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(1 - 14 of 14)
- Title
- MITOCHONDRIAL DNA ANALYSIS BY PYROSEQUENCING.
- Creator
-
Hastings, Patsy-Ann Susan, Ballantyne, Jack, University of Central Florida
- Abstract / Description
-
Mitochondrial DNA (deoxyribo nucleic acid) is typically used in forensic casework when small quantities of high molecular weight quality DNA is not expected to be present thus negating the chances of obtaining usable nuclear DNA. Typical samples that utilized mitochondrial DNA analysis are: hair, bones, teeth, ancient remains (samples or remains that are at least 100 years old) or very old samples (samples that are less than 100 but greater than 10 years old). The current method used to...
Show moreMitochondrial DNA (deoxyribo nucleic acid) is typically used in forensic casework when small quantities of high molecular weight quality DNA is not expected to be present thus negating the chances of obtaining usable nuclear DNA. Typical samples that utilized mitochondrial DNA analysis are: hair, bones, teeth, ancient remains (samples or remains that are at least 100 years old) or very old samples (samples that are less than 100 but greater than 10 years old). The current method used to evaluate mitochondrial DNA is Sanger sequencing. Although robust, it is also time consuming and labor intensive, on the other hand pyrosequencing is a nonelectrophoretic, rapid, reliable, and sensitive sequencing method which can be easily automated. Therefore pyrosequencing could enable the widespread use of mitochondrial DNA in forensic casework and reduce the amount of time spent on each sample without compromising quality.The aim of this study is to evaluate the efficacy of pyrosequencing for forensic DNA applications, in particular mitochondrial DNA. Two dispensation orders, cyclic and directed, were examined to determine if there is any effect on the sequence generated. The accuracy of pyrosequencing was evaluated by sequencing samples of known sequence provided by the FBI. The sensitivity of pyrosequencing was evaluated by sequencing samples at different DNA concentrations and inputs. Experiments were conducted to determine the ability of pyrosequencing to detect mixtures and heteroplasmy. Additionally, the ability of pyrosequencing to sequence damaged/degraded DNA was evaluated using blood, semen, and saliva samples that were subjected to three different environmental conditions. A blind study will be conducted to confirm the accuracy of pyrosequencing. Finally, a comparison study will be conducted in which pyrosequencing will be compared to Sanger sequencing.
Show less - Date Issued
- 2004
- Identifier
- CFE0000098, ucf:46116
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0000098
- Title
- THE USE OF PYROSEQUENCING FORTHE ANALYSIS OF Y CHROMOSOME SINGLE NUCLEOTIDE POLYMORPHISMS.
- Creator
-
Fletcher, Jeremy Charles, Ballantyne, Jack, University of Central Florida
- Abstract / Description
-
The potential value of the Y chromosome for forensic applications has been recognized for some time with the current work dedicated to Short Tandem Repeat analysis and Single Nucleotide Polymorphism (SNP) discovery. This study examined the ability of two different SNP analysis methods to determine if they could be utilized in forensic applications and ultimately be developed into an established system for Y chromosome SNP analysis. This study examined two principle SNP analysis systems:...
Show moreThe potential value of the Y chromosome for forensic applications has been recognized for some time with the current work dedicated to Short Tandem Repeat analysis and Single Nucleotide Polymorphism (SNP) discovery. This study examined the ability of two different SNP analysis methods to determine if they could be utilized in forensic applications and ultimately be developed into an established system for Y chromosome SNP analysis. This study examined two principle SNP analysis systems: single base extension and Pyrosequencing. Pyrosequencing was determined to be superior to single base extension, due to the wealth of information provided with sequencing and the flexibility of designing primers for analysis. Using Pyrosequencing, 50 Y chromosome loci were examined and the minimum loci required for maximum diversity for the development of a Y chromosome SNP analysis system were chosen. Thirteen loci were selected based on their ability to discriminate 60 different individuals from three different racial groups into 15 different haplogroups. The Y chromosome SNP analysis system developed utilized nested PCR for the amplification of all 13 loci. Then they were sequenced as groups, ranging from one to three loci, in a single reaction. The Y chromosome SNP analysis system developed here has the potential for forensic application since it has shown to be successful in the analysis of blood, buccal swabs, semen, and saliva, works with as little as 5 pg of starting DNA material, and will amplify only male DNA in the presence of male/female mixtures in which the female portion of the sample overwhelmed the male portion 30,000 to 1.
