Development of NASBA-primer search software for designing forensic saliva tandem repeat markers for mucin and amylase

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Ara, Andleeb.
McKillip, John L.
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Thesis (M.S.)
Department of Biology
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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.