copy number DNA samples examined. These results show that the modifications incorporated into the DOP-PCR technique allow for a more complete, balanced STR amplification from low-level DNA samples. However, in order to fully evaluate the utility of this newly described technique (dcDOP-PCR), the method was used to pre-amplify DNA from mock and non-probative casework samples, including aged and environmentally-exposed bloodstains, bones, teeth, hair shafts, dermal ridge fingerprints, and fired cartridge cases. The dcDOP-PCR method significantly improved STR allele success when compared to traditional STR analysis (without pre-amplification), producing strong partial or full profiles in many cases where little to no STR data was previously obtained. Further, dcDOP-PCR data quality was generally equivalent or superior to traditional STR analysis. This method will provide the forensic DNA community with a relatively easy, inexpensive alternative for analyzing compromised and/or low copy number DNA evidence.

Journal of Leukocyte Biology, Sep 9, 2009

Activation of the high-affinity receptor for IgE, FcRI, is known to elicit its rapid down-regulat... more Activation of the high-affinity receptor for IgE, FcRI, is known to elicit its rapid down-regulation through internalization and degradation. In keeping with this, expression of all three FcRI subunits is decreased at the protein level after cross-linkage of IgE with antigen. However, we find that the FcRI ␤-subunit is also selectively suppressed at the mRNA level, through a pathway primarily involving Fyn, Syk, PI3K, and NF-B. IgG or calcium ionophore, stimuli known to mimic portions of the IgE signaling cascade, similarly suppressed ␤-subunit expression. LPS, a NF-B-activating TLR ligand, did not alter ␤-subunit expression. As IgE increases FcRI expression, we examined the coordinated regulation of FcRI subunits during culture with IgE, followed by cross-linkage with antigen. IgE increased the expression of all three FcRI subunits and strikingly induced expression of the antagonistic ␤ T. The ratio of ␤:␤ T protein expression decreased significantly during culture with IgE and was reset to starting levels by antigen cross-linkage. These changes in protein levels were matched by similar fluctuations in ␤ and ␤ T mRNAs. FcRI␤ is a key regulator of IgER expression and function, a gene in which polymorphisms correlate with allergic disease prevalence. The ability of IgE and FcRI signaling to coordinate expression of the ␤ and ␤ T subunits may comprise a homeostatic feedback loop-one that could promote chronic inflammation and allergic disease if dysregulated.

Journal of Forensic Sciences, Sep 1, 2012

Journal of Forensic Sciences, Apr 6, 2017

DNA profiles have been obtained from fingerprints, but there is limited knowledge regarding DNA a... more 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 QIAampâ DNA Investigator Kit, and concentration with Centri-Sep TM 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.

Journal of Forensic Sciences, Jun 11, 2019

Evaluation of microRNA (miRNA) expression as a potential method for forensic body fluid identific... more Evaluation of microRNA (miRNA) expression as a potential method for forensic body fluid identification has been the subject of investigation over the past several years. Because of their size and encapsulation within proteins and lipids, miRNAs are inherently less susceptible to degradation than other RNAs. In this work, blood, urine, semen, and saliva were exposed to environmental and chemical conditions mimicking sample compromise at the crime scene. For many treated samples, including 100% of blood samples, miRNAs remained detectable, comparable to the untreated control. Sample degradation varied by body fluid and treatment, with blood remarkably resistant, while semen and saliva are more susceptible to environmental insult. Body fluid identification using relative miRNA expression of blood and semen of the exposed samples was 100% and 94%, respectively. Given the overall robust results herein, the case is strengthened for the use of miRNAs as a molecular method for body fluid identification.

