Publication Date

Summer 2016

Advisor(s) - Committee Chair

Dr. Moon-Soo Kim (Director), Dr. Kevin Williams, Dr. Blairanne Williams

Degree Program

Department of Chemistry

Degree Type

Master of Science

Abstract

RASSF1A is a tumor suppressor gene which loses its function due to methylation of CpG islands on its promoter region. Detection of methylation leads to early diagnosis of cancer.

Zinc finger proteins are capable of detecting a specific DNA sequence and Methyl binding domain can bind to the methyl group on the CpG, using this idea mCpG SEER- Lac system makes use of a split protein, β-lactamase. Lac A attached to the ZFP and Lac B attached to the MBD protein. On binding to the DNA, the Lac A and Lac B come in close proximity with each other causing a reassembly and activation of the enzyme. In the presence of a substrate, the activated β-lactamse enzyme hydrolyzes the β-lactam bond in the substrate and shows a color change from yellow to red in the presence of a methylated cognate DNA.

The study suggests that a solution based assay was not as specific in differentiating signal intensities between methylated and non-methylated DNA. It was also not sensitive in measuring dose dependent signals. Zinc finger array could successfully show relatively low signals for non-methylated DNA. The findings of the study show that MBD2 shows higher preference for mCpG than MBD1 in the mCpG SEER-Lac system and oligonucleotides with a 2 bp spacing between methylation and ZF target site shows higher signals than the 3 bp spacing. Due to it’s specificity and sensitivity, it serves as a potential diagnostic tool to detect cancer.

Disciplines

Biochemistry, Biophysics, and Structural Biology | Biological and Chemical Physics | Biophysics

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