摘要

本論文主要是以人類染色體20為主要的分析對象，而在2003年人類染色體14的序列被發表後，本研究也將人類染色體14也納入研究對象。本研究之所以選擇人類染色體20以及14當研究的材料，原因如下：首先，這是兩條已被定序完成的染色體；其次，染色體20是第一條被定序出來的metacentric chromosome，而染色體14則是人類染色體中五條acrocentric chromosome之一，因此，人類染色體20以及14，在其染色體的結構上各有其代表性；除此之外，本篇論文選擇人類染色體序列作為研究的對象，因為這是人類本身的遺傳載體，而由於基因體序列的解開，得以以分子的角度，來全面性的(除了目前定序技術尚未能突破的，而沒有解開的小部分區域—gap以外)探討染色質體上的核苷酸分布的情形。基於以上的理由，所以本研究選取人類染色體20以及14及來進行研究。

微衛星體是十分令人感興趣的研究目標，因為過去十餘年來，微衛星體一直是很好的分子與遺傳標誌(molecular and genetic makers)，利用微衛星體標誌，科學家可以將基因的位置標定出來(gene mapping)，研究基因的表現是linkage equilibrium 或是linkage disequilibrium，這可以進一步釐清基因與基因之間的相對位置，或是縮小對致病基因研究的範圍。在許多的研究中，更指出微衛星體可以參與基因的調控，例如加強子(enhancer)、蛋白質的結合位置(通常是一些轉譯因子的結合位置)。

本篇論文，是應用生物資訊學的方法，探討已被定序完成的20及14染色體序列中，微衛星體分布的情形以及相關的研究。利用已公開的染色體序列，以電腦程式來分析其上di-、tri-、tetra-nucleotide microsatellite分布的情形、GC content、以及基因密度。結果得到了的微衛星體序列情形，並且與前人發表的報告，以linkage map為基礎的實驗，相比較，驗證實驗之正確性。此外由本論文中所訂出的微衛星體與基因密度相互分析的結果，得知本研究的探索的方向與使用的方法，找到的微衛星體之位置與數目，可能可對後來的相關研究提供有關：分子標誌與遺傳標誌的線索，將有助於未來之研究。

Abstract

. A draft of the whole human genomic sequence has been completed and published in 2001. Researches on structural genomics, functional genomics, proteomics, evolutionary and medical sciences have just begun since. The existence of ninety percent repetitive, non-coding sequences in the human genome and in other mammalian species as well, indicating some potential but unaware functions of these repetitive regions within the mammalian genomes remained to be discovered.

Among all features in the genomic sequences, microsatellite has been characterized as one type of short tandem repeats (STRs), abundant in the human genome, which potentially plays some important roles in biological processes, such as makers for molecular biology, the enhancers for transcription, and protein-binding sites. Changes on microsatellite copy numbers also involve in the cause of a couple of diseases have been reported.

The sequence of human chromosome 20 has been finished in Dec. 2001, which provided us a valuable resource to study about gene density and the distribution of repetitive sequences such as microsatellties as well. To build a more detail chromosome 20 microsatellite map, including di-, tri- and tetra-nucleotide microsatellites, we therefore designed a sequence-based, molecular markers discovery system by bioinformatics approaches. The results indicate that these repetitive sequences distribute across the chromosome 20 evenly. We further analyzed the GC content of the human chromosome 20. A comparison of GC content, microsatellite distribution and gene density of each contig shows some correlation among them. The slippage rates of di-nucleotide microsatellite were predicted by the formula basing on Markov Chain. Some interesting results were found in this thesis. The same approaches were applied on human chromosome 14, which was being completed sequencing in Feb. 2003. The results implied that our strategies might be useful in the advanced genetic studied or medicine researches.