Show less - Date Issued
- 2004
- Identifier
- CFE0000071, ucf:46089
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0000071
- Title
- PHYSICAL CHARACTERISTICS OF AN INDIVIDUAL: THE IDENTIFICATION OF BIOMARKERS FOR BIOLOGICAL AGE DETERMINATION.
- Creator
-
Alvarez, Michelle, Ballantyne, Jack, University of Central Florida
- Abstract / Description
-
It is now a matter of routine for the forensic scientist to obtain the genetic profile of an individual from DNA recovered from a biological stain deposited at a crime scene. Potential contributors of the stain must either be known to investigators (i.e. a developed suspect) or the questioned profile must be searched against a database of DNA profiles such as those maintained in the CODIS National DNA database. However, in those instances where there is no developed suspect and no match is...
Show moreIt is now a matter of routine for the forensic scientist to obtain the genetic profile of an individual from DNA recovered from a biological stain deposited at a crime scene. Potential contributors of the stain must either be known to investigators (i.e. a developed suspect) or the questioned profile must be searched against a database of DNA profiles such as those maintained in the CODIS National DNA database. However, in those instances where there is no developed suspect and no match is obtained after interrogation of appropriate DNA databases, the DNA profile per se presently provides no meaningful information to investigators, with the notable exception of gender determination. In these situations it would be advantageous to the investigation, if additional probative information could be obtained from the biological stain. A useful biometric that could provide important probative information, and one that may be amenable to molecular genetic analysis, is the biological age of an individual. The ability to provide investigators with information as to whether a DNA donor is a newborn, infant, toddler, child, adolescent, adult, middle-aged or elderly individual could be useful in certain cases, particularly those involving young children such as kidnappings or in providing additional intelligence during terrorist investigations. Currently no validated molecular assays exist for age determination. Biological human ageing can be defined by two distinct processes, degenerative and developmental ageing. The degenerative process of ageing is based on theories which identify an increase or decrease in physiological conditions with increasing age. In contrast, the developmental process of ageing is based on the theory that as individuals increase in chronological age, there will be subtle corresponding molecular based biological changes, each requiring genes to be expressed or silenced, indicative of that particular stage of life. We investigated the degenerative process of chromosomal telomere shortening, as well as the developmental process of gene expression profiling analysis, in an attempt to identify biomarkers of biological age in a self-renewing tissue such as blood. While telomere length analysis was an ineffective method for age determination; gene expression analysis revealed three gene transcripts expressed in an age-dependent physiological manner. These species namely- COL1A2, HBE1 and IGFBP3, were found to be expressed at elevated levels in younger individuals, newborns, or post-pubertal individuals, respectively. The biological process of hemoglobin switching was also investigated for the possibility of determining human age. While experimenting with the potential of using the gamma-hemoglobin chains, as newborn specific gene candidates, we serendipitously discovered four novel truncated transcripts, which we have termed HBG1n1, HBG1n2, HBG2n2 and HBG2n3; whose expression was restricted to whole-blood newborn samples and specific fetal tissues. The molecular origin of these transcripts appears to be at the RNA level, being produced by specific rearrangement events occurring in the standard gamma hemoglobin transcripts (HBG1 and HBG2), which yield these new isoforms that are expressed in a highly regulated tissue specific manner.
Show less - Date Issued
- 2007
- Identifier
- CFE0001737, ucf:47297
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0001737
- Title
- ANALYSIS OF MITOCHONDRIAL DNA CODING REGION SNPS BY PYROSEQUENCING.
- Creator
-
Parker, Kyle, Ballantyne, Jack, University of Central Florida
- Abstract / Description
-
To date, the use of mitochondrial DNA in forensic analysis has relied on the presence of variations in the control region to differentiate between samples. One problem that this analysis has shown is the occurrence of common Haplogroup H haplotypes or identical sequences. Thus, there is a need to enhance the distinguishing power of this type of analysis. One option has been to investigate the mitochondrial coding region for polymorphisms that could differentiate between samples with identical...
