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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

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dc.contributor.advisor허진회-
dc.contributor.author최우리-
dc.date.accessioned2017-07-14T06:26:15Z-
dc.date.available2017-07-14T06:26:15Z-
dc.date.issued2015-02-
dc.identifier.other000000025597-
dc.identifier.urihttps://hdl.handle.net/10371/125569-
dc.description학위논문 (석사)-- 서울대학교 대학원 : 식물생산과학부, 2015. 2. 허진회.-
dc.description.abstractDNA 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.-
dc.description.tableofcontentsABSTRACT i
CONTENTS iii
LIST OF TABLES v
LIST OF FIGURES vi
LIST OF BBREVIATIONS viii

INTRODUCTION 1
LITERATURE REVIEWS 4
1. Plant epigenome
2. Principles of DNA methylation analysis
3. Epialleles and epimutants
4. Plant-specific DNA demethylase
MATERIAL AND METHODS 11
Purification of DNA demethylase, DEMETER (DME)
Preparation of in vitro-methylated plasmids
Plant materials
Cleaved Amplified Polymorphic Sequences (CAPS) Analysis
Bisulfite Sequencing
Preparation of quantitative real-time PCR templates
Analysis of DNA methylation using quantitative real-time PCR
RESULTS 17
CCGG methylation was detected by using DME, not McrBC
Proportion of CG-methylated pUC19 plasmids in total plasmid templates was measured by quantitative PCR after DME treatment
DME-qPCR distinguished epialleles from wild type alleles
DME-qPCR showed difference between hypermethylated wild type allele and hypomethylated fwa allele in F2 population from Arabidopsis
DISCUSSION 31
REFERENCES 34
ABSTRACT IN KOREAN 41
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dc.formatapplication/pdf-
dc.format.extent2091450 bytes-
dc.format.mediumapplication/pdf-
dc.language.isoen-
dc.publisher서울대학교 대학원-
dc.subjectEpiallele-
dc.subjectDNA methylation-
dc.subjectAnalysis of DNA methylation-
dc.subjectDEMETER-
dc.subject.ddc633-
dc.titleDetection of Epialleles by Measuring Differential DNA Methylation Levels Using Plant-Specific DNA Demethylase-
dc.title.alternative식물 특이적인 DNA 탈메틸화 효소를 이용하여 DNA 메틸화 수준을 파악함으로써 후성대립유전자를 탐지-
dc.typeThesis-
dc.contributor.AlternativeAuthorWOO LEE CHOI-
dc.description.degreeMaster-
dc.citation.pagesix, 42-
dc.contributor.affiliation농업생명과학대학 식물생산과학부-
dc.date.awarded2015-02-
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