RESUMO
HyBeacons are linear oligonucleotides which incorporate fluorescent dyes covalently linked to internal nucleotides. They have previously been used with PCR and isothermal amplification to interrogate SNPs and STRs in fields as diverse as clinical diagnostics, food authentication, and forensic DNA profiling. This work explores their use for the identification of expressed gene sequences through mRNA profiling. The use of mRNA is becoming increasingly common in forensic casework to identify body fluids on evidence items, as it offers higher specificity and fewer false positives than current chemical presumptive testing methods. The work presented here details the development of a single-step one-tube RT-PCR assay to detect the presence of body fluids of forensic interest (saliva, blood, seminal fluid, vaginal fluid and menstrual blood) using HyBeacon® probes and melt curve analysis. Each assay shows a high degree of specificity to the target body fluid mRNA suggesting there is no requirement to remove genomic DNA prior to analysis. Of the five assays developed, four were able to detect between 10 and 100 copies of target cDNA, the fifth 1000 copies of target. The results presented here demonstrate that such an approach can be optimised for non-expert users and further areas of work are discussed.
Assuntos
Líquidos Corporais/metabolismo , Modelos Biológicos , Sondas Moleculares/química , RNA Mensageiro/análise , Sequência de Bases , Biomarcadores/sangue , DNA/análise , Humanos , Polimorfismo de Nucleotídeo Único/genética , RNA Mensageiro/genética , Sensibilidade e Especificidade , Doadores de TecidosRESUMO
The ability to obtain DNA 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 a donor's hands to an object or person during direct physical contact. Current methods for the recovery of trace DNA employ swabs or adhesive tape to sample an area of interest. While of practical utility, such 'blind-swabbing' approaches will necessarily co-sample cellular material from the different individuals whose cells are present on the item, even though the individuals' cells are principally located in topographically dispersed, but distinct, locations on the item. Thus the act of swabbing itself artifactually creates some of the DNA mixtures encountered in touch DNA samples. In some instances involving transient contact between an assailant and victim, the victim's DNA may be found in such significant excess as to preclude the detection and typing of the perpetrator's DNA. In order to circumvent the challenges with standard recovery and analysis methods for touch DNA evidence, we reported previously the development of a 'smart analysis' single cell recovery and DNA analysis method that results in enhanced genetic analysis of touch DNA evidence. Here we use the smart single cell analysis method to recover probative single source profiles from individual and agglomerated cells from various touched objects and clothing items belonging to known donors. We then use the same approach for the detection of single source male donor DNA in simulated physical contact/assault mixture samples (i.e. male 'assailant' grabbing the wrist, neck or clothing from the female 'victim', or being in transient contact with bedding from the 'victim'). DNA profiles attributable to the male or female known donors were obtained from 31% and 35% of the single and agglomerated bio-particles (putative cells) tested. The known male donor 'assailant' DNA profile was identified in the cell sampling from every mixture type tested. The results of this work demonstrate the efficacy of an alternative strategy to recover single source perpetrator DNA profiles in physical contact/assault cases involving trace perpetrator/victim cellular admixtures.
Assuntos
Vestuário , Impressões Digitais de DNA/métodos , DNA/isolamento & purificação , Abuso Físico , Pele/química , Feminino , Humanos , Masculino , Micromanipulação , Repetições de Microssatélites , Pele/citologia , TatoRESUMO
We report the identification of sensitive and specific miRNA biomarkers for menstrual blood, a tissue that might provide probative information in certain specialized instances. We incorporated these biomarkers into qPCR assays and developed a quantitative statistical model using logistic regression that permits the prediction of menstrual blood in a forensic sample with a high, and measurable, degree of accuracy. Using the developed model, we achieved 100% accuracy in determining the body fluid of interest for a set of test samples (i.e. samples not used in model development). The development, and details, of the logistic regression model are described. Testing and evaluation of the finalized logistic regression modeled assay using a small number of samples was carried out to preliminarily estimate the limit of detection (LOD), specificity in admixed samples and expression of the menstrual blood miRNA biomarkers throughout the menstrual cycle (25-28 days). The LOD was <1 ng of total RNA, the assay performed as expected with admixed samples and menstrual blood was identified only during the menses phase of the female reproductive cycle in two donors.
