Title

Capillary electrophoresis with laser induced fluorescence for DNA damage analysis

Date of Completion

January 2007

Keywords

Chemistry, Analytical

Degree

Ph.D.

Abstract

Due to the advancements in organic chemistry, millions of new compound are created everyday. Some of these compounds when absorbed by the body can be activated by cytochromes p450 and react with DNA. Therefore, these compounds have to be assessed for possible genotoxicity before its release to the general public. Abasic sites are excellent biomarkers to quantify damage from reactions of DNA with genotoxic compounds and/or their metabolites. Abasic sites may occur in very small amounts. Therefore, a quick and sensitive analytical method like capillary electrophoresis with laser induced fluorescence (CE-LW) is required to measure them.^ In the first part of this thesis, a method to detect abasic sites was developed using CE-LIF. In this method a fluorescent tag that reacts specifically to abasic sites is incubated DNA. Even after ethanol precipitation and extensive washing excess unreacted probe is still present with DNA. This is probably due to intercalation of the fluorescent tag with the double stranded DNA. The unreacted tagged was however separated from tag attached to the abasic site by capillary electrophoresis. Thus the tagged and untagged probe elided as 2 different peaks in the electropherogram. Using this method we were able to get a limit of detection of 20 attomoles abasic sites.^ Damage to cellular DNA results in cell death or cancer. Of the 2 consequences, it is the latter which is more deadly. Cancer results when damage occurs at specific points within the DNA. In the second part of this thesis, we developed a method to determine sequence specificity of DNA damage using CE-LIF array. This is done by determining the exact location of the abasic site which was formed by the removal of the damaged base. A 5' fluoropore tagged DNA fragment of known sequence was incubated with styrene oxide. Heating the DNA fragment under optimized conditions releases the adenine and guanine adducts while leaving the unmodified guanines and adenines intact. This procedure leaves a piperidine cleavable abasic site at the point where the damaged nucleobase existed. Using correction tools we were able to measure the exact size of the fragments which helped us determine the exact location of the damaged point. ^