Browsing by Subject "polymerase chain reaction"
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Item Obesity Genetics: The Prevalence of DRD2, DAT1 and DBH Genes in the Obese Individual(1998-08-01) Davis, Karla R.; Eisenberg, Arthur; Agarwal, Neeraj; Sherman, MarkDavis, Karla R., Obesity Genetics: The prevalence of DRD2, DAT1 and DBH Genes in the obese individual. Master of Science (Biomedical Sciences), August, 1998, 106 pp., 3 tables, 14 illustrations, reference, 44 titles. Obesity has been presented in research literature as a polygenic or multiple gene disorder. Currently, 3 genes have been associated with obesity, dopamine receptor D2 (DRD2), dopamine transporter (DAT1), and dopamine beta hydroxylase (DBH). The primary objective of this study is to analyze the DRD2, DAT1 and DBH genes to determine if a correlation exists between certain allelic variations of these 3 genes and the body mass index of obese individuals. We have developed an assay for the DRD2, DAT1 and DBH genes, utilizing polymerase chain reaction (PCR) technology. Within the DRD2 gene, 2 allelic variants have been identified, the A1 and A2 alleles. The A1 allele consists of a 310 bp fragment in which the Taq 1 restriction site has been deleted. The A2 allele consists of 180 bp fragment and a 130 bp fragment. The presence of the A1 allele after enzyme digestion has shown a strong correlation to obesity in prior studies. With respect to the DAT1 gene, a VNTR of 40 bp’s has been correlated to other disorders within the ‘reward deficiency syndrome’. The fragment length identified most often is 440 or 480 bp, with 480 as the primary fragment in obesity. The DBH gene is similar to the DRD2 in that it also contains a Taq I restriction. Two allelic variants are also identified, B1 and B2. The B1 allele contains no Taq I site and produces a 316 bp fragment while the B2 does cleave, exhibiting an 86 bp and a 230 bp fragment after enzyme digestion. The presence of one or more of the aberrant alleles could be associated with and a predisposing factor to obesity.Item Validation of Four Multiplex SNP Panels for Forensic DNA Testing: An Assessment of the Sensitivity and Reproducibility of the SNaPshot Primer Extension Assay(2003-05-01) Dutton, Kristi R.; John Planz; Arthur Eisenberg; Joseph WarrenOrchid Cellmark provided four proprietary multiplex SNP primer panels, each of which has been developed to identify 10-12 SNPs using a modified version of the SNaPshot Mutliplex protocol. The commercially available STR typing kits routinely used in forensic testing require an input of between 0.5 to 2ng of DNA. Orchid Cellmark has suggested using 2ng of DNA with each of their multiplex SNP primer panels. However, preliminary data has indicated that as little as 100pg of DNA can yield results with many of the SNP markers. Several methods of SNP detection exist. This project relied on the use of multiplex SNP extension primers in conjunction with the ABI SNaPshot Multiplex Kit (Applied Biosystems, Foster City, CA) to identify SNPs in the nuclear genome. Analysis of 50 or more SNPs would be very laborious if single-tube polymerase chain reaction (PCR) was used for sample testing. Multiplexing SNP primers to include 10-12 per reaction tube will increase the throughput of SNP analysis. The cost of analysis can also be reduced using multiplexes since the amount of reagents per SNP is decreased. This project determined if the SNaPshot extension assay used in conjunction with the Orchid Cellmark multiplex panels could accurately detect SNPs at quantities less than 2ng DNA on a capillary electrophoresis (CE) platform.