目錄
中文摘要
英文摘要
誌謝
表目錄....................................................................................I
圖目錄................................................................................III
第一章 文獻回顧.................................................................1
第一節 人類基因體計劃.........………......................................1
第二節 人類染色體重複序列..........................................….1-4
第一項 Long Interspersed Element (LINE)……………………2
第二項 Short Interspersed Elements (SINE)…………………..2
第三項 衛星體(Satellite)………………………………………2-3
第四項 小衛星體(Minisatellite)…………………………………3
第五項 微衛星體(Microsatellite)………………………………..4
第三節 微衛星………………………………...........................4
第一項 微衛星體在細胞中的功能…………………………….4-6
第二項 微衛星體的多型性(polymorphism)產生的可能假說….7
第四節 人類染色體20……………………………………………...7-8
第五節 人類染色體14………………………………………………..9
第六節 馬可夫鏈(Markov Chain)…………………………………10
第二章 序言......……....................................................11-12
第三章 材料與方法...........................................................13
第一節 微衛星體在人類染色體20上分布的分析……….................................................................13-14
第二節 WHY Computer Program...............………….........15
第三節 人類染色體20上GC content的分析……………16
第四節 人類染色體20上，基因密度與微衛星體密度的分布
比較………………………………………………….16
第五節 人類染色體20上，以sequence-based method 找到的微衛星體與linkage-based 微衛星體之比較…...17
第六節 人類染色體20上，微衛星體滑脫率(Slippage rate)的推算……………………………………………..17-18
第七節 人類染色體14上，微衛星體的分析比較…………19
第八節 DNA序列擷取的相關生物資訊中心…………………...20-21
第四章 結果.......................................................................22
第一節 微衛星體在人類染色體20上分布的分析..............22
第二節 人類染色體20上GC content的分析………......22-23
第三節 人類染色體20上Sequence-based microsatellites與linkage-based microsatellites 的比較.............................23-24
第四節 人類染色體20上，基因密度與sequence-based microsatellite密度的比較………………………………...…24
第五節 人類染色體20上，微衛星體滑脫率的比較.......….25
第六節 人類染色體14的分析..........................................…..25
第一項. 人類染色體14上sequence-based microsatellites的分析…………………………………………………………….25
第二項. 人類染色體14上sequence-based GC content的分析…………………………………………………………….26
第三項. 比較人類染色體14上sequence-based microsatellites與linkage-based microsatellites…………………………………26
第四項. 綜合比較人類染色體14及20上基因密度與sequence-based microsatellite密度…………………………..27
第五項. 人類染色體14上，di-nucleotide microsatellite 的滑脫率……………………………………………………………..27
第五章 三核甘酸重複序列於人類第20、21與22條染色體之分析結果比較...............................................28
第六章 討論..................................................................29-34
參考文獻........................................................................35-39





表目錄

表一 人類染色體20上微衛星體重複次數(frequency)之比較………………………………………………………………….40
表二 人類染色體14上微衛星體重複次數之比較……………41
表三 人類染色體20及14 上，各contig之GC content與基因密度之比較………………………………………………42
表四 人類染色體20上，每1Mb的長度中的基因數目與疾病基因……………………………………………………43-45
表五 人類染色體20上，含ACG與TCG的片段的 GC content與contig GC content的比較……………………46-47
表六 人類染色體20上，linkage-based的微衛星體資料整理……………………………………………………48-51
表七 人類染色體14上，linkage-based的微衛星體資料整理……………………………………………………52-59
表八 本論文人類染色體20 sequence-based microsatellit與linkage-based microsatellites之比較………60-62
表九 人類染色體20上， sequence-based di-nucleotide microsatellites較linkage-based microsatellites多出的倍數………………………………………………62
表十 人類染色體20上，較長的tri-nucleotide microsatellites在contig上的位置與重複次數，以及所在的位置相關的基因……………………………63-64
表十一 人類染色體20及14上，不同的di-nucleotide microsatellites滑脫率的比較……………………..65

圖目錄

圖一 人類染色體20及其七個contigs………………….66
圖二 人類染色體14及其contig………………………...67
圖三 人類染色體20上tri-nucleotide microsatellites定位
圖四 人類染色體20上，基因密度與di-nucleotide microsatellites密度的分析……………………68-75
圖五 人類染色體20上，基因密度與tri-nucleotide microsatellite密度的分析………………………..76
圖六 人類染色體20上，基因密度與tetra-nucleotide microsatellite密度的分析………………………..77
圖七 人類染色體20上，di-，tri-，tetra-nucleotide microsatellites的分布…………………………….77
圖八 人類染色體14上，基因密度與di-nucleotide repeat microsatellite密度的比較………………………..78
圖九 人類染色體14上，基因密度與tri-nucleotide microsatellite密度的分析………………………..79
圖十 人類染色體14上，基因密度與tetra-nucleotide microsatellites密度的分析………………………80
圖十一 人類染色體14上，di-，tri-，tetra-nucleotide microsatellites的分布…………………………….80
圖十二 人類染色體20及14上，di-，tri-，tetra-nucleotide microsatellites的密度比較……………………….81
圖十三 人類染色體14上，各種di-nucleotide microsatellite所佔的比例……………………………………….82
圖十四 人類染色體14上，各種tri-nucleotide repeats所佔的比例………………………………………….83
圖十五 人類染色體14上，各種tetra-nucleotide microsatellites所佔的比例……………………….84
圖十六 人類染色體20上，各種di-nucleotide microsatellites所佔的比例……………………… 85
圖十七 人類染色體20上，tri-nucleotide microsatellites所佔的比例………………………………………….86
圖十八 人類染色體20上，各種tetra-nucleotide microsatellites所佔的比例……………………….87

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