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Hanson E.K.,National Center for Forensic Science | Ballantyne J.,National Center for Forensic Science | Ballantyne J.,University of Central Florida
Methods in Molecular Biology | Year: 2013

In forensic casework analysis it is sometimes necessary to obtain genetic profiles from increasingly smaller amounts of biological material left behind by persons involved in criminal offenses. The ability to obtain profiles from trace biological evidence is routinely demonstrated with the so-called touch DNA evidence (generally perceived to be the result of DNA obtained from shed skin cells transferred from donor to an object or a person during physical contact). The current method of recovery of trace DNA employs cotton swabs or adhesive tape to sample an area of interest. While of practical utility, such a "blind-swabbing" approach will necessarily co-sample cellular material from the different individuals whose cells are present on the item, even if the individuals' cells are located in geographically distinct locations on the item. Thus some of the DNA mixtures encountered in such touch DNA samples are artificially created by the swabbing itself. Therefore, a specialized approach for the isolation of single or few cells from "touch DNA evidence" is necessary in order to improve the analysis and interpretation of profiles recovered from these samples. Here, we describe an optimized and efficient removal strategy for the collection of cellular microparticles present in "touch DNA" samples, as well as enhanced amplification strategies to permit the recovery of short tandem repeat profiles of the donor(s) of the recovered microparticles. © 2013 Springer Science+Business Media, New York.


Hanson E.K.,National Center for Forensic Science | Ballantyne J.,National Center for Forensic Science | Ballantyne J.,University of Central Florida
Science and Justice | Year: 2013

The inability to definitively determine the tissue source of origin of forensically relevant biological fluids could result in a failure to provide crucial information necessary to the investigation and prosecution of the case. For example, in instances of sexual assault with a foreign object or digital penetration, the identification of vaginal secretions (VS) transferred to such objects or the perpetrators might be critical in establishing the circumstances of the assault. Conventional serological and immunological methods for body fluid identification can confirm the presence of human blood and semen. However, currently none of the routinely used biochemical tests can definitively identify the presence of human saliva or VS. It has been demonstrated that mRNA (or miRNA) profiling of body fluid stains can provide a degree of identification specificity of tissue and body fluids heretofore unattainable by conventional means. Early promising VS candidate RNA biomarkers, however, failed to exhibit the required degree of specificity or sensitivity and thus, at present, it is not possible for the forensic scientist to definitively identify VS using molecular genetics techniques.The aim of this work was to find novel highly specific RNA biomarkers for the identification of VS. Whole transcriptome profiling (RNA-Seq) of vaginal swab samples from different donors resulted in the identification of a number of putative VS-specific mRNA candidates. After detailed evaluation of >200 candidates from the tens of thousands of mRNA species found in VS, six promising candidates were identified. From these, two gene transcripts, namely CYP2B7P1 and MYOZ1, consistently demonstrated high specificity and sensitivity for VS when used in a qualitative capillary electrophoresis-based assay. Importantly these two biomarkers are able to differentiate between VS and other body fluids containing significant numbers of epithelia, particularly saliva and skin. Significantly, CYP2B7P1 is exceedingly specific with no detectable cross reactivity with other forensically relevant body fluids/tissues noted to date. The other four putatively specific biomarkers are expressed at higher levels in VS compared with saliva and will be more suitable for use with a quantitative (i.e. qRT-PCR) assay format. © 2012 Forensic Science Society.


Hanson E.K.,National Center for Forensic Science | Ballantyne J.,National Center for Forensic Science | Ballantyne J.,University of Central Florida
Methods in Molecular Biology | Year: 2013

The development of molecular genetics-based body fluid identification methods in forensic science has become necessary to provide greater sensitivity and specificity than that obtained using conventional serological and immunological methods. Numerous studies have demonstrated the ability to identify the body fluid origin of forensically relevant biological stains using messenger RNA expression analysis. However, the length of the amplified products used in these assays may not be ideal for use with highly degraded or environmentally compromised forensic casework samples. Therefore a novel approach to body fluid identification using small RNA profiling (e.g., microRNA or miRNA profiling) was developed in an attempt to improve the success of analysis with highly degraded samples. We have identified a set of nine differentially expressed miRNAs that permit the identification of the body fluid origin of forensic biological stains and in this chapter provide the detailed procedures for performing these assays. © Springer Science+Business Media New York 2013.


The ability to determine the time since deposition of a bloodstain found at a crime scene could prove invaluable to law enforcement investigators, defining the time frame in which the individual depositing the evidence was present. Although various methods of accomplishing this have been proposed, none has gained widespread use due to poor time resolution and weak age correlation. We have developed a method for the estimation of the time since deposition (TSD) of dried bloodstains using UV-VIS spectrophotometric analysis of hemoglobin (Hb) that is based upon its characteristic oxidation chemistry. A detailed study of the Hb Soret band (λmax=412 nm) in aged bloodstains revealed a blue shift (shift to shorter wavelength) as the age of the stain increases. The extent of this shift permits, for the first time, a distinction to be made between bloodstains that were deposited minutes, hours, days and weeks prior to recovery and analysis. The extent of the blue shift was found to be a function of ambient relative humidity and temperature. The method is extremely sensitive, requiring as little as a 1 μl dried bloodstain for analysis. We demonstrate that it might be possible to perform TSD measurements at the crime scene using a portable low-sample-volume spectrophotometer.