Assuntos
Análise Química do Sangue/métodos , Líquidos Corporais/química , Genética Forense/métodos , Menstruação , MicroRNAs/genética , Feminino , Marcadores Genéticos , Humanos , Limite de Detecção , Modelos Logísticos , Reprodutibilidade dos TestesRESUMO
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.
Assuntos
Muco do Colo Uterino/química , RNA Mensageiro/metabolismo , Estupro , Vagina/química , Análise Química do Sangue , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Família 2 do Citocromo P450 , Feminino , Perfilação da Expressão Gênica , Marcadores Genéticos , Humanos , Masculino , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Saliva/química , Sêmen/químicaRESUMO
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.
Assuntos
Impressões Digitais de DNA/métodos , Lasers , Funções Verossimilhança , Técnicas de Cocultura , Genótipo , Humanos , Repetições de Microssatélites , Reação em Cadeia da Polimerase , SoftwareRESUMO
Recent studies have examined the influence on patterns of human genetic variation of a variety of cultural practices. In India, centuries-old marriage customs have introduced extensive social structuring into the contemporary population, potentially with significant consequences for genetic variation. Social stratification in India is evident as social classes that are defined by endogamous groups known as castes. Within a caste, there exist endogamous groups known as gols (marriage circles), each of which comprises a small number of exogamous gotra (lineages). Thus, while consanguinity is strictly avoided and some randomness in mate selection occurs within the gol, gene flow is limited with groups outside the gol. Gujarati Patels practice this form of "exogamic endogamy." We have analyzed genetic variation in one such group of Gujarati Patels, the Chha Gaam Patels (CGP), who comprise individuals from six villages. Population structure analysis of 1,200 autosomal loci offers support for the existence of distinctive multilocus genotypes in the CGP with respect to both non-Gujaratis and other Gujaratis, and indicates that CGP individuals are genetically very similar. Analysis of Y-chromosomal and mitochondrial haplotypes provides support for both patrilocal and patrilineal practices within the gol, and a low-level of female gene flow into the gol. Our study illustrates how the practice of gol endogamy has introduced fine-scale genetic structure into the population of India, and contributes more generally to an understanding of the way in which marriage practices affect patterns of genetic variation.
Assuntos
Cromossomos Humanos Y , Fluxo Gênico , Casamento/etnologia , Análise por Conglomerados , Evolução Molecular , Variação Genética , Genoma Mitocondrial/genética , Haplótipos , Humanos , Índia , Masculino , Modelos GenéticosRESUMO
The serology-based methods routinely used in forensic casework for the identification of biological fluids are costly in terms of time and sample and have varying degrees of sensitivity and specificity. Recently, the use of a molecular genetics-based approach using messenger RNA (mRNA) profiling has been proposed to supplant conventional methods for body fluid identification. However, the size of the amplification products used in these mRNA assays (approximately 200-300 nt) might not be ideal for use with degraded or compromised samples frequently encountered in forensic casework. Recently, there has been an explosion of interest in a novel class of small noncoding RNAs, microRNAs (miRNAs, approximately 20-25 bases in length), with numerous published studies reporting that some miRNAs are expressed in a tissue-specific manner. In this article, we provide the first comprehensive evaluation of miRNA expression in dried, forensically relevant biological fluids--blood, semen, saliva, vaginal secretions, and menstrual blood--in an attempt to identify putative body fluid-specific miRNAs. Most of the 452 human miRNAs tested (approximately 67% of the known miRNAome) were either expressed in multiple body fluids or not expressed at all. Nevertheless, we have identified a panel of nine miRNAs--miR451, miR16, miR135b, miR10b, miR658, miR205, miR124a, miR372, and miR412--that are differentially expressed to such a degree as to permit the identification of the body fluid origin of forensic biological stains using as little as 50 pg of total RNA. The miRNA-based body fluid identification assays were highly specific because the miRNA expression profile for each body fluid was different from that obtained from 21 human tissues. The results of this study provide an initial indication that miRNA profiling may provide a promising alternative approach to body fluid identification for forensic casework.