Show moreTo date, the use of mitochondrial DNA in forensic analysis has relied on the presence of variations in the control region to differentiate between samples. One problem that this analysis has shown is the occurrence of common Haplogroup H haplotypes or identical sequences. Thus, there is a need to enhance the distinguishing power of this type of analysis. One option has been to investigate the mitochondrial coding region for polymorphisms that could differentiate between samples with identical control region haplotypes. The goal of this study has been to identify polymorphic coding region sites for development in a Pyrosequencing assay that would effectively enhance the discriminatory power of mitochondrial DNA analysis. With this goal in mind, five duplexes have been successfully developed and tested, utilizing the ten polymorphic sites that had been selected, with most sites being specific to Caucasians. Validation studies were performed to test the durability of the assay. The specificity of the assay to primate and non-primate species was determined to be limited to primate species only. Sample variations, including mixtures, dilutions and environmental exposure, were utilized to assess the sensitivity of the Pyrosequencing method. It was found that a minimum initial DNA input of 10fg was necessary for reliable results. The Pyrosequencing assay was able to detect mixtures at a 1:1 ratio and environmental samples exposed to the elements from up to 1 week for blood and 6 weeks for semen. Samples designed to simulate typical casework materials were analyzed and found to provide for consistent results, including trace fingerprints and digested hair shafts. These validation results provide the conclusion that this assay is suitable for use in forensic casework and demonstrate that the mitochondrial coding region provides a viable alternative to hypervariable region analysis.
Show less - Date Issued
- 2007
- Identifier
- CFE0001562, ucf:47132
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0001562
- Title
- DEVELOPMENT AND FORENSIC APPLICATION OF DYE PROBE FLUORESCENCE RESONANCE ENERGY TRANSFER FOR IMPROVED DETECTION OF CHANGES IN DNA SEQUENCE.
- Creator
-
Halpern, Micah, Ballantyne, Jack, University of Central Florida
- Abstract / Description
-
Discovering, screening, and associating changes in DNA sequence are important to a broad range of disciplines and play a central role in Forensic Science. The typical types of changes include sequence variations [single nucleotide polymorphisms (SNP)] and length variations [short tandem repeats (STR)]. The steps for forensic DNA sample processing are similar for both types of changes but diverge at the point of detection. A number of approaches are being explored for SNP genotyping while STR...
Show moreDiscovering, screening, and associating changes in DNA sequence are important to a broad range of disciplines and play a central role in Forensic Science. The typical types of changes include sequence variations [single nucleotide polymorphisms (SNP)] and length variations [short tandem repeats (STR)]. The steps for forensic DNA sample processing are similar for both types of changes but diverge at the point of detection. A number of approaches are being explored for SNP genotyping while STR analysis primarily consists of size-based analysis by capillary electrophoresis. Limitations exist for all current detection methods that pose significant impacts to forensic analysis. Bi-allelic SNPs result in three possible genotypes with a minimal amount of information generated per marker. Limitations for SNP analysis are due to the inability to amplify a suitable number of SNP markers from low DNA content samples to provide an appropriate level of discrimination. Multi-allelic STR markers are currently the marker of choice for forensic typing but a variety of experimental artifacts are possible that consist of either biology or technology related causes. Molecular genotyping methods developed across other disciplines have potential to alleviate some of these shortcomings but no current approach is capable of genotyping both SNP and STR loci with a single chemistry. The need for a more effective, efficient, and generalized approach led to development of a unique method called Dye Probe Fluorescence Resonance Energy Transfer (dpFRET) and determination of its suitability for forensic analysis. The development phase of the research consisted of synthetic testing to establish proof of concept for the chemistry followed by polymerase chain reaction (PCR) based assays to demonstrate real world applications. Following successful development, the boundaries and limitations for the technology were established (sensitivity, allelic dropout, mixed samples) and efforts were made to improve the approach. In the process, parallel testing for other fields including molecular pathology and conservation biology were incorporated to explore potential widespread application of this new approach. The overall goal of this project was to develop and explore the limitations for a unique approach to genotyping both SNPs and STRs. A majority of the work involved development of the method itself with the ultimate objective of application for forensic science. The focus of this project was to address and alleviate some of the shortcomings of current approaches that result in potential limitations for forensic analysis. It is expected that future applications of this technology might impact a wide range of disciplines to aid in discovery, screening and association of changes in DNA sequence.
Show less - Date Issued
- 2008
- Identifier
- CFE0002385, ucf:47752
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0002385
- Title
- STABILITY AND RECOVERY OF RNA IN BIOLOGICAL STAINS.
- Creator
-
Setzer, Mindy Eileen, Jack Ballantyne, Dr, University of Central Florida
- Abstract / Description
-
In theory, RNA expression patterns, including the presence and relative abundance of particular RNA species, provide cell and tissue specific information that could be of use to forensic scientists. An mRNA based approach could allow the facile identification of the tissue components present in a body fluid stain and conceivably could supplant the battery of serological and biochemical tests currently employed in the forensic serology laboratory. Some of the potential advantages include...
