Methylation-Dependent Rolling-Circle Amplification: A Novel Method for Detecting Dna Methylation

Date of Award

2012

Document Type

Thesis

Degree Name

Bachelors

Department

Natural Sciences

First Advisor

Hart, Christopher

Keywords

Methylation, DNA, Molecular Biology

Area of Concentration

Biology

Abstract

Epigenetics describes the complex network of covalent and noncovalent modifications to DNA and histone proteins, which cooperatively act to regulate gene expression without changes to the Watson-Crick base pairing of the DNA sequence. DNA methylation, the addition of a methyl group to cytosine residues in the DNA sequence, is an epigenetic regulatory mechanism that primarily influences transcription. Aberrant methylation has been discovered in numerous human diseases, giving the study of DNA methylation tremendous biomedical importance. Currently numerous assays exist for identifying methylation but no current methods are able to detect methylation at single-nucleotide resolution in situ. This study describes a novel method for detecting site-specific methylation on single DNA molecules. This novel technique, called methylation-dependent rolling-circle amplification (MD-RCA), uses methyl-sensitive restriction enzymes to discriminate between methylated and unmethylated targets followed by the use of padlock probes to create circularized templates for replication using rolling-circle amplification. After confirming that methylation-dependent target degradation and ligation-dependent rolling-circle amplification of synthetic targets was possible, methylation-dependent rolling-circle amplification was validated in vitro. Results demonstrated that preferential amplification of methylated targets allowed accurate detection of methylated loci. Conformation of this technique in vitro has established that the basic design is functional and ready for development as an in situ single-CpG methylation detection assay.

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