Assuntos
Líquidos Corporais/química , Medicina Legal , MicroRNAs/análise , Feminino , Humanos , Menstruação , MicroRNAs/sangue , Reação em Cadeia da Polimerase , Saliva/química , Sêmen/química , Vagina/químicaRESUMO
Identifying the biological origin of forensic traces can provide crucial evidence to aid criminal investigations. Current forensic practice for the identification of body fluids mostly uses protein-based presumptive tests. Such tests cannot identify all of the forensically relevant fluids and have issues of cross-reactivity. More recently, messenger RNA methods have been developed that have expanded the range of body fluids that can be positively identified. However, these methods are slow and require expert scientists to run the processes and interpret the results. The ParaDNA® Body Fluid ID System has been designed to provide a simple, fast and robust way to identify forensically relevant body fluids in a lab or field-deployable manner. The system can analyse and detect mRNA targets for six different body fluids: vaginal fluid, seminal fluid, sperm cells, saliva, menstrual blood and peripheral blood. Utilising the ParaDNA Sample Collector and instruments, minimal training is required to enable both forensic scientists and non-specialist personnel to analyse biological samples directly, without prior sample processing, in approximately 90 min. The developmental validation studies described here were designed to address requirements of end users, based on the Scientific Working Group on DNA Analysis Methods (SWGDAM) guidelines, and tested the sensitivity, reproducibility, accuracy, inhibitor tolerance, and performance of the ParaDNA Body Fluid ID System on a range of mock evidence items. The data collected demonstrate that the Body Fluid ID System can automatically determine the presence of specific body fluid mRNA markers in single-source and mixed samples on multiple substrate types and body fluids could be identified with as little as 0.05ng total RNA and 1µl of the relevant fluid. Results can either be used to support confirmation of source from previously obtained STR DNA profiling results or to improve sample success rates by making better informed evidential submissions.
Assuntos
Análise Química do Sangue , Muco do Colo Uterino/química , Genética Forense/instrumentação , RNA Mensageiro/genética , Saliva/química , Sêmen/química , Espermatozoides/química , Animais , Feminino , Marcadores Genéticos , Humanos , Masculino , Menstruação , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Software , Especificidade da EspécieRESUMO
In forensic casework, compromised samples often possess limited or degraded nuclear DNA, rendering mitochondrial DNA a more feasible option for forensic DNA analyses. The emergence of massively parallel sequencing (MPS) has enabled the recovery of extensive sequence information from very low quantities of DNA. We have developed a multiplex PCR method that amplifies the complete mitochondrial genome in a range of forensically relevant samples including single cells, cremated remains, bone, maggot and hairs isolated from dust bunnies. Following library preparation, MPS yields complete or nearly complete mitochondrial genome coverage for all samples. To confirm concordance between sample types and between sequencing platforms, we compared sequencing results from hair and buccal swabs from two references. Low initial DNA input into the multiplex PCR allows for conservation of precious DNA while MPS maximizes recovery of genetic information.