Show moreIn theory, RNA expression patterns, including the presence and relative abundance of particular RNA species, provide cell and tissue specific information that could be of use to forensic scientists. An mRNA based approach could allow the facile identification of the tissue components present in a body fluid stain and conceivably could supplant the battery of serological and biochemical tests currently employed in the forensic serology laboratory. Some of the potential advantages include greater test specificity, and the ability to perform simultaneous analysis using a common assay format for the presence of all body fluids of forensic interest. In this report, the recovery and stability of RNA in forensic samples was evaluated by conducting an in-depth study on the persistence of RNA in biological stains. Stains were prepared from blood, saliva, semen, and vaginal secretions, and were exposed to a range of environmental conditions so that the affects of different light sources, temperatures, and environments could be assessed. Using the results from quantitation and sensitivity studies performed with pristine forensic stains, the RNA stability of samples which were collected over a period of 1 day to 1 year for blood, saliva, and vaginal secretion stains and for up to 6 months for semen stains were analyzed. The extent of RNA degradation within each type of body fluid stain was determined using quantitation of total RNA and reverse transcriptase polymerase chain reaction (RT-PCR) with selected housekeeping and tissue-specific genes. The results show that RNA can be recovered from biological stains in sufficient quantity and quality for mRNA analysis. The results also show that mRNA is detectable in samples stored at room temperature for at least one year, but that heat and humidity appear to be very detrimental to the stability of RNA.
Show less - Date Issued
- 2004
- Identifier
- CFE0000077, ucf:46141
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0000077
- Title
- THE USE AND DEVELOPMENT OF LASER MICRODISSECTION TO SEPARATE SPERMATOZOA FROM EPITHELIAL CELLS FOR STR ANALYSIS.
- Creator
-
Sanders, Christine, Ballantyne, Jack, University of Central Florida
- Abstract / Description
-
Short Tandem Repeat (STR) analysis has become a valuable tool in identifying the source of biological stains, particularly from the investigation of sexual assault crimes. Difficulties in analysis arise primarily in the interpretation of mixed genotypes when cell separation of the sexual assailant's sperm from the victim's cells is incomplete. The forensic community continues to seek improvements in cell separation methods from mixtures for DNA typing. This report describes the use of laser...
Show moreShort Tandem Repeat (STR) analysis has become a valuable tool in identifying the source of biological stains, particularly from the investigation of sexual assault crimes. Difficulties in analysis arise primarily in the interpretation of mixed genotypes when cell separation of the sexual assailant's sperm from the victim's cells is incomplete. The forensic community continues to seek improvements in cell separation methods from mixtures for DNA typing. This report describes the use of laser microdissection (LMD) for the separation of pure populations of spermatozoa from two-donor cell mixtures. In this study, cell separation was demonstrated by microscopic identification of histologically stained spermatozoa and female buccal cell mixtures, and STR analysis of DNA obtained from the separated sperm cells. Clear profiles of the male donor were obtained with the absence of any additional alleles from the female donor. Five histological stains were evaluated for use with LMD and DNA analysis: hematoxylin/eosin, nuclear fast red/picroindigocarmine, methyl green, Wright's stain, and acridine orange. Hematoxylin/eosin out-performed all other stains however nuclear fast red/picroindigocarmine could be used satisfactorily with STR analysis. In addition, three DNA isolation methods were evaluated for LMD collected cells: QIAamp (Qiagen), microLYSIS (Microzone Ltd.) and Lyse-N-Go (Pierce Chemical Co.). MicroLYSIS performed poorly, yielding low levels of PCR product. Lyse-N-Go extraction was effective for the recovery of DNA from LMD collected sperm cells while QIAamp isolation performed best for the recovery of DNA from LMD collected epithelial cells. LMD is shown to be an effective, low-manipulation separation method that enables the recovery of sperm while excluding epithelial cell DNA.
Show less - Date Issued
- 2005
- Identifier
- CFE0000876, ucf:46652
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0000876
- Title
- ASSESSMENT AND IN VITRO REPAIR OF DAMAGED DNA TEMPLATES FROM FORENSIC STAINS.
- Creator
-
Hall, Ashley, Ballantyne, Jack, University of Central Florida
- Abstract / Description
-
DNA extracted from biological stains is often intractable to analysis. This may due to a number of factors including a low copy number (LCN) of starting molecules, the presence of soluble inhibitors or damaged DNA templates. Remedies may be available to the forensic scientist to deal with LCN templates and soluble inhibitors but none presently exist for damaged DNA. In fact, only recently has the biochemical nature, the extent of DNA damage in physiological stains and the point at which the...
