臺灣博碩士論文加值系統

胚胎著床前基因診斷(PGD)配合HLA配型試驗進行雙重篩檢胚胎，是近年來新興之基因檢測技術，其目的是幫助具有急迫致病之遺傳疾病家族，渴望生一個不具此遺傳疾患的小孩，且同時可以臍帶血或骨髓移植治療罹病之兄姐；礙於時間之急迫及檢體DNA之微量，我們必須研發出更快速準確之技術方可成功。
傳統之HLA配型試驗無法對微量細胞之DNA做檢測，本研究是利用檢測遍佈HLA之短序列重複片段 (STR)來進行連鎖分析 (Linkage analysis)，因不同族群會有不同之HLA-STR基因頻率及盛行率，所以當務之急先篩選17個STR標誌檢測台灣族群之盛行率，隨機選取台灣族群100個檢體進行HLA-STR分析，統計其異質性比率 (Heterozygosity rate) 及對偶基因頻率 (Allele frequency)，利用異質性比率較高之至少八個STR標誌進行單一淋巴細胞之檢測得到良好的分析結果，將此17個STR標誌依其異質性高低及PCR的條件予以分組，分成四組 (Panel A-D)，方便於進行胚胎著床前基因診斷(PGD)時可快速精確之應用。
對於單一細胞之放大過程中經常發生Allele drop-out (ADO) 的情形，即只有一股被放大的困擾，本研究同時利用HLA-STR來檢測ADO的比率，而微量細胞放大技術之研發亦可改善ADO的情形，本實驗使用全基因體放大技術 (WGA)，可大量放大此單一細胞DNA，並可重複檢測不會互相干擾，且目前胚胎著床前基因診斷(PGD)之胚胎切片技術除了單一胚葉細胞之抽取外，另可以進行囊胚期切片技術抽取較多細胞，所以比較單一淋巴細胞與五顆淋巴細胞在進行全基因體放大技術 (WGA) 後，利用HLA-STR檢測其ADO比率各為34.0%、20.9%，有明顯減少之情況；以傳統放大技術Nested PCR進行單一淋巴細胞與五顆淋巴細胞之倍增，再利用HLA-STR測其ADO比率各為45.4%、27.2%，根據這些結果顯示以全基因放大技術 (WGA) 進行HLA-STR配型試驗於微量細胞之檢測是可行的，且囊胚期切片技術抽取較多的細胞數 (約五顆細胞) 可大大增加檢測之準確度，是未來發展之趨勢，利用此技術平台之建立，可實際應用於胚胎著床前基因檢測，達到最準確之檢測結果。

Preimplantation HLA matching has recently emerged as a tool for couples desiring to conceive a potential donor progeny for transplantation in a sibling with a life-threatening disorder . DNA in single blastomeres removed from 8-cell embryos by embryo biopsy following in vitro fertilization (IVF) was analyzed for short tandem repeats (STR) in the HLA region to select and transfer only those embryos that were HLA matching to affected siblings .
In this study , the allele frequency and the prevalence rate of seventeen short tandem repeats (STR) makers which are located in HLA complex region (6p21.3) in 100 Taiwan individuals were analyzed . The selected STR makers displayed high heterozygosity, broad distribution of alleles and identifiable allelic size ranges ; thus , obtained the accurate result for a single lymphocyte .
The allele drop-out (ADO) remains a significant problem for single cell PCR and diagnosis of single-gene disorders . Biopsy is done by removing one blastomere from a cleavage-stage embryo having 6-8 cells , and can also be done at the blastocyte stage , involving the removal of multiple trophectoderm cells . In the second part of this study , we further evaluate the performance of the newly developed whole genome amplification (WGA) in DNA amplification and compared with the traditional Nested PCR strategy . The ADO rates of whole genome amplification (WGA) for a single lymphocyte and five lymphocytes were 34.0% and 20.9% , accordingly . The ADO rates of Nested PCR for a single lymphocyte and five lymphocytes were 45.4% and 27.2% , accordingly . Our data suggested that diagnostic accuracy for DNA analysis in minimal cell would be benefit significantly from the biopsy of larger number of cells (from one cell to 5 cells) and the performance could be further improved by using whole genome amplification (WGA) technique .