RESUMO
Individual Y-chromosome STR locus allele frequencies and gene diversity values were determined for 48 Y-STR loci (DYS444; DYS450; DYS458; DYS481; DYS485; DYS490; DYS494; DYS495; DYS497; DYS503; DYS505; DYS508; DYS510; DYS511; DYS520; DYS521; DYS525; DYS533; DYS534; DYS535; DYS540; DYS543; DYS551; DYS552; DYS556; DYS562; DYS572; DYS578; DYS591; DYS593; DYS596; DYS617; DYS622; DYS627; DYS630; DYS631; DYS634; DYS636; DYS638; DYS641; DYS643; DYS657; DYS660; DYS685; DYS688; DYS698; DYS703; DYS707). Data was obtained from a sampling of 198 male individuals, from the Caucasian American (n=98) and African American (n=100) populations.
Assuntos
Negro ou Afro-Americano/genética , Cromossomos Humanos Y/genética , Frequência do Gene , Variação Genética , Genética Populacional , Genótipo , Haplótipos , Repetições de Microssatélites , População Branca/genética , Alelos , Humanos , Masculino , Reação em Cadeia da Polimerase , Estados UnidosRESUMO
POPULATION: POPULATION samples for gene diversity studies were obtained from the Virginia Division of Forensic Science (bloodstains), Richmond, VA. Ninety-eight Caucasian and 100 African American samples were included in this study.
Assuntos
Cromossomos Humanos Y , Genética Populacional , Sequências de Repetição em Tandem , População Negra/genética , Impressões Digitais de DNA , Frequência do Gene , Humanos , Masculino , Reação em Cadeia da Polimerase , Virginia , População Branca/genéticaRESUMO
A plethora of Y-STR markers from diverse sources have been deposited in public databases and represent potential candidates for incorporation into the next generation of Y-STR multiplexes for forensic use. Here, based upon all of the Y-STR loci that have been deposited in the human genome database (>400), we have sequentially positioned each one along the Y chromosome using the most current human genome sequencing data (NCBI Build 35). The information derived from this work defines the number and relative position of all potentially forensically relevant Y-STR loci, their location within the physical linkage map of the Y chromosome and their relationship to structural genes. We conclude that there exists at present at least 417 separate Y-STR markers available for potential forensic use, although many of these will be found to be unsuitable for other reasons. However, from this data, we were able to identify 28 pairs of duplicated loci that were given separate DYS designations and four pairs of loci with overlapping flanking regions. Removing one locus from each set of duplicates reduced the number of potentially useful loci from 417 to 389. The derived information should be useful for workers who are designing novel Y-STR multiplexes to ensure the presence of non-synonymous loci and, if so desired, to avoid loci that lie within structural genes. It may also be useful for forensic casework practitioners (or molecular anthropologists) to aid in distinguishing between chromosomal rearrangements (such as duplications and deletions) and bona fide DNA admixtures or null alleles caused by primer binding site mutations. We illustrate the practical usefulness of the chromosomal positioning data in the design of eight multiplex systems using 94 Y-STR loci.
Assuntos
Mapeamento Cromossômico , Cromossomos Humanos Y , Sequências de Repetição em Tandem , Bases de Dados Factuais , Marcadores Genéticos , Humanos , Transcrição GênicaRESUMO
A developmental validation study was performed on three Y-STR multiplex systems, Multiplex III (MPIII), Multiplex IV (MPIV), and Multiplex V (MPV), to ascertain their potential applicability to forensic casework. MPIII contains eight Y-STRs, including DYS426, DYS435, DYS436, DYS441, DYS442, DYS446, DYS462, and Y-GATA-A10, and one InDel, YAP (DYS287). MPIV contains 21 Y-STR loci, including DYS443, DYS444, DYS445, DYS447, DYS448, DYS449, DYS452, DYS453, DYS454, DYS455, DYS456, DYS458, DYS463, DYS464, DYS468, DYS484, DYS522, DYS527, DYS531 DYS557, and DYS588. MPV contains 13 Y-STR loci, including DYS459, DYS476, DYS488, DYS513, DYS549, DYS561, DYS570, DYS575, DYS576, DYS590, DYS594, DYS598, and DYS607. Full genetic profiles were consistently obtained for all three multiplexes with 25-50 pg of male DNA. No significant amplification was observed with 1 mug of female DNA. Each multiplex permitted the determination of the number of male donors in male:male DNA admixtures. Species specificity studies demonstrated some cross-reactivity with some primate samples. Environmentally compromised blood samples produced full or partial profiles after exposure to various conditions for up to 1 year. Full profiles were recovered from simulated casework specimens including cigarette butts and postcoital cervicovaginal swabs. Population data were collected to determine individual loci gene diversity and multiplex discriminatory capacity.