Show moreDNA extracted from biological stains is often intractable to analysis. This may due to a number of factors including a low copy number (LCN) of starting molecules, the presence of soluble inhibitors or damaged DNA templates. Remedies may be available to the forensic scientist to deal with LCN templates and soluble inhibitors but none presently exist for damaged DNA. In fact, only recently has the biochemical nature, the extent of DNA damage in physiological stains and the point at which the damage inflicted upon a particular sample precludes the ability to obtain a genetic profile for purposes of identification been examined. The primary aims of this work were first to ascertain the types of DNA damage encountered in forensically relevant stains, correlating the occurrence this damage with the partial or total loss of a genotype, and then to attempt the repair of the damage by means of in vitro DNA repair systems. The initial focus of the work was the detection of damage caused by exogenous, environmental sources, primarily UV irradiation, but also factors such as heat, humidity and microorganism growth. Results showed that the primary causes of the damage that resulted in profile loss were strand breaks, both single and double stranded, as well as modifications to the DNA structure that inhibited its amplification. Armed with this knowledge, the next focus was the repair of the damage by means of in vitro DNA systems. Efforts have been concentrated on single strand break/gap repair and translesion synthesis assays. By modifying the assays and employing various combinations of the systems, a genetic signature has been recovered from previously intractable samples. Additionally, the effects that various storage conditions have on the DNA in physiological stains stored in a laboratory were examined. The optimal long term storage conditions for biological evidence has been a matter of debate in the forensic community for some time. But, no comprehensive study had previously been undertaken to describe the effects of dehydration and temperature on degradation and the ability to obtain a genetic profile on bloodstains kept in different types of storage media at a range of temperatures. To examine this, bloodstains were either allowed to dry overnight or placed in the storage medium while still wet and were stored at room temperature, 4oC or 30oC for up to four years. Results showed that specimens dehydrated prior to storage were very stable, and these bloodstains showed no degradation or loss of a genetic profile for up to four years.
Show less - Date Issued
- 2005
- Identifier
- CFE0000878, ucf:46647
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0000878
- Title
- MESSENGER RNA PROFILING: A PROTOTYPE METHOD FOR BODY FLUIDAND TISSUE IDENTIFICATION.
- Creator
-
Juusola, Jane, Ballantyne, Jack, University of Central Florida
- Abstract / Description
-
Conventional methods of body fluid identification use labor-intensive, technologically diverse techniques that are performed in a series, not parallel, manner and are costly in terms of time and sample. Furthermore, for some frequently encountered body fluids, such as saliva or vaginal secretions, no confirmatory technique exists. Terminally differentiated cells, such as blood lymphocytes or epithelial cells lining the oral cavity, have a unique pattern of gene expression, which is evinced by...
Show moreConventional methods of body fluid identification use labor-intensive, technologically diverse techniques that are performed in a series, not parallel, manner and are costly in terms of time and sample. Furthermore, for some frequently encountered body fluids, such as saliva or vaginal secretions, no confirmatory technique exists. Terminally differentiated cells, such as blood lymphocytes or epithelial cells lining the oral cavity, have a unique pattern of gene expression, which is evinced by the presence and relative abundance of specific mRNA species. If the type and abundance of mRNAs can be determined in a stain or tissue sample recovered at the crime scene, it would be possible to definitively identify the tissue or body fluid in question. Advantages of an mRNA-based approach, compared to conventional biochemical analysis, include greater specificity, simultaneous and semi-automated analysis though a common assay format, improved timeliness, decreased sample consumption and compatibility with DNA extraction methodologies. In this report, we demonstrate that RNA is stable in biological stains and can be recovered in sufficient quantity and quality for analysis using reverse transcriptasepolymerase chain reaction assay (RT-PCR). We have identified sets of candidate tissuespecific genes for body fluids and tissues of forensic interest, namely blood, saliva, semen, vaginal secretions, menstrual blood, urine, skin, muscle, adipose, and brain. We also report the identification of a new housekeeping gene for use in mRNA based assays. Select body fluid-specific genes have been incorporated into multiplex PCR and real-time PCR assays. These assays allow for the positive identification of blood, saliva, semen,vaginal secretions, and/or menstrual blood in a stain. The final task of this work was the molecular characterization of mRNA degradation patterns in biological stains, which not only has fundamental importance in possibly revealing mRNA degradation pathways in dried biological stains, but may ultimately lead to better assay design strategies for mRNA markers for forensic use. An mRNA-based approach described in this report could allow the facile identification of the tissue components present in a body fluid stain and could conceivably supplant the battery of serological and biochemical tests currently employed in the forensic serology laboratory.
Show less - Date Issued
- 2005
- Identifier
- CFE0000862, ucf:46668
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0000862
- Title
- SIMPLIFIED LOW COPY NUMBER DNA ANALYSIS BY POST PCR PURIFICATION.
