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Nucleic Acid Sequence Based Amplification (NASBA) is a powerful in vitro technique for amplification. NASBA is routinely used in many fields of microbiology, including food microbiology, and most recently in the identification of forensic body secretions (saliva, tears, sweat, semen, vaginal secretions). NASBA has many advantages over the traditional Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) including speed, high sample throughput and increased sensitivity. Proper selection of the sequence of importance and the designe of NASBA primers precisely for that sequence are the two most critical steps for any NASBA assay. Proper designe of NASBA primers includes important considerations such as product (amplicon) length, the addition of a T7 RNA polymerase promoter sequence at the 5’end of one primer, and a 3’AT rich region. Primer designing is, therefore, laborious and error-prone. Currently, no such software is available that facilitates primer designing for NASBA. In this study Java-based software for designing NASBA primers was developed which will enable rapid and specific NASBA primer designing for gene expression studies. The designed program focused on scripting minimum Java coding lines to reduce the time and storage space. Two codes were scripted for this software, a pseudo-code (for Java Program Developers) and Byte-code (for Windows operating system). Our results showed that Java is an efficient tool in searching sequences of interest within a gene (or mRNA), allowing for NASBA primers to be designed more quickly and effortlessly. The program has maximum memory storage capacity and allows the users to retrieve old data with reference to date or time. To test the practicality of the newly developed program, gene sequences of salivary mucin and amylase were examined to facilitate extraction of novel RNA tandem repeat element NASBA markers for human saliva forensic identification. Tandem repeats of non-coding portion with in the of human genome are highly polymorphic and considered best for forensic use. Currently, only 13 Short Tandem Repeat (STR) markers are available for forensic case work, which are not enough to establish a definitive link between the victim and suspects. Identification and validation of a new human body fluid tandem repeat markers is cruicial for achieving high through put results and to exonerate the innocent. / Access to thesis permanently restricted to Ball State community only / Department of Biology

Mucin was isolated for incorporation in bacteriological media by reduction and proteolysis of mucous gel from porcine small intestine. Mucin prepared in this study contained (by weight) 37.2% protein, 58.7% carbohydrate (4.6% fucose, 9.4% mannose, 10.7% sialic acid, 13.5% galactose, and 20.5% hexosamine), and 3.7% ester sulfate. Fractionation of mucin on Sepharose CL-4B yielded one peak which eluted at the void volume. However, sodium dodecyl sulfate gel electrophoretic patterns contained 9 polypeptide bands of which 6 stained with periodic acid—Schiff reagent. Proline, serine, and threonine residues accounted for 26% (by weight) of the total protein in the preparation. Half-cysteine residues made up another 1%. Intrinsic viscosity of mucin prepared by reduction and proteolysis was 135 ml/g. Mucin was incorporated into a minimal bacteriological medium as the sole-source of carbon and nitrogen. Enterotoxigenic and non-enterotoxigenic Escherichia coli grew equally well in mucin medium at levels comparable to growth in 3 mM glucose medium. Growth did not appear to be limited by availability of metabolizable substrates. Spent mucin medium supported growth in comparable numbers. E. coli P-155 produced heat-stable and heat-labile enterotoxins during growth in both mucin medium and fresh mucosal scrapings medium. E. coli utilized total hexose and protein in mucin medium at comparable levels (6 to 10%). Increases in reducing end groups (0.28 μ moles/ml) and free amino sugar end groups (0.04 μmoles/ml) during growth were detected. E. coli used approximately 15% of the total carbohydrate in mucin medium including 36% of the galactose, 15% of the fucose, and 27% of the mannose. Utilization of mucin by QL ggli produced minor changes in gel filtration patterns on Sepharose CL-4B. Twelve strains of E. coli were examined for glycosidase activity during growth on mucin. All twelve produced a cell-bound and an extracellular α-fucosidase although the majority of activity was cell-bound. Although α-fucosidase was a constitutive enzyme of E. coli P-155, maximum activity was observed during exponential growth in mucin medium. Eleven strains produced cell-bound α-galactosidase. No extracellular activity of this enzyme was detected. Maximum levels of induced α-galactosidase activity were obtained in late exponential to early stationary growth of E. coli. E. coli ATCC 23723, a mutant of E. coli K12 lacking the galactoside permease gene, did not produce α-galactosidase activity during growth on mucin. No α-mannosidase activity was detected using nitrophenylmannoside as substrate. Porcine small intestinal mucin was a positive chemoattractant for E. coli in capillary assays. Optimal chemotactic response by E. coli P-155 in capillary experiments was obtained at a mucin concentration of 1 mg dry wt/ml at a pH of 7.0. Spent mucin was still a positive chemoattractant for E. coli P-155 and 123 despite losing 15% of the total mucin carbohydrate. / Ph. D.