Assuntos
Cromossomos Humanos Y , Impressões Digitais de DNA/métodos , Marcadores Genéticos , Sequências de Repetição em Tandem , Animais , Sangue , DNA/isolamento & purificação , Primers do DNA , DNA Bacteriano/análise , Feminino , Genética Forense , Humanos , Masculino , Reação em Cadeia da Polimerase , Especificidade da Espécie , EspermatozoidesRESUMO
In some cases of sexual assault the victim may not report the assault for several days after the incident due to various factors. The ability to obtain an autosomal STR profile of the semen donor from a living victim rapidly diminishes as the post-coital interval is extended due to the presence of only a small amount of male DNA amidst an overwhelming amount of female DNA. Previously, we have utilized various technological tools to overcome the limitations of male DNA profiling in extended interval post-coital samples including the use of Y-chromosome STR profiling, cervical sample, and post-PCR purification permitting the recovery of Y-STR profiles of the male DNA from samples collected 5-6 days after intercourse. Despite this success, the reproductive biology literature reports the presence of spermatozoa in the human cervix up to 7-10 days post-coitus. Therefore, novel and improved methods for recovery of male profiles in extended interval post-coital samples were required. Here, we describe enhanced strategies, including Y-chromosome-targeted pre-amplification and next generation Y-STR amplification kits, that have resulted in the ability to obtain probative male profiles from samples collected 6-9 days after intercourse.
Assuntos
Cromossomos Humanos Y , Genética Forense , Genoma Mitocondrial , Repetições de Microssatélites/genética , Hibridização de Ácido Nucleico/métodos , Estupro , Sêmen , Eletroforese Capilar , Feminino , Humanos , Masculino , Reação em Cadeia da Polimerase/métodosRESUMO
DNA profiles can be obtained from 'touch DNA' evidence, which comprises microscopic traces of human biological material. Current methods for the recovery of trace DNA employ cotton swabs or adhesive tape to sample an area of interest. However, such a 'blind-swabbing' approach will co-sample cellular material from the different individuals, even if the individuals' cells are located in geographically distinct locations on the item. Thus, some of the DNA mixtures encountered in touch DNA samples are artificially created by the swabbing itself. In some instances, a victim's DNA may be found in significant excess thus masking any potential perpetrator's DNA. In order to circumvent the challenges with standard recovery and analysis methods, we have developed a lower cost, 'smart analysis' method that results in enhanced genetic analysis of touch DNA evidence. We describe an optimized and efficient micromanipulation recovery strategy for the collection of bio-particles present in touch DNA samples, as well as an enhanced amplification strategy involving a one-step 5 µl microvolume lysis/STR amplification to permit the recovery of STR profiles from the bio-particle donor(s). The use of individual or few (i.e., "clumps") bioparticles results in the ability to obtain single source profiles. These procedures represent alternative enhanced techniques for the isolation and analysis of single bioparticles from forensic touch DNA evidence. While not necessary in every forensic investigation, the method could be highly beneficial for the recovery of a single source perpetrator DNA profile in cases involving physical assault (e.g., strangulation) that may not be possible using standard analysis techniques. Additionally, the strategies developed here offer an opportunity to obtain genetic information at the single cell level from a variety of other non-forensic trace biological material.