- Creator
-
Smith, Pamela, Ballantyne, Jack, University of Central Florida
- Abstract / Description
-
Frequently evidentiary items contain an insufficient quantity of DNA to obtain complete or even partial DNA profiles using standard forensic gentotyping techniques. Here, various methods of post PCR purification were evaluated for their effects on the sensitivity of fluophore-based allelic detection. A method of post PCR purification is described which increases the sensitivity of standard 28 cycle PCR such that low copy number DNA templates (
Show moreFrequently evidentiary items contain an insufficient quantity of DNA to obtain complete or even partial DNA profiles using standard forensic gentotyping techniques. Here, various methods of post PCR purification were evaluated for their effects on the sensitivity of fluophore-based allelic detection. A method of post PCR purification is described which increases the sensitivity of standard 28 cycle PCR such that low copy number DNA templates (<100 pg DNA) can be analyzed. Full profiles were consistently obtained with as little as 20 pg template DNA without increased cycle number. In mock case type samples with dermal ridge fingerprints, genetic profiles were obtained by amplification with 28 cycles followed by post-PCR purification whereas no profiles were obtained without purification of the PCR product. Allele drop-out, increased stutter, and contamination (allele drop-in) typical of LCN analysis were observed. A single incident of contamination was observed in a reagent blank (not duplicated upon re-amplification) however, no contamination was observed in negative amplification controls.
Show less - Date Issued
- 2006
- Identifier
- CFE0001003, ucf:46841
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0001003
- Title
- PERFORMANCE EFFICACY USING A COMPARISON OF COMMERCIAL AND IN-HOUSE Y-STR MULTIPLEX SYSTEMS FOR OPERATIONAL USE.
- Creator
-
Mayntz-Press, Kathleen, Ballantyne, Jack, University of Central Florida
- Abstract / Description
-
It is routine for the forensic scientist to obtain a genetic profile of an individual from DNA recovered from a biological stain deposited at a crime scene. In contrast, only a limited number of laboratories in the United States have the capability of performing Y-STR analysis in casework. In order to aid in facilitating the transfer of Y-STR technology to the crime laboratory community for operational use, a comparison between commercial products from three main vendors (Applied Biosystems...
Show moreIt is routine for the forensic scientist to obtain a genetic profile of an individual from DNA recovered from a biological stain deposited at a crime scene. In contrast, only a limited number of laboratories in the United States have the capability of performing Y-STR analysis in casework. In order to aid in facilitating the transfer of Y-STR technology to the crime laboratory community for operational use, a comparison between commercial products from three main vendors (Applied Biosystems AmpFℓSTR® Yfiler™ PCR Amplification Kit, Promega PowerPlex® – Y System, Reliagene Y-PLEX™ 12) and two in-house Y-STR multiplexes (MPI and MPB) commenced. The main intention for this comparison was to ascertain whether commercial Y-STR kits are able to obtain a male profile from difficult samples which have been accomplished with our in-house Y-STR multiplexes; such as mixtures, post coital specimens, and environmental insults. To aid the crime laboratory community an in depth comparison of the three main commercial Y-STR kits began in hopes to glean information in circumstances where Y chromosome polymorphisms may need to be employed. For example, the ability to provide investigators with the numbers of semen donors in multiple rape cases, identification of the genetic profile of the male component in a male/female mixture, and identification of the genetic profile of the male component in an extended interval post-coital sample. The capability of typing Y-STR loci by the crime laboratory community could dramatically affect the admissibility of Y-STR evidence. Therefore, the comparison of commercially available kits is an imperative process by which the scientific community acquires the necessary information to assess the ability of a procedure to obtain reliable results, determine the conditions under which such results can be obtained and define the limitations of the procedure. Thus the information for the study could lend itself to a standard being established amongst Y-STR kits for operational use and/or the production of a new Y-STR kit. One example of how the comparison of the three main commercial Y-STR kits could directly impact a new standard being established is by examining post-coital samples and their extreme limits (>48 hrs) for each kit in which a full male genetic profile was observed and comparing it to other commercial Y-STR kit and in-house Y-STR multiplexes. This would help establish the types of cases where specific Y-STR kits would be most useful, and the parameters in which each kit is able to perform. Thus leading to the development of a highly sensitive Y-STR kit that would be more sufficient to perform with the variety of samples an operational crime laboratory would routinely analyze. The capability of typing Y-STR loci by the crime laboratory community could dramatically affect the admissibility of Y-STR evidence. Therefore, the comparison of commercially available kits is an imperative process in order to inform the forensic community of different Y-STR kits available and their performance through direct comparison using modified SWGDAM validation guidelines.
