Browsing by Subject "SNP"
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Item Analysis of Low Copy Number DNA Using Profiler Plus at Increased Amplification Cycles and Modifications in Sample Injection Parameters(2003-08-01) Hynds, Jody Lynn; Arthur Eisenberg; John Planz; Joseph WarrenThere are many DNA testing techniques that can be utilized for samples with low quantities of DNA. Mitochondrial DNA testing is designed for successful DNA sequencing of hair shafts, degraded and burned samples. Newly developed SNP (single nucleotide polymorphisms) testing is also designed for the analysis of challenging samples. The increased interest in the analysis of low copy number DNA samples using STR testing is necessitated since the national database CODIS (Combined Data Index System) currently only accepts the DNA profiles analyzed with the 13 core STR loci. CODIS contains DNA profiles of evidence found at crime scenes, convicted offender and missing persons DNA profiles (4). The goal of this project is to develop methodologies to increase the success rate of LCN DNA samples using STR testing. The experimental design for this study involved the amplification of DNA isolated from buccal swabs using the Profiler Plus multiplex kit at two different DNA input quantities: 0/0156ng (15.6pg) and 0.0312ng (31.2pg). Four separate amplifications of these DNA samples were done at: 28, 30, 32 and 34 cycles. The manufacturer’s recommended cycle number for AmpFISTR Profiler Plus is 28 cycles. These samples were analyzed on both the ABI Prism 310 Genetic Analyzer and the ABI Prism 3100 Genetic Analyzer using OCD standard protocols for loading samples. The injection time and voltage were modified for each of the number of PCR cycles. The best combination of cycle number and injection parameters was chosen for the low copy number reproducibility study.Item Development of a Comprehensive Massively Parallel Sequencing Panel of Single Nucleotide Polymorphism and Short Tandem Repeat Markers for Human Identification(2015-08-01) Warshauer, David H.; Bruce Budowle; Ranajit Chakraborty; Bobby L. LaRueMassively parallel sequencing (MPS) technologies allow for the detection of an unparalleled amount of genetic information with unprecedented speed and relative ease. These qualities make the technology desirable for generating DNA profiles that may be uploaded into forensic offender, arrestee, and family reference database files. This doctoral dissertation research was conducted under the hypothesis that MPS, with its exquisitely high throughput, can provide a system whereby reference samples can be typed for a large battery of markers, providing more discrimination power for forensic DNA typing and offering increased opportunities to develop investigative leads. The design and implementation of large marker panels for the typing of reference samples will reduce debates on the best core markers for forensic utility, generate innovation because focus will not be solely on a core set of autosomal STRs, promote the development of better systems that can analyze more challenging samples, and enable sharing of data across laboratories worldwide. The primary goal of this project was to develop the capability of typing reference samples for a large battery of markers: 84 autosomal, Y-chromosome, and X-chromosome short tandem repeats (STRs), Amelogenin, and 275 human identity single nucleotide polymorphisms (SNPs), in a single multiplex analysis. To that end, a bioinformatic software package, STRait Razor, was developed to detect STR alleles in raw MPS data. A proof-of-concept study was performed to evaluate the efficacy of using MPS to type forensically relevant markers, using a PCR multiplex-based SNP assay. The proposed comprehensive capture-based MPS panel then was designed and extensively tested. Finally, the benefits of the additional genetic data afforded by MPS, as opposed to traditional methods, were illustrated through the characterization of intra-repeat nucleotide variation within Y-chromosome STR alleles. The results of this dissertation research indicate that MPS is capable of providing robust genetic data from a wide variety of forensically-relevant STR and SNP loci in a single analysis. To date, the comprehensive MPS panel developed during the course of these studies is the most potentially informative assay for reference sample testing for human identification.Item Sequencing Long Amplicon Microsatellite Loci Using the Oxford Nanopore Technologies MinION[TM] Device(2019-05) Hall, Courtney L.; Planz, John V.; Zascavage, Roxanne R.; Phillips, Nicole R.; Menegaz, Rachel A.Forensic DNA typing utilizes highly variable short tandem repeat (STR) markers to differentiate individuals. Despite the power and reliability of current techniques, sequence-level variations are masked in the length-based profiles generated. Nanopore sequencing has the ability to provide long-read data, allowing for accurate alignment and identification of single nucleotide polymorphisms (SNPs) within and around microsatellite loci. To evaluate the applicability of nanopore sequencing to forensically-relevant autosomal and Y chromosome markers, selected STRs and their flanking regions (~800 bp) were amplified using custom primer sets, barcoded by sample, and sequenced on the MinION[TM] device. High quality sequencing data were obtained for all 24 samples at the 45 STRs interrogated using a customized data analysis pipeline. This project sets the foundation for future development of STRs for potential forensic applications as well as biomedically-relevant regions.Item Single Nucleotide Polymorphisms and Haplotype Analyses of Complex Medical Disorders(2008-05-01) Gonzalez, Suzanne D.; Arthur Eisenberg; Robert Luedtke; Rustin ReevesGonzalez, Suzanne D., Doctor of Philosophy. Cell Biology and Genetics. Single Nucleotide Polymorphisms and Haplotype Analyses of Complex Medical Disorders. Number of Pages: 129. Number of Tables: 25. Number of Illustrations: 5. Number of Titles Included in References: 197. There has been great difficulty in identifying genes involved in complex disorders. The complex genetic basis of these diseases indicates that either several genes act together to cause disease, or genetic heterogeneity is present in the population. This dissertation was aimed at developing new assays to identify polymorphisms in novel candidate genes that potentially