Assuntos
DNA/análise , DNA/genética , Genética Forense/métodos , Pele/química , Vestuário , Eletroforese Capilar/métodos , Humanos , Masculino , Micromanipulação/métodos , Repetições de Microssatélites , Pele/citologia , TatoRESUMO
A DNA profile from the perpetrator does not reveal, per se, the circumstances by which it was transferred. Body fluid identification by mRNA profiling may allow extraction of contextual 'activity level' information from forensic samples. Here we describe the development of a prototype multiplex digital gene expression (DGE) method for forensic body fluid/tissue identification based upon solution hybridization of color-coded NanoString(®) probes to 23 mRNA targets. The method identifies peripheral blood, semen, saliva, vaginal secretions, menstrual blood and skin. We showed that a simple 5 min room temperature cellular lysis protocol gave equivalent results to standard RNA isolation from the same source material, greatly enhancing the ease-of-use of this method in forensic sample processing. We first describe a model for gene expression in a sample from a single body fluid and then extend that model to mixtures of body fluids. We then describe calculation of maximum likelihood estimates (MLEs) of body fluid quantities in a sample, and we describe the use of likelihood ratios to test for the presence of each body fluid in a sample. Known single source samples of blood, semen, vaginal secretions, menstrual blood and skin all demonstrated the expected tissue-specific gene expression for at least two of the chosen biomarkers. Saliva samples were more problematic, with their previously identified characteristic genes exhibiting poor specificity. Nonetheless the most specific saliva biomarker, HTN3, was expressed at a higher level in saliva than in any of the other tissues. Crucially, our algorithm produced zero false positives across this study's 89 unique samples. As a preliminary indication of the ability of the method to discern admixtures of body fluids, five mixtures were prepared. The identities of the component fluids were evident from the gene expression profiles of four of the five mixtures. Further optimization of the biomarker 'CodeSet' will be required before it can be used in casework, particularly with respect to increasing the signal-to-noise ratio of the saliva biomarkers. With suitable modifications, this simplified protocol with minimal hands on requirement should facilitate routine use of mRNA profiling in casework laboratories.
Assuntos
Automação , Líquidos Corporais/metabolismo , Genética Forense , Hibridização de Ácido Nucleico , Sondas de Oligonucleotídeos , RNA Mensageiro/genética , Humanos , RNA Mensageiro/isolamento & purificaçãoRESUMO
DNA profiling through the analysis of STRs remains one of the most widely used tools in human identification across the world. Current laboratory STR analysis is slow, costly and requires expert users and interpretation which can lead to instances of delayed investigations or non-testing of evidence on budget grounds. The ParaDNA(®) Intelligence System has been designed to provide a simple, fast and robust way to profile DNA samples in a lab or field-deployable manner. The system analyses 5-STRs plus amelogenin to deliver a DNA profile that enables users to gain rapid investigative leads and intelligent prioritisation of samples in human identity testing applications. Utilising an innovative sample collector, minimal training is required to enable both DNA analysts and nonspecialist personnel to analyse biological samples directly, without prior processing, in approximately 75min. The test uses direct PCR with fluorescent HyBeacon(®) detection of STR allele lengths to provide a DNA profile. The developmental validation study described here followed the Scientific Working Group on DNA Analysis Methods (SWGDAM) guidelines and tested the sensitivity, reproducibility, accuracy, inhibitor tolerance, and performance of the ParaDNA Intelligence System on a range of mock evidence items. The data collected demonstrate that the ParaDNA Intelligence System displays useful DNA profiles when sampling a variety of evidence items including blood, saliva, semen and touch DNA items indicating the potential to benefit a number of applications in fields such as forensic, military and disaster victim identification (DVI).