Show less - Date Issued
- 2006
- Identifier
- CFE0001026, ucf:46824
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0001026
- Title
- THE DEVELOPMENT OF A "GENETIC EYEWITNESS" PROFILING SYSTEM FOR LOW TEMPLATE FORENSIC SPECIMENS: IDENTIFICATION OF NOVEL PROTEIN, RNA, DNA BIOMARKERS.
- Creator
-
Hanson, Erin, Ballantyne, Jack, University of Central Florida
- Abstract / Description
-
In many criminal investigations, valuable information regarding the physical appearance of suspected perpetrators or the time and order of events that transpired are provided by eyewitness accounts. However, the information obtained from eyewitnesses is often constrained by human recollection or subjective accounts and provides a biased description of the perpetrator's appearance or an inaccurate time line of events. Additionally, in numerous situations eyewitness accounts may not be...
Show moreIn many criminal investigations, valuable information regarding the physical appearance of suspected perpetrators or the time and order of events that transpired are provided by eyewitness accounts. However, the information obtained from eyewitnesses is often constrained by human recollection or subjective accounts and provides a biased description of the perpetrator's appearance or an inaccurate time line of events. Additionally, in numerous situations eyewitness accounts may not be available. An increasing reliance therefore is placed on the biological evidence recovered during criminal investigations to act as a silent witness, providing unbiased and scientific information that may aid in the resolution of criminal investigations. While the current capabilities of operational forensic crime laboratories include analytical methods to allow for a determination of the origin of a biological stain and for the recovery of a genetic profile of the donor, the sensitivity of such methods is not always sufficient to accommodate the limited amounts of biological material often recovered in forensic casework, Therefore, it is critical that continual advancements in the analysis of low template samples be made. In this report, we have sought to identify novel protein, RNA and DNA biomarkers that, in combination with enhanced profiling strategies, would allow for a determination of the time since deposition, the body fluid of origin and the genetic profile of the donor ("genetic eyewitness") of forensic low template specimens. First, we have developed a novel strategy for the determination of the time since deposition of dried bloodstains using spectrophotometric analysis of hemoglobin. An examination of the Soret band (lambda max = 414nm) in aged bloodstains has revealed a previously unidentified hypsochromic shift as the age of the stain increases. The extent of this shift permits a distinction to be made between stains that differ in age by only minutes, hours, days and months thus providing the highest resolution of any previously developed method. We also demonstrate that it may be possible to utilize a decline in enzyme activity to determine the age of a forensic biological stain. Second, we demonstrate that the differential expression of a panel of nine miRNAs allows for the identification of the body fluid origin of forensic biological stains using as little as 50pg of total RNA. This is the highest reported sensitivity of any RNA-based approach and this assay has demonstrated a high degree of specificity for each body fluid tested. The final task of this work was to identify novel DNA biomarkers and to develop enhanced profiling strategies to allow for greater sensitivity and reliability in the genetic profiling of low template samples. We demonstrate that the use of laser capture micro-dissection and enhanced amplification strategies resulted in the ability to obtain genetic profiles from as few as 2-5 epithelial cells and 5-10 sperm cells with greater reproducibility than previously reported studies. The use of a novel whole genome amplification method provided the ability to not only increase the quantity of genetic material obtained from micro-dissected cells but also the ability to recover additional genetic information from individual samples using novel DNA biomarkers. The novel biomarkers and profiling strategies described in this report provide the basis for the establishment of a molecular "genetic eyewitness" from low template forensic samples and demonstrate the future potential for routine and reliable analysis of trace amounts of genetic material recovered from low template biological evidence.
Show less - Date Issued
- 2008
- Identifier
- CFE0002373, ucf:47785
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0002373
- Title
- THE RELATIVE RECOVERABILITY OF DNA AND RNA PROFILES FROM FORENSICALLY RELEVANT BODY FLUID STAINS.
- Creator
-
Parker, Charly, Ballantyne, Jack, University of Central Florida
- Abstract / Description
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Biological material (fluids or tissues) whether from the victim or suspect is often collected as forensic evidence, and methods to obtain and analyze the DNA found in that material have been well established. The type of body fluid (i.e. blood, saliva, semen, vaginal secretions, and menstrual blood) from which the DNA originated is also of interest, and messenger RNA typing provides a specific and sensitive means of body fluid identification. In order for mRNA profiling to be utilized in...
