S-Space College of Agriculture and Life Sciences (농업생명과학대학) Dept. of Plant Science (식물생산과학부) Theses (Master's Degree_식물생산과학부)
Detection of Epialleles by Measuring Differential DNA Methylation Levels Using Plant-Specific DNA Demethylase
식물 특이적인 DNA 탈메틸화 효소를 이용하여 DNA 메틸화 수준을 파악함으로써 후성대립유전자를 탐지
- 농업생명과학대학 식물생산과학부
- Issue Date
- 서울대학교 대학원
- 학위논문 (석사)-- 서울대학교 대학원 : 식물생산과학부, 2015. 2. 허진회.
- DNA methylation is one of the crucial epigenetic factors to control gene expression in plants and mammals. Aberrant changes in DNA methylation often lead to the formation of epialleles in plants, and the epimutants that harbor such epialleles sometimes display stable transgenerational inheritance. Therefore, DNA methylation analysis is required to detect epialleles. However, conventional DNA methylation analyses sometimes are not sufficient to detect epialleles. Here we report a novel DNA methylation analysis method combining both 5-methylcytosine (5mC) excision activity of plant-specific DNA demethylase DEMETER (DME) protein and a quantitative real-time PCR (qRT-PCR) technique. Because DME induces a nick in a sequence non-specific manner at the position where 5mC is present, heavily methylated targets cannot be PCR-amplified due to a number of DNA strand breaks, whereas unmethylated or less methylated regions can be easily amplified. We demonstrate the feasibility of DME-qPCR by successfully distinguishing between wild type and two epialleles, fwa and Cnr, derived from Arabidopsis and tomato, respectively. This novel method has versatility over other enzyme-based tools, and at the same time, may overcome several problems that current DNA methylation analysis principles have – such as pretreatment and intrinsic technical bias.