Date of Award

12-2011

Document Type

Campus Access Thesis

Degree Name

Master of Science (MS)

Department

Biology

First Advisor

Kenneth L. Campbell

Second Advisor

Alexey Veraksa

Third Advisor

Kenneth C. Kleene

Abstract

Apoptotic detection methods were explored with emphases on fluorescence-based assays, and improvements upon current DNA amplification protocols such as ligase-mediated polymerase chain reaction, LMPCR, and multiple displacement amplification, MDA. Current methods detecting any aspect of apoptosis are not ideal as they may be labor intensive, time consuming, unsuitable for high throughput testing, or not specific or sensitive to apoptosis. Methods utilizing fluorescent intercalating dyes (i.e. PicoGreen, SYBR Green I, Syto 13, and Syto 82) in real-time enzymatic assays were explored. Fluorescence visualization assays are advantageous because of their exquisite sensitivity to tiny masses of DNA. However, these popular commercially available reagents have limitations, especially in the context of enzymatic assays. Differences in molecular size among DNA samples as well as reagents within enzymatic assays prevented these four intercalating dyes from detecting low nanogram masses of DNA. The fluorescence intensity (FI) of DNA/dye complexes were high, stable, and detected small masses of DNA if limited use to DNA quantitation at room temperature, as incubation at 37°C decreased FI over 30 minutes. The presence of cations suppressed the FI of DNA/dye complexes with Cr+3 being more effective than Mg+2, Ca+2, or Mn+2, which were more effective than Na+, or K+. Presence of proteins (i.e. bovine serum albumin, sheep IgG or bovine insulin) markedly decreased FI of DNA/dye complexes and produced fluorescent in absence of DNA. Immobilization of DNA on surfaces decreased the FI of DNA/dye complexes in comparison to DNA free in-solution. While capillary electrophoresis based methods using fluorescently-labeled DNA samples proved unsuccessful using current conditions, tests have indicated a fruitful improvement maybe found. Likewise, optimization of LMPCR and MDA provide additional means to assess apoptosis using no more than 3 ng target DNA. Since LMPCR exhibits a biased amplification of DNA lengths up to 3 kbp and MDA exhibits a biased DNA amplification towards DNA greater than 3 kbp, an optimal approach would combine these two methods in a single DNA assessment prior to agarose gels electrophoresis. Resulting from a variety of different dsDNA samples suggests this approach may generate a good representation of the apoptotic DNA in a small sample.

Comments

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