F1000Research, Jan 27, 2016

Journal of Forensic Sciences, Apr 6, 2011

Journal of Forensic Sciences, Dec 7, 2020

DNA extractions of semen samples commonly utilize dithiothreitol (DTT) to reduce and disrupt disu... more DNA extractions of semen samples commonly utilize dithiothreitol (DTT) to reduce and disrupt disulfide bonds. Although traditional extraction techniques remove DTT before downstream analyses, the forensic DNA community has recently explored Y-screening, direct amplification, and direct cell lysis assays that omit purification but employ reducing agents to lyse spermatozoa. This study examined the impact of residual DTT on downstream processes involving fluorescent dyes. Quantification using Investigator ® Quantiplex HYres revealed a significant increase in the male DNA yield (p = 0.00056) and a >150,000,000-fold increase in the male:human DNA ratio when DTT remained in extracts versus when it was filtered out using a traditional purification method. When DTT was present with Quantifiler™ Trio, the true mean DNA yield for the large autosomal target significantly increased (p = 0.038) and the average reported DNA yields increased 1.1-fold, >9.5-fold, and 1.3-fold for the small autosomal, large autosomal, and male targets, respectively. DTT-spiked DNA standards from both kits were impacted similarly to samples with residual DTT, demonstrating that observed effects were related to DTT and not the extraction method. This study corroborates other reports that DTT adversely affects multiple dyes (e.g., Cy5, Quasar 670, SYBR Green I, TMR, and Mustang Purple ®). Overall, DTT causes inaccurate quantities and, consequently, inaccurate calculated male:female ratios when used in conjunction with these kits. Thus, implementation of newer direct-to-PCR assays incorporating DTT should either be avoided or used only with carefully evaluated, compatible dyes.

Electrophoresis, Oct 21, 2016

This work describes the development of a novel microdevice for forensic DNA processing of referen... more This work describes the development of a novel microdevice for forensic DNA processing of reference swabs. This microdevice incorporates an enzyme-based assay for DNA preparation, which allows for faster processing times and reduced sample handling. Infrared-mediated PCR (IR-PCR) is used for STR amplification using a custom reaction mixture, allowing for amplification of STR loci in 45 min while circumventing the limitations of traditional block thermocyclers. Uniquely positioned valves coupled with a simple rotational platform are used to exert fluidic control, eliminating the need for bulky external equipment. All microdevices were fabricated using laser ablation and thermal bonding of PMMA layers. Using this microdevice, the enzyme-mediated DNA liberation module produced DNA yields similar to or higher than those produced using the traditional (tube-based) protocol. Initial microdevice IR-PCR experiments to test the amplification module and reaction (using Phusion Flash/SpeedSTAR) generated near-full profiles that suffered from interlocus peak imbalance and poor adenylation (significant -A). However, subsequent attempts using KAPA 2G and Pfu Ultra polymerases generated full STR profiles with improved interlocus balance and the expected adenylated product. A fully integrated run designed to test microfluidic control successfully generated CE-ready STR amplicons in less than 2 h (&amp;amp;amp;amp;amp;lt;1 h of hands-on time). Using this approach, high-quality STR profiles were developed that were consistent with those produced using conventional DNA purification and STR amplification methods. This method is a smaller, more elegant solution than current microdevice methods and offers a cheaper, hands-free, closed-system alternative to traditional forensic methods.

Legal Medicine, Nov 1, 2012

A thirteen year old boy was murdered by a gunshot wound to the head. In order to confirm identity... more 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.

Forensic sciences research, Aug 20, 2019

Sexual assault evidence often contains sperm cells, which are typically separated from nonsperm c... more Sexual assault evidence often contains sperm cells, which are typically separated from nonsperm cells using manual differential lysis procedures. The goal of this study was to evaluate the automated QIAGEN QIAcube for this purpose and to compare it to manual QIAGEN and manual organic differential methods using DNA yields and STR profile data for assessment. DNA yields were determined by qPCR, followed by multiplex STR amplification, CE analysis, and mixture interpretation. The automated method was capable of effective cell separation, producing DNA yields sufficient for STR amplification. Further, sperm fraction human:male DNA ratios from the QIAcube samples were consistently closer to the desired 1:1 and STR profiles were less likely to result in mixtures, with 6-8Â fewer female alleles detected (median 1.5 alleles). Ultimately, using the QIAcube for automated differential processing of semen-containing mixtures reduces the need for downstream mixture interpretation and improves STR profile quality with substantially less hands-on time.

Psychoneuroendocrinology, Nov 1, 2011

Journal of Forensic Sciences, 2009

Screening methods capable of identifying DNA samples that will not yield short tandem repeat (STR... more 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 QuantifilerÔ 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 ⁄ lL. Samples were analyzed once with QuantifilerÔ, followed by Profiler PlusÔ 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 QuantifilerÔ. However, no STR alleles were detected in 73% of these ''undetected'' samples, indicating that QuantifilerÔ data may be useful for predicting STR typing success.

