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研究生:謝瑞煒
研究生(外文):Jui-Wei Hsieh
論文名稱:以即時聚合酶鏈鎖反應篩檢單一核苷酸變異方法之研發
論文名稱(外文):High Throughput Detection of Single Nucleotide Variation with Real-Time PCR
指導教授:張正張正引用關係詹爾昌
指導教授(外文):C. Allen ChangErr-Cheng Chan
學位類別:碩士
校院名稱:國立交通大學
系所名稱:生物科技研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2001
畢業學年度:90
語文別:中文
論文頁數:88
中文關鍵詞:點突變多形性基因變異偵測方法
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基因變異是基因體中去氧核醣核酸上的永久改變,而這種改變可能是導因於染色體結構的變異或者是基因中核苷酸序列的改變。在基因中的變異有些對於個人、家庭或者整個族群健康狀態可能不會有任何直接明顯的影響,但是也有的可能造成立即、顯著的影響,例如人類鈉碘共運器基因中的特定位置核苷酸的點突變導致第354個胺基酸由蘇胺酸變成脯胺酸,造成蛋白失去或降低其從血液中運送碘離子至甲狀腺細胞內的活性,進而影響整個甲狀腺素的合成。除此之外,多形性也屬於基因變異的一種,特別是單一核苷酸多形性在藥物遺傳學上扮演相當重要的角色,目前有些研究顯示,個體間單一核苷酸多形性的資訊似乎和每個人對於藥物之不同反應程度有關。
現今常用於偵測去氧核醣核酸上的單點變異有下列方法:Single Strand Conformation Polymorphism(SSCP), Denaturing Gradient Gel Electrophoresis(DGGE), Heteroduplex Analysis(HA), Protein Truncation Tests(PTT)和 Cleavage Methods(chemical, enzymatic)。上述方法各有其特色、應用範圍與優缺點,以至於無法歸納出哪一種為最佳偵測方法,不過在愈來愈強調方便性、靈敏度與低成本之考量下,這些方法顯然無法滿足這些需求。
本實驗的目的乃針對上述方法使用上的限制,發展一套簡單、快速與高效率的基因單點變異偵測方法,其主要的特點如下:除了聚合酶鏈鎖反應所需的一對引子外,我們設計了另外一條引子,這條引子除了 3’ 端最後一個核苷酸外,其餘的序列和 DNA 模板完全互補,而這 3’ 端最後一個核苷酸依據欲篩檢之變異核苷酸種類而定;再者,我們於反應中加入 SYBR® Green I dye 做為染劑,能夠在PCR 產物形成的同時與其結合達到迅速偵測的目的;除此之外,GeneAmp® 5700 Sequence Detection System 提供了高效率的作業平台。接著我們應用這套方法偵測病人或其家屬之鈉碘共運器基因與纖維母細胞生長因子23基因是否存在基因變異,結果除了在纖維母細胞生長因子23基因中發現716C/T(239T/M)變異外,在鈉碘共運器基因中並沒有發現任何的基因變異。
雖然我們無法在鈉碘共運器基因中發現任何的基因變異,以推測或驗證特定的臨床症狀是否與特定位置的突變有關,但是就這套方法而言,在某種程度的應用範圍上,其簡單、高效率的特性是極佳的偵測基因單一核苷酸變異之方法。
Gene variation is permanent alterations in DNA, due to either a structure chromosome aberration or change in the nucleotide sequence of a gene. Such heritable changes in DNA may have little effect or a significant effect on the health of an individual or family, for example, human sodium iodide symporter(hNIS)gene T354P mutation. Besides some DNA variance called polymorphism, especially single nucleotide polymorphism(SNP), may play an important role on pharmacogenetics. With the SNP information, it may be possible to solve the mysteries of individual responses to medicines.
Current most common methods for the detection of DNA point variation are Single Strand Conformation Polymorphism(SSCP), Denaturing Gradient Gel Electrophoresis(DGGE), Heteroduplex Analysis(HA), Protein Truncation Tests(PTT)and Cleavage Methods(chemical, enzymatic), but they are unsatisfactory due to a range of reasons, particularly due to cost, sensitivity and lack of convenience.
