臺灣博碩士論文加值系統

阿茲海默症 (Alzheimer's disease, AD) 是一種中樞神經退化疾病，被世界衛生組織認為是全球公共衛生重點。載脂蛋白E (Apolipoprotein E, ApoE)基因突變位於19號染色體上，在晚發性阿茲海默症 (Late-Onset Alzheimer's Disease, LOAD) 中扮演著相當重要的角色。研究指出有40%的LOAD患者ApoE基因屬於ɛ4型，也就是說ɛ4等位基因是罹患LOAD的主要遺傳風險因素。ApoE-ɛ4 (Arg112 CGC，Arg158 CGC) 序列是由兩個單核苷酸多型性 (Single-nucleotide polymorphisms, SNPs) 決定，即rs429358和rs7412。現行基因檢測方法為桑格定序法，然而其價格昂貴(如ABI 3730約550,000美金)又耗時。因此本實驗建立-單一限制酶無標記螢光感測平台，檢測阿茲海默症患者ApoE基因之核苷酸變異。
該技術採用5’端生物素 (Biotin) 修飾之前置引子擴增目標基因後，利用單一HinP1I限制性內切酶消化反應，雙重檢測ApoE之兩種不同SNP，並藉由鏈黴親和素磁珠 (Streptavidin magnetic beads, SMB) 磁力分離目標基因並收集上清液，於上清液加入核酸染劑後，測定其螢光訊號，藉由訊號的強弱判斷其基因型。研究結果顯示所得之最適化條件為個別於rs429358和rs7412添加0.6X和0.5X稀釋的Cyber™ Green，鏈黴親和素磁珠添加量20 μg，並與目標基因接合時間設定為15和20分鐘，HinP1I限制性內切酶濃度5 U。實驗結果利用R值判斷突變狀況，R值設定為F-F0/F0，F0為空白組螢光訊號，F為野生型 (WT) 或突變型 (MT) 螢光訊號；D值則表示野生型及突變型R值之間的差值，D值設定rs429358為R ( WT-MT ) 或 rs7412為R ( MT-WT )。結果顯示rs429358和rs7412之野生型及突變型序列的R值範圍分別落在2.28±0.11、7.59±0.07和8.90±0.13、3.53±0.08，對於突變型序列之偵測極限分別為10.15%和7.96%。將此方法實際應用於臨床檢品，結果與定序結果一致，成功區分ε3/ε3、ε2/ε3和ε3/ε4基因型，因此本法適用於臨床中的快速分析。

Alzheimer's disease (AD) is a neurodegenerative disease of the central nervous system that has been identified by the World Health Organization (WHO) as a global public health priority. A mutation in the apolipoprotein E (ApoE) gene, located on chromosome 19, plays an important role in late-onset Alzheimer's disease (LOAD). Studies have shown that 40% of LOAD patients have the ɛ4 type of ApoE gene, making the ɛ4 allele a major genetic risk factor for developing LOAD. The ApoE-ɛ4 (Arg112 CGC, Arg158 CGC) sequence is determined by two single nucleotide polymorphisms (SNPs), rs429358 and rs7412. The current gene detection method is Sanger sequencing, which is expensive (e.g., ABI 3730 about 550,000 USD) and time-consuming. Therefore, we developed a single restriction enzyme label-free fluorescence sensing platform to detect nucleotide variations in the ApoE gene of Alzheimer's disease patients.
The technique uses a 5'-biotin-modified forward primer to amplify the target gene and a single restriction endonuclease, HinP1I, for dual detection of two different ApoE SNPs, collecting the supernatant after magnetic separation using streptavidin magnetic beads (SMB). Finally, Cyber™ Green nucleic acid stain is added to the supernatant and the fluorescence intensity is measured to determine the type of gene sequence. The results showed that the ratio value (R value) of wild type and mutant type sequences of rs429358 and rs7412 were in the range of 2.28±0.11, 7.59±0.07 and 8.90±0.13, 3.53±0.08, respectively. The detection limits for mutant sequences were 10.15% and 7.96%, respectively. Practical application of this method in clinical testing showed that the results were consistent with sequencing results and successfully discriminated between ε3/ε3, ε2/ε3, and ε3/ε4 genotypes, making this method suitable for rapid clinical analysis.

中文摘要 i
Abstract iii
Graphical abstract v
目錄 vi
圖目錄 x
表目錄 xiv
第壹章 緒論 1
第一節 阿茲海默症 1
一、 阿茲海默症的病理原因 1
二、 阿茲海默症相關遺傳基因 2
第二節 載脂蛋白E (Apolipoprotein E, ApoE) 3
一、 ApoE蛋白質 3
二、 ApoE基因 5
第三節 檢測ApoE基因文獻回顧 7
第四節 現行基因檢測方法 9
一、 基因放大技術 10
1. 聚合酶連鎖反應 (Polymerase chain reaction, PCR) 10
2. 巢式聚合酶鏈式反應 (Nested Polymerase Chain Reaction) 12
二、 基因分離技術 13
1. 膠體電泳 (Gel electrophoresis) 13
2. 毛細管膠體電泳法 (Capillary gel electrophoresis, CGE) 14
3. 定序法 (Sequencing) 16
4. 多重連接探針擴增技術(Multiplex ligation-dependent probe amplification, MLPA) 17
5. 限制性片段長度多態性(Restriction fragment length polymorphism, RFLP) 17
第五節 實驗目的與機制 19
第貳章 實驗器材及方法 21
第一節 儀器設備 21
第二節 試藥及材料 22
一、 聚合酶連鎖反應 22
二、 PCR核酸純化 23
三、 限制性內切酶反應 23
四、 毛細管電泳儀分析 23
五、 螢光生物感測器 24
第三節 試藥溶液之配製與反應條件 25
一、 儲備溶液之配製 25
二、 聚合酶連鎖反應之擴增反應 26
1. 聚合酶連鎖反應之配製方法 26
2. 聚合酶連鎖反應條件 28
2.1 ApoE基因多型性位置及DNA模板 28
2.2 ApoE基因之引子設計 29
2.3 PCR條件 31
三、 PCR核酸純化 32
四、 限制性內切酶消化條件 33
五、 螢光生物感測器 34
第四節 實驗操作步驟 35
一、 Template之操作步驟 35
二、 真實樣品之操作步驟 35
第五節 毛細管電泳 36
一、 毛細管電泳分析 36
二、 毛細管清洗方式 36
三、 DNA分離條件 37
第參章 結果與討論 38
第一節 鏈黴親和素磁珠與生物素 (Streptavidin magnetic beads & Biotin) 38
第二節 CGE確認PCR產物 40
一、 利用CGE確認Template之產物 40
二、 利用CGE確認真實樣品之產物 42
第三節 反應條件最適化探討 43
一、 核酸染劑添加濃度 43
二、 鏈黴親和素磁珠添加量 46
三、 鏈黴親和素磁珠接合DNA反應時間 48
四、 限制性內切酶添加濃度 50
五、 限制性內切酶消化時間 51
第四節 野生型(WT)/突變型(MT)比例與回收率之試驗 53
第五節 真實樣品之應用 58
一、 檢品來源 58
二、 DNA 萃取之純度及濃度測定 58
三、 真實樣品之螢光感測器應用 59
四、 不同比例之真實樣品應用 61
第肆章 結論 62
參考文獻 63
附錄 72

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