contribute to two classes of common complex disorders: psychiatric diseases and metabolic disorders. Genotyping assays were developed to investigate single nucleotide polymorphisms (SNPs) and haplotypes in complex genetic disorders using multiplexed SNP panels, restriction fragment length polymorphism technology, and cycle sequencing platforms. An introduction to the study is provided in Chapter 1. Manuscripts focus on association studies of candidate genes in Bipolar Disorder and Schizophrenia (Chapter 2), Type 2 Diabetes, Hypertension and Metabolic Syndrome (Chapter 3), and baseline blood pressure in African Americans (Chapter 4). The summary of these manuscripts (Chapter 5) describes the significant associations made between SNPs/haplotypes in psychiatric and metabolic complex genetic disorders. Significant genetic associations of SNPs within the PHLPP gene were detected among schizophrenics (Chapter 2). The G allele of SNP rs8087170 was associated with the control population with the T allele of SNP rs12966002 was found only in schizophrenics. A significant variance was detected at SNP rs12457020 between bipolar and schizophrenic datasets, as there was a 10 fold increase in the A allele in the bipolar group. Significant associations of ATP1A2 5’ SNPs C-1489T and G-1253A were detected in metabolic syndrome and hypertensive groups (Chapter 3). Haplotypes based on these 3 SNPs were significantly associated with metabolic syndrome and hypertensive populations. Four linked ATP1A2 SNPs, G3756C, G3853A, C3913T and C3915T, were associated with baseline blood pressure (Chapter 4). Haplotypes associated with blood pressure in an ethnic specific manner. GGCC associated with lower blood pressures, while haplotype GGTT associated with higher blood pressures in African Americans. These studies provide new mechanisms to identify mutations and provide evidence supporting the pathophysiology of these disorders.Item STRspy-ing hidden variation in forensic DNA profiles using the Oxford Nanopore Technologies MinION device(2022-12) Hall, Courtney L.; Phillips, Nicole R.Forensic DNA examinations harness the high degree of repeat length variation characteristic of short tandem repeats (STRs) for human identification. Conventional approaches to STR profiling consist of PCR amplification followed by length-based separation and detection via capillary electrophoresis (CE). These well-established methods are used in forensic laboratories throughout the world to generate robust and reliable profiles that can discriminate between individuals based on differences in STR repeat length alone. The power of discrimination achieved with length-based allele designations across established panels of autosomal and YSTRs is often sufficient for routine DNA examinations. However, nucleotide-level variation within and around STRs has been shown to increase resolution and facilitate interpretation in more challenging casework scenarios such as those involving partial and mixed DNA profiles. The MinION is a DNA sequencer from Oxford Nanopore Technologies (ONT) that is small in both size and price tag. This portable device could provide an alternative for STR sequencing in forensic laboratories that cannot afford the initial investment or commitment of common next-generation sequencing (NGS) platforms. Despite this potential, the relatively high error rate and lack of STR analysis software have precluded accurate forensic profiling with nanopore sequencing in previous studies. This project aims to determine whether STRs amplified with a commercial kit can be sequenced and profiled on the ONT MinION device. To achieve our overall objective, we developed and tested a novel bioinformatic method known as STRspy that is designed to produce forensic STR profiles from third-generation sequencing data. The results presented herein demonstrate that STRspy can predict the correct sequence- and length-based allele designations across an entire panel of autosomal and Y-STRs using error-prone ONT reads as well as detect variation in the flanking regions with a high level of accuracy. Moreover, these data provide novel insight into how PCR-induced stutter and sample multiplexing impact STR profiling on the MinION. Ultimately, this work increases the feasibility of nanopore sequencing in forensic investigations and provides the foundation for future efforts that aim to harness the big potential of the small MinION device.Item Study of 5-HTTLPR and rs25531 to Identify Allele Variants of Offenders Charged with Driving while Intoxicated and Driving under the Influence(2013-07-01) Gudep, Ravali; Rhonda RobySerotonin, also referred to as 5-hydroxytryptamine, is a neurotransmitter which plays a critical role in behavioral and genetics studies. The 5-hydroxytryptamine-transporter-linked polymorphic region (5-HTTLPR), present in the promoter region of the serotonin transporter gene, can have varying number of repeats. In addition, a single nucleotide polymorphism (SNP), rs25531, is present within the repeats. A novel assay was designed to genotype 5-HTTLPR and its SNP variant in a single capillary electrophoretic injection. A total of 1 00 samples received from the Center for Alcohol and Drug Studies at San Diego State University Research Foundation were successfully genotyped.Item The Applicability of Whole Genome Amplification to Minute Quantities of DNA for Forensic Use(2004-08-01) Leal, Barbara L.; Joseph Warren; John Planz; Arthur EisenbergThis research study was performed at DNAPrint™ in Sarasota. FL. DNAPrint™ is a private laboratory specializing in Single Nucleotide Polymorphism (SNP) technology. Through the use of this technology, they have been able to map SNPs termed Ancestry Informative Markers. This has allowed them to perform additional research with regards to biogeographical ancestry, identification of an individual's physical traits, as well as pharmacogenomics research. In addition, DNAPrint™ also performs paternity and forensic testing using STR markers. This research study was a preliminary investigation of the applicability of Whole genome amplification (WGA) using the GenomiPhi™ DNA Amplification assay in conjunction with available STR kits. The GenomiPhi™ assay was utilized due to its ease and efficiency. Depending upon the outcome of this project, further studies may be conducted to incorporate WGA for use with both their SNP and STR technology.