Show moreBiological material (fluids or tissues) whether from the victim or suspect is often collected as forensic evidence, and methods to obtain and analyze the DNA found in that material have been well established. The type of body fluid (i.e. blood, saliva, semen, vaginal secretions, and menstrual blood) from which the DNA originated is also of interest, and messenger RNA typing provides a specific and sensitive means of body fluid identification. In order for mRNA profiling to be utilized in routine forensic casework, RNA of sufficient quantity and quality must be obtained from biological fluid stains and the methods used for RNA analysis must be fully compatible with current DNA analysis methodologies. Several DNA/RNA co-extraction methods were evaluated based on the quantity and quality of DNA and RNA recovered and were also compared to standard non-co-extraction methods. The two most promising methods, the in-house developed NCFS co-extraction and the commercially available AllPrep DNA/RNA Mini kit, were then optimized by improving nucleic acid recovery and consistency of CE (capillary electrophoresis) detection results. The sensitivity of the two methods was also evaluated, and DNA and RNA profiles could be obtained for the lowest amount of blood (0.2 µL) and saliva and semen (1 µL) tested. Both extraction methods were found to be acceptable for use with forensic samples, and the ability to obtain full DNA profiles was not hindered by the co-extraction of RNA. It is generally believed that RNA is less stable than DNA which may prevent its use in forensic casework. However, the degradation rates of DNA and RNA in the same biological fluid stain have not been directly compared. To determine the relative stability of DNA and RNA, the optimized NCFS co-extraction protocol was used to isolate DNA and RNA from environmentally compromised stains. Dried blood, saliva, and semen stains and vaginal secretions swabs were incubated at set temperatures and outside for up to 1 year. Even at 56°C, DNA and RNA were both stable out to 1 year in the blood and semen stains, out to 3 months (DNA) and 1 year (RNA) in the saliva stains, and out to 6 months (DNA) and 3 months (RNA) in the vaginal secretions swabs. The recoverability of both nucleic acids was reduced when the samples were exposed to increased humidity, sunlight, and rain. In general, DNA and RNA stability was found to be similar with a loss in ability to obtain a DNA or RNA profile occurring at the same time point; however, there were instances where RNA body fluid markers were detected when a poor/no DNA profile was obtained, indicating that RNA in dried stains is sufficiently stable for mRNA body fluid typing to be used in forensic casework.
Show less - Date Issued
- 2011
- Identifier
- CFE0003596, ucf:48849
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0003596
- Title
- MICROMANIPULATION AND GENETIC ANALYSIS OF INDIVIDUAL SPERM CELLS FOR SEXUAL ASSAULT INVESTIGATIONS.
- Creator
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Penn, Amanda, Borgon, Robert, Ballantyne, Jack, University of Central Florida
- Abstract / Description
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Sexual assault investigations utilize both physical and biological evidence to aid in the investigation. Physical evidence may include fingerprints, hair, fibers, stains, soil, and glass. Biological evidence may include semen, saliva, vaginal secretions, menstrual blood, and skin. Semen, often found in small or trace quantities, is of great importance when trying to identify the perpetrator. From the semen sample, DNA profiles using autosomal short tandem repeats (aSTRs) (gold standard in...
Show moreSexual assault investigations utilize both physical and biological evidence to aid in the investigation. Physical evidence may include fingerprints, hair, fibers, stains, soil, and glass. Biological evidence may include semen, saliva, vaginal secretions, menstrual blood, and skin. Semen, often found in small or trace quantities, is of great importance when trying to identify the perpetrator. From the semen sample, DNA profiles using autosomal short tandem repeats (aSTRs) (gold standard in forensic science) or Y-chromosome short tandem repeats (Y-STRs) can be obtained and can be used to identify a perpetrator through comparison to suspect reference samples or by searching the profile against a DNA database (CODIS). Obtaining DNA profiles can be challenging when assaults are reported many days after the incident. The amount of semen will decrease as the time frame increases due to various factors such as drainage from the vagina. To potentially overcome this obstacle and improve the recovery of profiles from extended interval samples, it may be possible to develop novel collection and analysis methods using individual or few sperm cells. Small quantities of sperm cells may still be present in extended interval samples that may otherwise fail to provide a DNA profile using conventional methods. The work presented here focuses on the development of these novel analysis methods using micromanipulation techniques and enhanced amplification strategies for the analysis of individual sperm cells to determine if a full DNA profile is present. The developed methods will be applied to the analysis of extended interval post coital samples.
Show less - Date Issued
- 2019
- Identifier
- CFH2000522, ucf:45624
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFH2000522