F1000Research, Nov 30, 2015

Mixture interpretation continues to be a challenge for the forensic community. While methods exis... more Mixture interpretation continues to be a challenge for the forensic community. While methods exist to separate cells from different contributors prior to DNA extraction, most can only be used on mixtures of different cell types. Yet, an increasing proportion of evidentiary samples submitted for casework are contact mixtures in which two or more individuals have contributed epidermal cells. Resolving these mixtures is more problematic due to the consistent morphology and size of epidermal cells across individuals. Protein biochemistry offers one promising avenue for differentiating contributors in a mixture since individuals may vary in the abundance of certain structural alleles within epidermal cells. The objective of this research was to survey biochemical profiles of contact epidermal cells across individuals by labeling them with allele-specific antibodies. Observed differences in the protein abundance and/or diversity would then be used to separate individual cell populations from the mixture using flow cytometry.

F1000Research, Nov 18, 2015

Medicine and Science in Sports and Exercise, May 1, 2010

Acute psychological stress alters physiological homeostasis and induces transient endothelial dys... more Acute psychological stress alters physiological homeostasis and induces transient endothelial dysfunction. One potential mechanism that links acute psychological stress to endothelial dysfunction is a change in circulating levels of pro-inflammatory cytokines such as TNF-a and IL-6, but the direct effects of psychological stress on pro-inflammatory cytokines are unclear. PURPOSE: The purpose of this study was to examine the effect of acute psychological stress on lipopolysaccharide (LPS)-stimulated TNF-a and IL-6 cytokines and mRNA expression. METHODS: Twenty one healthy male subjects participated in 20 minutes of acute stress. Blood samples for norepinephrine and LPS-stimulated TNF-a and IL-6 cytokines and mRNA were drawn prior to, immediately after and one-hour after stress. RESULTS: Stress-induced increases in anxiety scores, plasma norepinephrine, and heart rate confirmed the stress model. LPS-stimulated TNF-a mRNA decreased significantly immediately post-stress and partially recovered at one hour post-stress, whereas LPS-stimulated IL-6 mRNA exhibited a significant change across time, with an increase immediately after stress and a decrease one hour after stress. Although changes in LPS-stimulated TNF-a cytokine concentrations were not statistically significant, trends followed the pattern of TNF-a expression. Similarly, LPS-stimulated IL-6 cytokine levels followed the pattern of IL-6 mRNA, with a small but not significant increase immediately after stress and a significant decrease from post-stress to one hour of recovery. A negative correlation of BMI and percentage change of LPS-stimulated TNF-a mRNA was observed immediately post-stress, and BMI positively correlated with percentage change of LPS-stimulated IL-6 cytokine levels immediately following stress. CONCLUSION: These findings are the first direct evidence that acute psychological stress affects IL-6 and TNF-a gene expression. These results also indicate that BMI may play a role in alterations of TNF-a mRNA and IL-6 cytokine expression in response to acute stress. 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 complete representation of the cytokine responses to acute psychological stress.

American Journal of Forensic Medicine and Pathology, Jun 1, 2009

This study investigated whether a difference exists in the ability to obtain quality mitochondria... more 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.

PLOS ONE, Feb 7, 2019

A single focus optical tweezer is formed when a laser beam is launched through a high numerical a... more A single focus optical tweezer is formed when a laser beam is launched through a high numerical aperture immersion objective. This objective focuses the beam down to a diffraction-limited spot, which creates an optical trap where cells suspended in aqueous solutions can be held fixed. Spermatozoa, an often probative cell type in forensic investigations, can be captured inside this optical trap and dragged one by one across millimeter-length distances in order to create a cluster of cells which can be subsequently drawn up into a capillary for collection. Sperm cells are then ejected onto a sterile cover slip, counted, and transferred to a tube for DNA analysis workflow. The objective of this research was to optimize sperm cell collection for maximum DNA yield, and to determine the number of trapped sperm cells necessary to produce a full STR profile. A varying number of sperm cells from both a single-source semen sample and a mock sexual assault sample were isolated utilizing optical tweezers and processed using conventional STR analysis methods. Results demonstrated that approximately 50 trapped spermatozoa were required to obtain a consistently full DNA profile. A complete, single-source DNA profile was also achieved by isolating sperm cells via optical trapping from a mixture of sperm and vaginal epithelial cells. Based on these results, optical tweezers are a viable option for forensic applications such as separation of mixed populations of cells in forensic evidence.