Hanson E.K.,National Center for Forensic Science | Ballantyne J.,National Center for Forensic Science | Ballantyne J.,University of Central Florida
PLoS ONE | Year: 2010

The ability to determine the time since deposition of a bloodstain found at a crime scene could prove invaluable to law enforcement investigators, defining the time frame in which the individual depositing the evidence was present. Although various methods of accomplishing this have been proposed, none has gained widespread use due to poor time resolution and weak age correlation. We have developed a method for the estimation of the time since deposition (TSD) of dried bloodstains using UV-VIS spectrophotometric analysis of hemoglobin (Hb) that is based upon its characteristic oxidation chemistry. A detailed study of the Hb Soret band (λmax = 412 nm) in aged bloodstains revealed a blue shift (shift to shorter wavelength) as the age of the stain increases. The extent of this shift permits, for the first time, a distinction to be made between bloodstains that were deposited minutes, hours, days and weeks prior to recovery and analysis. The extent of the blue shift was found to be a function of ambient relative humidity and temperature. The method is extremely sensitive, requiring as little as a 1 μl dried bloodstain for analysis. We demonstrate that it might be possible to perform TSD measurements at the crime scene using a portable low-sample-volume spectrophotometer. © 2010 Hanson, Ballantyne.


Gerasimova Y.V.,University of Central Florida | Hayson A.,University of Central Florida | Ballantyne J.,University of Central Florida | Ballantyne J.,National Center for Forensic Science | Kolpashchikov D.M.,University of Central Florida
ChemBioChem | Year: 2010

Molecular beacon (MB) probes are dual-labeled hairpin-shaped oligodeoxyribonucleotides that are extensively used for real-time detection of specific RNA/DNA analytes. In the MB probe, the loop fragment is complementary to the analyte: therefore, a unique probe is required for the analysis of each new analyte sequence. The conjugation of an oligonucleotide with two dyes and subsequent purification procedures add to the cost of MB probes, thus reducing their application in multiplex formats. Here we demonstrate how one MB probe can be used for the analysis of an arbitrary nucleic acid. The approach takes advantage of two oligonucleotide adaptor strands, each of which contains a fragment complementary to the analyte and a fragment complementary to an MB probe. The presence of the analyte leads to association of MB probe and the two DNA strands in quadripartite complex. The MB probe fluorescently reports the formation of this complex. In this design, the MB does not bind the analyte directly; therefore, the MB sequence is independent of the analyte. In this study one universal MB probe was used to genotype three human polymorphic sites. This approach promises to reduce the cost of multiplex real-time assays and improve the accuracy of single-nucleotide polymorphism genotyping. © 2010 Wiley-VCH Verlag GmbH & Co. KGaA.


Ballantyne J.,National Center for Forensic Science | Ballantyne J.,University of Central Florida | Hanson E.K.,National Center for Forensic Science | Perlin M.W.,Cybergenetics
Science and Justice | Year: 2013

Two person DNA admixtures are frequently encountered in criminal cases and their interpretation can be challenging, particularly if the amount of DNA contributed by both individuals is approximately equal. Due to an inevitable degree of uncertainty in the constituent genotypes, reduced statistical weight is given to the mixture evidence compared to that expected from the constituent single source contributors. The ultimate goal of mixture analysis, then, is to precisely discern the constituent genotypes and here we posit a novel strategy to accomplish this. We hypothesised that LCM-mediated isolation of multiple groups of cells ('binomial sampling') from the admixture would create separate cell sub-populations with differing constituent weight ratios. Furthermore we predicted that interpreting the resulting DNA profiling data by the quantitative computer-based TrueAllele® interpretation system would result in an efficient recovery of the constituent genotypes due to newfound abilities to compute a maximum LR from sub-samples with skewed weight ratios, and to jointly interpret all possible pairings of sub-samples using a joint likelihood function. As a proof of concept, 10 separate cell samplings of size 20 recovered by LCM from each of two 1:1 buccal cell mixtures were DNA-STR profiled using a specifically developed LCN methodology, with the data analyzed by the TrueAllele® Casework system. In accordance with the binomial sampling hypothesis, the sub-samples exhibited weight ratios that were well dispersed from the 50% center value (50 ± 35% at the 95% level). The maximum log(LR) information for a genotype inferred from a single 20 cell sample was 18.5 ban, with an average log(LR) information of 11.7 ban. Co-inferring genotypes using a joint likelihood function with two sub-samples essentially recovered the full genotype information. We demonstrate that a similar gain in genotype information can be obtained with standard (28-cycle) PCR conditions using the same joint interpretation methods. Finally, we discuss the implications of this work for routine forensic practice. © 2012 Forensic Science Society.