Assuntos
Impressões Digitais de DNA/métodos , Repetições de Microssatélites , Alelos , Animais , DNA/análise , DNA/sangue , DNA/genética , Impressões Digitais de DNA/instrumentação , Furões , Genética Forense/instrumentação , Genética Forense/métodos , Humanos , Reação em Cadeia da Polimerase , Reprodutibilidade dos Testes , Especificidade da Espécie , Manejo de Espécimes/métodosRESUMO
In order to increase significantly the discriminatory potential of Y-STR systems available to the forensic community, we have developed and validated a 21-locus Y-STR multiplex system. Since the system was designed specifically to augment the European Y chromosome typing community's "minimal haplotype" Y-STR set (MHL) for forensic casework, it contains a novel constellation of markers not contained therein. The system, which we refer to as Multiplex IV (MPIV), permits the co-amplification of DYS 443, DYS 444, DYS 445, DYS 447, DYS 448, DYS 449, DYS 452, DYS 453, DYS 454, DYS 455, DYS 456, DYS 458, DYS 463, DYS 464, DYS 468, DYS 484, DYS 522, DYS 527, DYS 531, DYS 557, and DYS 588. Although the multiplex contains 21 Y-STR loci, of which one is bi-local and one is tetra-local, there are actually 25 sites exhibiting allelic variation, and this has prompted us to use the descriptor "megaplex" to describe the system. This report describes a number of performance checks that were employed to characterize the system including sensitivity, specificity, discriminatory capacity, and nonprobative casework studies. Although 1 ng of male DNA was found to be the optimal amount of input template, a complete 21-locus profile was obtained with as little as 50 pg of male DNA (i.e., approximately 8 to 9 diploid cells). The specificity of the system was demonstrated by the lack of significant female DNA derived artifacts when tested using either 300 ng of female DNA alone or an admixture of male/female DNA in which the female component was present in a 100-fold excess. The ability of the system to determine the number of male donors was demonstrated by testing different admixtures of DNA at different ratios from two male donors. Cervicovaginal samples taken up to 48 h post coitus yielded a complete 21-locus Y-STR profile of the semen donor, thus confirming the potential utility of the system for forensic casework. Preliminary estimates of the gene diversity (h) of the individual loci for the Caucasian and African-American population indicated that 15 of the 21 loci possessed an h of > or = 0.5 in at least one population. Multi-locus haplotype analysis revealed that the 21-plex system could augment the use of the minimal haplotype loci and increase significantly the discriminatory capacity of Y-STR analysis.
Assuntos
Cromossomos Humanos Y , Impressões Digitais de DNA/métodos , DNA/isolamento & purificação , Haplótipos , Sequências de Repetição em Tandem , Coito , DNA/sangue , Primers do DNA , Eletroforese Capilar , Etnicidade/genética , Feminino , Frequência do Gene , Marcadores Genéticos , Genética Populacional , Humanos , Masculino , Reação em Cadeia da Polimerase , Sêmen/química , Sensibilidade e Especificidade , Vagina/químicaRESUMO
Current assessment of whether a forensic evidence item should be submitted for STR profiling is largely based on the personal experience of the Crime Scene Investigator (CSI) and the submissions policy of the law enforcement authority involved. While there are chemical tests that can infer the presence of DNA through the detection of biological stains, the process remains mostly subjective and leads to many samples being submitted that give no profile or not being submitted although DNA is present. The ParaDNA(®) Screening System was developed to address this issue. It consists of a sampling device, pre-loaded reaction plates and detection instrument. The test uses direct PCR with fluorescent HyBeacon™ detection of PCR amplicons to identify the presence and relative amount of DNA on an evidence item and also provides a gender identification result in approximately 75 minutes. This simple-to-use design allows objective data to be acquired by both DNA analyst and non-specialist personnel, to enable a more informed submission decision to be made. The developmental validation study described here tested the sensitivity, reproducibility, accuracy, inhibitor tolerance, and performance of the ParaDNA Screening System on a range of mock evidence items. The data collected demonstrates that the ParaDNA Screening System identifies the presence of DNA on a variety of evidence items including blood, saliva and touch DNA items.
Assuntos
DNA/genética , Genética Forense , Corantes Fluorescentes , Humanos , Reação em Cadeia da PolimeraseRESUMO
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.