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研究生:蘇家興
研究生(外文):Chia-Hsin Su
論文名稱:利用Alu-PCR指紋分析及Bisulfite定序尋找及選殖小鼠組織基因體DNA中的甲基化的片段:p53啟動子到s27基因之間組織特異性的CpG甲基化的分析
論文名稱(外文):Dvelopment of a Novel Method for Cloning and Characterization of Methylated DNA sequence in Mouse Tissues: Analysis of Tissue-Specificity of CpG Methylation Sites from p53 Promoter to s27 Gene
指導教授:徐明達徐明達引用關係
指導教授(外文):Ming-Ta Hsu
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:生物化學研究所
學門:生命科學學門
學類:生物化學學類
論文種類:學術論文
論文出版年:2001
畢業學年度:89
語文別:中文
論文頁數:78
中文關鍵詞:甲基化組織特異性
外文關鍵詞:Alu-PCRBusulfite sequenceTissue-specificity methtlation
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摘 要
利用共價修飾核酸(DNA)的機制來調控基因的表現是一種直接且強而有力的方法,此機制亦稱之epigenetic,也就是不改變核甘酸序列即能改變基因的表現。核酸甲基化就是其中一種epigenetic的機制,在真核生物中它主要是利用甲基化酵素(methyltransferase)將甲基共價在5’-CG-3’序列上cytosine環上的第五號碳的位置。核酸甲基化這種特殊的核酸標記已知和調控一些基本基因的表現和組織特異性的基因有密切的關係,並且也調節一些細胞功能的表現如不活化的X染色體和染色體印跡基的表現、還有核酸的突變、癌症的形成越來越多證據都指出和核酸甲基化有關。僅管核酸甲基化是那麼重要,但是即將完成的基因體計畫序列卻不能告訴我們任何核酸甲基化的任何訊息又加上過去的甲基化研究都只限於在已知的基因。有鑑於此,我們發展一種新的技術用甲基化敏感inter-Alu PCR的方法能夠廣泛地在基因組中來搜尋組織特異性甲基化片段和胚胎組織中甲基化片段。我們利用這種技術成功地選殖到一些甲基化片段其中包括了有組織特異性甲基化片段和不同階段的甲基化片段。同時我們也利用bisulfite定序方法來偵測p53基因和s27基因所有CpG甲基化的情形,結果發現在p53基因啟動子內所有的CpG都沒有甲基化而相反地在p53基因啟動子之前直到s27基因之間的CpG都甲基化。另外,我們感到興趣的是在甲基化和沒有甲基化之間的界線,經我們bisulfite定序結果發現有組織特異性的CpG甲基化。現在,我們更進一步發展出更能廣泛地在基因組中尋找甲基化片段以期待能夠找出更多的組織特異性和不同時其的甲基化片段。
Abstract
The covalent modification of DNA provides a direct and powerful mechanism to regulate gene expression. DNA methylation in eukaryotes involves addition of a methyl group to the carbon 5 position of the cytosine ring. This reaction is catalyzed by DNA methyltransferase in the context of the sequence 5’-CG-3’, which is also referred to as a CpG dinucleotide. It’s the most common eukaryotic DNA modification and is one of the many epigenetic mechanism. This special DNA marking has been closely associated with controlling the expression of housekeeping genes and possibly also tissue-specific genes, as well as several important cellular functions such as X chromosome inactivation and genomic imprinting , as well as mutagenesis and tumorigenesis. Unfortunately, the ongoing genomic sequencing projects are unable to provide information on this important area of gene regulation and all current investigations on changes in methylation status have been limited to known genes. To initiate a genome-wide search for differentially methylation pattern in a tissue- or development-specific manner, we developed a methylation-sensitive Alu-PCR technique to examine the mathylated DNA sequence in different mouse tissues at different stage of development. Using this technique we have cloned and sequenced several methylated sequences, some tissue-specific. We have then examined in detail the methylation status of each CpG sites in a 6 kb region surrounding the s27-p53 gene using bisulfite DNA sequencing technique. The results revealed an interesting methylation transition boundary that demarcated the full methylated region from the unmethylation region and a tissue-specific methylated site in the transition boundary. Currently, we are developing a more global methodology for fishing out tissue- and developmental stage-specific methylated sites.
目錄
中文摘要………………………………………………………1
英文摘要………………………………………………………2
背景……………………………………………………………3
材料和方法…………………………………………………...11
實驗結果……………………………………………………...21
討論…………………………………………………………...27
附圖…………………………………………………………...33
附錄…………………………………………………………..54
參考文獻……………………………………………………...68
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