In this study, we introduce a fast and simple modification of the polymerase chain reaction, in which the sequence of forward primer is complementary to DNA template except one different base in the last one at the 3’end, and SYBR® Green I dye on GeneAmp® 5700 Sequence Detection System(Applied Biosystem)to detect known point variations of hNIS gene and FGF23(Fibroblast growth factor 23)gene. We attempt to find out whether there are some relationship between the clinical features of iodide transport defect(ITD)patients and the defined hNIS gene mutation, and a phosphate wasting disorder is associated with mutation in FGF23 gene, in addition to screening all exons of hNIS gene and coding sequence of FGF23 gene of some patients for unknown DNA variation. Unfortunately, we can’t find out any known and unknown point variation of the two genes except a 716C/T(239T/M)variation of FGF23 gene on three specific patients with phosphate wasting disorder.
第一章 緒論----------------------------------------------1
1.1 研究目的---------------------------------1
1.2 現行偵測方法說明-------------------------2
1.2.1 Heteroduplex Analysis--------------------2
1.2.2 RNAse A方法------------------------------3
1.2.3 Single Strand Conformation Polymorphism--3
1.2.4 Denaturing Gradient Gel Electrophoresis--3
1.2.5 Chemical Cleavage of Mismatch------------4
1.2.6 Enzyme mismatch cleavage-----------------5
1.3 實驗偵測原理介紹-------------------------5
1.3.1 Modified method of PCR-------------------5
1.3.2 SYBR® Green I Dye------------------------6
1.3.3 GeneAmp® 5700 Sequence Detection System--6
1.3.4 結果說明----------------------------------7
1.4 研究策略---------------------------------8
1.4.1 鈉碘共運器介紹---------------------------8
1.4.2 纖維母細胞生長因子23介紹-----------------9
1.4.3 實驗流程---------------------------------10
第二章 材料與方法----------------------------------------12
2.1 儀器-------------------------------------12
2.2 材料-------------------------------------13
2.2.1 檢體來源與取樣方法-----------------------13
2.2.2 菌株-------------------------------------13
2.2.3 載體-------------------------------------13
2.2.4 培養液與緩衝液---------------------------13
2.3 方法-------------------------------------16
2.3.1 基因體 DNA 的製備------------------------16
2.3.2 聚合酶鏈鎖反應---------------------------17
2.3.3 電泳分析---------------------------------19
2.3.4 PCR產物的純化----------------------------20
2.3.5 接合反應---------------------------------21
2.3.6 勝任細胞之製備---------------------------21
2.3.7 轉殖-------------------------------------22
2.3.8 質體 DNA的製備---------------------------22
2.3.9 以限制酵素進行質體篩選-------------------23
2.3.10 DNA 定量分析與純度測定-------------------24
2.3.11 Mutant type DNA 的製備-------------------24
2.3.12 NIS基因已知變異之篩選--------------------29
2.3.13 NIS基因未知變異之篩選--------------------31
2.3.14 FGF23基因未知變異之篩選------------------35
2.3.15 FGF23基因變異之確認----------------------36
2.3.16 FGF23基因已知變異之篩選------------------37
第三章 結果----------------------------------------------39
3.1 基因體DNA的製備--------------------------39
3.2 特定基因片段的放大-聚合酶鏈鎖反應-------39
3.3 構築 PCR 產物於 pGEM®-T Easy Vector------39
3.4 Mutant type DNA 的製備-------------------40
3.5 建立基因變異篩選方法---------------------40
3.6 NIS基因已知變異之篩選--------------------41
3.7 NIS基因未知變異之篩選--------------------41
3.8 FGF23基因未知變異之篩選------------------41
3.9 FGF23基因已知變異之篩選------------------42
第四章 討論----------------------------------------------43
參考文獻 --------------------------------------------------67
附錄一 NIS 基因序列及 Exons 相關位置---------------------72
附錄二 FGF23 基因之 cDNA 及胺基酸序列--------------------86
附錄三 pGEM®-T Easy Vector 圖譜--------------------------88
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