Quantitative PCR methods for assessing epichlorohydrin damage within chicken erythroid cells

CHED 894

Megan L. Watts, mlwatts@colby.edu and Julie T. Millard, jtmillar@colby.edu. Department of Chemistry, Colby College, 5750 Mayflower Hill Drive, Waterville, ME 04901
Epichlorohydrin (ECH), common in the synthetic polymer industry, is a bifunctional alkylating agent that undergoes reaction with DNA. To assess whether DNA structure affects ECH reactivity in vivo, we are developing quantitative PCR techniques to monitor damage at different loci of 6C2 chicken erythroid cells. We are targeting mitochondrial DNA and distinct regions of nuclear DNA near the beta-globin domain that differ in their degree of packaging. Because the folate receptor gene is expressed in 6C2 cells, we are designing primer sets to amplify within this locus to assess ECH targeting of “open” chromatin. Targeting of this locus will be compared to that of a highly condensed, unexpressed region to the 3' end of the beta-globin gene. Micrococcal nuclease digestion will be used to verify DNA accessibility at these sites. Our ultimate goal is to provide insight into the DNA damage arising from ECH exposure.