Hanson E.,National Center for Forensic Science | Haas C.,University of Zürich | Jucker R.,University of Zürich | Ballantyne J.,National Center for Forensic Science | Ballantyne J.,University of Central Florida
Forensic Science International: Genetics | Year: 2012

In forensic casework analysis it is often necessary to attempt to obtain DNA profiles from microscopic amounts of biological material left behind by perpetrators of crime. The ability to obtain profiles from trace biological evidence is routinely demonstrated with so-called 'touch DNA' evidence, which is generally perceived to be the result of DNA obtained from shed skin cells transferred from donor to an object or person during physical contact. Although a genetic profile from trace biological evidence is routinely obtained, the tissue source of the profile is rarely known. This merely perpetuates the 'mystery' of the nature of 'touch DNA' evidence allowing the significance or meaningfulness of genetic profiles obtained from these samples to be challenged. Numerous reports state that the tissue source of origin of 'touch DNA' evidence cannot be determined due to the small amount of biological material present, while others conclude that the DNA profiles are obtained from shed skin cells (as opposed to, say, buccal epithelial cells present in saliva traces) without any scientific basis for this assertion. Proper identification of the biological material present might be crucial to the investigation and prosecution of a criminal offense and a misrepresentation of the nature of the evidence can have undue influence on the perception of the circumstance of the crime. Thus far, research has failed to provide forensic scientists with feasible, definitive methods to identify the tissue origin of 'touch DNA'. In the present work, we sought to identify novel highly specific and sensitive messenger RNA (mRNA) biomarkers for the identification of skin. Gene candidates were identified using both literature searches and whole transcriptome deep sequencing (RNA-Seq). Utilizing this dual approach, we identified and evaluated over 100 gene candidates. Five mRNA markers were identified that demonstrated a high degree of specificity for skin. Using these markers, we have been able to successfully detect and identify skin using as little as 5-25 pg of input total RNA from skin and, significantly, in swabs of human skin and various touched objects. One of the markers, LCE1C, is particularly highly sensitive and was detected in the majority of skin samples tested including touched objects. We have been successful in incorporating the five skin biomarkers into two multiplex systems. Although further work is needed to optimize the assay for routine casework, the initial studies demonstrate that a molecular-based characterization of the biological material recovered from touch samples is possible. © 2012 Elsevier Ireland Ltd. All rights reserved.


Marrone A.,University of Central Florida | Marrone A.,National Center for Forensic Science | Ballantyne J.,University of Central Florida | Ballantyne J.,National Center for Forensic Science
Forensic Science International: Genetics | Year: 2010

The biochemistry of dry state DNA is of interest to the field of forensic biology. The precise chemical nature of the hydrolytic degradation products of the DNA molecule in the dry state has not been previously investigated in detail. In this study we found that the mechanistic chemistry of DNA hydrolysis appears to be the same for the hydrated and dry states. The thermodynamic parameters are also similar in both states and the activation energies for base hydrolysis are indistinguishable. The principal difference between the two states is the rate at which hydrolytic degradation occurs. The duplex configuration of dry state DNA offers much more protection for the molecule than is offered in single strand and nucleotide species. Single strand breaks of dry state duplex DNA occur with a half life of 24 ± 2 days at 65 °C and appear to occur in a mechanistic manner. © 2009 Elsevier Ireland Ltd. All rights reserved.


Haas C.,University of Zürich | Hanson E.,National Center for Forensic Science | Kratzer A.,University of Zürich | Bar W.,University of Zürich | And 2 more authors.
Forensic Science International: Genetics | Year: 2011

In the present work, we have evaluated eight reportedly blood-specific mRNA markers (HBB, HBA, ALAS2, CD3G, ANK1, PBGD, SPTB, AQP9) in an attempt to determine the most suitable ones for use in forensic applications based on their sensitivities, specificities and performance with casework samples. While varying levels of expression were observed, all markers were relatively sensitive requiring as little as 1 ng of RNA input into the reverse transcription (RT) reaction. In singleplex reactions, seven of the eight analyzed blood markers (all except AQP9) demonstrated a high degree of specificity for blood. In multiplex reactions, non-reproducible cross-reactivity was observed for several of the mRNA markers, which was reduced and, in most cases, eliminated when less input total RNA was used. Additionally, some cross-reactivity was observed with tissue and animal samples. Despite differences in the observed sensitivity and specificity of the blood markers examined in this study, a number of the candidates appear to be suitable for inclusion in appropriately validated multiplex mRNA-based body fluid identification systems. © 2010 Elsevier Ireland Ltd.

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