圖目錄……………………………………………………………………i
表目錄…………………………………………………………………ii
中文摘要…………………………………………………………… iii
英文摘要………………………………………………………………iv
專有名詞中英文對照表…………………………………………………v
一、緒論…………………………………………………………………1
1.1簡介…………………………………………………………………1
1.2 HLA之特性…………………………………………………………3
1.2.1分子特性…………………………………………….…………4
1.2.2遺傳模式…………………………………………………………5
1.2.3 HLA功能…………………………………………………………5
1.3 HLA檢驗方式………………………………………………………6
1.3.1序列特異引子聚合酶連鎖反應法(sequence-specific primer；PCR-SSP)….6
1.3.2序列特異寡核苷酸探針法(sequence-specific oligonucleotide probe；PCR-SSOP)………………………………7
1.3.3定序分型法(Sequence-Based Typing；SBT )………………7
1.4 HLA-STR配型試驗…………………………………………………8
1.4.1 STR簡介…………………………………………………………8
1.4.2 STR之臨床應用…………………………………………………8
1.4.3 HLA-STR配型試驗檢測原理……………………………………10
1.5研究動機……………………………………………………………10
二、實驗材料與儀器…………………………………………………12
2.1實驗材料……………………………………………………………12
2.1.1隨機抽取100個台灣族群之血液檢體…………………………12
2.1.2引子對……………………………………………………………12
2.1.3聚合酶連鎖反應試劑……………………………………………12
2.1.4洋菜凝膠電泳試劑…………………….………………………12
2.1.5毛細管電泳自動分析試劑組…………………..……………12
2.1.6其他試劑……………………………………………………….12
2.2實驗儀器……………………………………………………………13
2.2.1 PCR熱循環器……………………………………………………13
2.2.2水平式電泳槽……………………………………………………13
2.2.3離心機……………………………………………………………13
2.2.4 毛細管電泳自動分析儀………………………………………13
三、實驗方法…………………………………………………………13
3.1 DNA檢體之抽取……………………………………………………13
3.2分離淋巴球…………………………………………………………13
3.3挑出單一淋巴球及五顆淋巴球……………………………………13
3.4全基因體放大技術(WGA)…………………………………………14
3.5 STR之選取…………………………………………………………14
3.6聚合酶連鎖反應……………………………………………………14
3.7 Nested PCR………………………………………………………15
3.8洋菜凝膠電泳(Agarose gel electrophoresis )………………15
3.9毛細管電泳自動分析………………………………………………15
四、實驗結果…………………………………………………………17
4.1 異質性比率(Heterozygosity rate)分析結果…………………17
4.2 測試HLA-STR配型試驗於家族之連鎖分析………………………17
4.3 Allele frequency統計結果………………………………………17
4.4 HLA-STR運用於單一淋巴細胞的檢測……………………………18
4.5比較一顆淋巴細胞與五顆淋巴細胞之ADO Rate…………………18
4.6比較Nested PCR與WGA之ADO Rate………………………………19
五、討論………………………………………………………………20
六、救人寶寶之倫理爭議……………………………………………23
七、參考文獻…………………………………………………………25

圖目錄

圖一、HLA 於第六號染色體之基因分佈圖---------------------30
圖二、HLA class 1 & HLA class 2之功能--------------------31
圖三、HLA之遺傳模式示意圖--------------------------------32
圖四、STR示意圖------------------------------------------33
圖五、各個不同STR makers遍佈HLA基因區域圖----------------34
圖六、HLA-STR檢測原理------------------------------------35圖七、利用STR進行Linkage analysis------------------------36
圖八、分析台灣族群的十七個HLA-STR makers之電泳圖---------37
圖九、列舉八個異質性較高之HLA-STR makers經自動分析儀所得之圖形---------38
圖十、列舉八個異質性較高之HLA-STR makers測試家族父母與病童之連鎖分析-----------------------------------------------39
圖十一、測試單一淋巴細胞進行HLA-STR之分析圖形------------43
圖十二、測試五顆淋巴細胞進行HLA-STR之分析圖形------------44
圖十三、抽取胚胎細胞之方式-------------------------------45


表目錄

表一、列舉可應用胚胎著床前基因診斷之單基因遺傳疾病-------46
表二、HLA-STR makers於HLA之位置--------------------------47
表三、HLA-STR makers進行螢光標的聚合酶連鎖反應所需之引子對序列表---------48
表四、PCR理想之反應試劑條件------------------------------49
表五、二次放大之外在引子序列-----------------------------50
表六、台灣族群各個HLA-STR makers之分析結果---------------51
表七、台灣族群HLA-STR makers之Allele Frequency統計表-----52
表八、以WGA進行單一淋巴細胞之放大成功率及ADO的比率-------54
表九、以WGA進行五個淋巴細胞之放大成功率及ADO的比率-------55
表十、比較單一淋巴細胞與五個淋巴細胞之放大成功率及ADO比率-56
表十一、以Nested PCR進行單一淋巴細胞之放大成功率及ADO的比率--------------57
表十二、以Nested PCR進行五個淋巴細胞之放大成功率及ADO的比率--------------58
表十三、比較WGA及Nested PCR其單一淋巴細胞與五個淋巴細胞之放大成功率及ADO比率----------------------------------------59
表十四、各種胚胎切片技術優缺點比較-----------------------60

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