Browsing by Subject "allele"
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Item Construction of a Cost Effective Nested-PCR Reaction for Use with the Applied Biosystems AmpFLSTR Identifiler Kit(2005-12-01) Mikeska, Margo M.; John Planz; Joseph Warren; Arthur EisenbergHuman STR analysis has greatly increased the ability to perform identity testing for many different situations. These situations include, but are not limited to, the identification of individuals involved in violent crimes, establishing paternity, and identification of unknown human remains. The most common type of DNA information currently used for identity testing is the short tandem repeat, or STR. STR testing utilizes the number of repeating units in the DNA to assign an allele. Alleles from several different loci are used to establish a genetic profile. Currently, the United States used a standard of 13 different DNA loci to establish identity. These 13 loci can be typed by using a number of different multiplex kits such as the Applied Biosystems Profiler Plus, Cofiler, and Identifiler Kits [1,2]. The 13 loci were selected based on a number of parameters. Each locus was required to be polymorphyic, and a tetranucleotide repeat. The loci also could not display any linkage between each other and extensive population studies had to be conducted to both verify the absence of linkage and to establish allelic frequencies [1]. The goal of this research was the construction of a more cost effective method of utilizing the Applied Biosystems Identifiler Kit. Across the country there is a large backlog of samples that need to be processed in order to obtain a genetic profile. If these samples could be tested using a more cost effective method, more funding could be directed to other endeavors. Paternity testing, as well as some research endeavors could be conducted at a fraction of the cost, leaving resources for other projects or additional staff. Although it would be inadvisable to use this technique on forensic samples, the implications on paternity and research samples would be positive. This research attempted to design a nested PCR reaction and subsequently dilute the Applied Biosystems Primers in order to reduce the cost. The first step was to design new primers for the first round of PCR, followed by testing of those primers. The new primers then required optimization so that they all worked effectively together. After optimization was accomplished, the Identifiler primers were diluted until loci began dropping out of the genetic profile.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.