Date Approved
2022
Degree Type
Open Access Thesis
Degree Name
Master of Science (MS)
Department or School
Biology
Committee Member
Anne Casper, Ph.D.
Committee Member
Robert Winning, Ph.D.
Committee Member
Andrew Ross, Ph.D.
Abstract
Dynamic Molecular Combing (DMC) is a method of making visible single molecules of DNA. The method stretches the molecules, attaching them to a glass coverslip; labels them with an intercalating dye or fluorescent antibodies; and images the molecules with fluorescent microscopy. After combing, fluorescent in situ hybridization (FISH) can be used to inspect regions on the DNA molecule. DMC is especially technically challenging for undergraduate research laboratories. To make the technique accessible to an undergraduate laboratory this project addresses three challenges: pH of buffer solution, NaOH exposure time, and halogenated thymidine exposure time in yeast (Saccharomyces cerevisiae). A serial pH experiment is necessary to determine optimal pH in any given laboratory. The optimal NaOH exposure time is 20 minutes. Despite using flow cytometry to determine the amount of time spent in S phase, the expected correlation between halogenated thymidine exposure and labeled tract length was not found, leaving the third challenge for further optimization.
Recommended Citation
Mishler, Jeanmarie, "The promise and perils of molecular combing" (2022). Master's Theses and Doctoral Dissertations. 1174.
https://commons.emich.edu/theses/1174