Real time ribonucleic acid based amplification allows for sensitive forensic blood evidence analysis

The purpose of this experiment was to determine if nucleic acid sequence based amplification (NASBA) is a suitable application for the differentiation of body fluids that might comprise a forensic evidence sample. NASBA is a sensitive RNA transcription based amplification system. NASBA could theorhetically be used for bodily fluid identification based upon amplification of tissue-specific mRNA transcripts present in a given forensic sample.Amplification of both Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and Matrix Metalloproteinase 1 1 (MMPmRNA transcripts were used to determine that NASBA could amplify body fluid transcripts and whether it could distinguish between menstrual and non-menstrual blood, respectively. GAPDH is a housekeeping gene that is constituently expressed and its mRNA transcripts could therefore be used to determine whether non-menstrual blood could be amplified using the NASBA procedure. MMP 11 is a menstrual cycle-specific gene associated with endometrial breakdown. Using the mRNA transcripts from MMP 11, NASBA could be utilized for menstrual blood identification. In this study, non-menstrual and menstrual blood samples were analyzed with NASBA both in the presence and absence of chemical contamination. Contaminants utilized ranged from commercial automotive wax, transmission fluid, brake fluid, artificial tears, hand soap, 10% bleach, and the luminol blood detecting reagent. Non-menstrual blood was aliquoted onto a 1 cm x 1 cm cotton cloth for contamination, while menstrual blood was provided on a 1 cm x 1 cm area of sterile menstrual pad. All samples underwent Tri reagent extraction to obtain RNA samples for NASBA amplification.With respect to NASBA amplification data, non-menstrual blood data (from extracted RNA and unextracted blood samples) revealed the highest levels of amplification as shown in relative fluorescence units (RFU). Uncontaminated menstrual blood revealed the second highest amplification data. In the presence of chemical contamination, high levels of amplification were observed when samples were contaminated with brake fluid and commercial hand soap. Moderately low amplification was observed with samples contaminated with transmission fluid, 10% bleach, and artificial tears. NASBA amplification was completely inhibited in the presence of automotive wax and luminol. Cycle threshold (CO values for each amplification result were also obtained from each reaction. Smaller Ct values correspond to a higher NASBAreaction efficiency and therefore larger amplification values. The Ct values obtained for each amplified sample correlate strongly with the amount of amplification observed from reaction. Based upon the results of this experiment, NASBA should be considered as a novel tool for forensic evidence analysis.Department of BiologyThesis (M.S.