臺灣博碩士論文加值系統

針對單一基因疾病的胚胎著床前診斷，主要的方法以聚合酶連鎖反應為主，將特定基因片段放大，進而偵測單一基因突變點。而單一細胞聚合酶連鎖反應步驟中會出現許多問題，像是污染、放大失敗、或是只優先放大其中一股，而另一股完全沒有被放大，造成ADO 的情形，但是單一細胞聚合酶連鎖反應，仍是作為胚胎著床前偵測單一基因突變的唯一方法。單一細胞的DNA 含量是很有限的，使用整體染色體放大技術，可以增加DNA 的含量，並且能將整段染色體完整的放大出來。因此，整體染色體放大技術對於單一細胞基因診斷，是一種非常好用的方法。本論文主要利用兩種整體染色體放大的方法: Linker-adaptor 方法與MDA 方法，利用整體染色體放大技術，將染色體中的基因體DNA，進行非專一性的放大，再使用有乙型海洋性貧血基因常見的兩個SNP 個體中淋巴球的整體DNA，針對這兩個SNP 設計專一性引子，進行聚合酶連鎖反應和DNA 定序，評估整體染色體放大技術的過程中造成ADO 的現象。本實驗共做了222 個檢體，分別使用Linker-adaptor 和MDA 方法放大整體染色體後，利用聚合酶連鎖反應放大乙型海洋性貧血常見的兩個SNP 基因，共有174 個檢體被放大出來，DNA 序列分析結果顯示有91 個檢體兩股allele 皆有放大出來，83 個檢體有ADO的情形。Linker-adaptor和MDA 方法倍增速率皆為90 ％，ADO 比率則分別是26.67 ％和28.89 ％。這兩種整體染色體放大方法仍然有超過25 ％ ADO 情形。因此，ADO 對於單一基因疾病診斷所使用的單一細胞聚合酶連鎖反應，仍然是一個主要的問題，需要更進一步去克服它。

Preimplantation genetic diagnosis (PGD) of single gene disorders relies on PCR-based tests performed on single cells (polar bodies or blastomeres). Single cell PCR protocols are subject to serious difficulties, including contamination, amplification failure, and preferential amplification or the complete absence of one allele (allele dropout, ADO) in heterozygous loci, but it remains the only tool for detecting specific mutation in PGD. The DNA content of single cell was limited. Different whole genome amplification (WGA) techniques have been developed to increase the DNA quantities from clinical samples with limited DNA contents. Therefore, the utilization of genomic amplification methods will be a useful tool in single-cell genetic diagnosis. In this study, the complete genome DNA of all chromosome could be non-specific amplify by two WGA methods, Linker adaptor and the multiple displacement amplification (MDA) method, without the loss of genomic regions or preferential amplification of genomic loci or alleles. The WGA technique was not only increase the original DNA content, but also retained the integrity of whole genome DNA. The two common SNP of β-thalassemia was amplified from the longer or shorter primer set from these amplified genome DNA. DNA sequencing of the PCR product was performed to evaluate the ADO effect during two WGA methods. Total of 222 single cells were picked up and performed the Linker-adaptor and MDA method to amplify the whole genome DNA, respectively. DNA sequencing results of the PCR product from 174 samples found that 91 samples had the complete allele, and 83 samples had the allele drop-out (ADO) effect. The amplification efficiency of linker adaptor and MDA two methods are 90 ％. The ADO rate was 26.67 ％ and 28.89 ％, respectively. These two methods still had higher than 25 ％ of ADO rate. Therefore, the ADO problem was the biggest obstacle for single cell PCR and diagnosis of single-gene disorders.

總目錄
目錄……………………………………………………………...……I
圖目錄…………………………………………………………….…III
表目錄…………………………………………………………………IV
中文摘要…………………………………………………………….…V
英文摘要………………………………………………………………VI
一、緒論………………………………………………………...……1
1-1 簡介……………………………………………………….....…1
1-2 單一細胞聚合酶連鎖反應之特徵………………………….……2
1-2-1 污染(contamination)………………………………........2
1-2-2 倍增速率失敗(Amplification efficiency failure).....3
1-2-3 Allele drop-out（ADO）…………………………………….3
1-3 單一細胞之檢測方法……………………….....………………3
1-3-1 傳統聚合酶連鎖反應法（Polymerase Chain Reaction，PCR）....................................................4
1-3-2 二次聚合酶連鎖反應（Nested Polymerase Chain Reaction, Nested PCR）…........................…...……5
1-3-3 螢光聚合酶連鎖反應法（Fluorescent Polymerase Chain Reaction，F-PCR）……..................................…5
1-3-4 整體染色體放大(Whole Genome Amplification)………...5
1-4 單一細胞檢測方法的重要性…...………………………………7
1-5 研究動機與方向…...……………………………………………8
二、實驗材料與儀器…………………………………………………10
2-1 實驗材料……..…………………………………………………10
2-1-1 人類基因體DNA (Human Genomic DNA).……………………10
2-1-2 引子……………………………………………………………10
2-1-3 其他反應試劑…………………………………………………10
2-2 實驗儀器…………………………………………………………11
三、實驗方法…………………………………………………………11
3-1 分離淋巴球………………………………………………………11
3-2 挑出單一淋巴球…………………………………………………11
3-3 整體染色體放大(Whole Genome Amplification)..……....12
3-4 聚合酶連鎖反應（Polymerase Chain Reaction，PCR）……12
3-5 洋菜凝膠電泳（Agarose gel electrophoresis）……….…12
3-6 直接序列分析（Direct DNA sequencing）………………….12
四、結果…...……………………………………………………….13
4-1 整體染色體放大（WGA）的結果……………………………….13
4-2 聚合酶連鎖反應的結果………………………………………..13
4-3 直接DNA 序列分析的結果………………………………………14
4-5 資料分析………………………………………………………..14
五、討論………………………………………………………………16
六、總結…………….……………………………………………….18
參考文獻………………………………………………………………20

圖目錄
圖一、聚合酶連鎖反應之簡單原理示意圖……………………....25
圖二、Linker-adaptor amplification 之簡單原理示意圖….…26
圖三、Multiple displacement amplification （MDA）之簡單原理示意圖…..............................................…27
圖四、血球分離後分層示意圖…………….……………………….28
圖五、Linker adaptor 方法： GenomePlex 的Protocol….……29
圖六、Multiple Displacement Amplification, MDA 方法：REPLI-g 的Protocol…..........................................30
圖七、洋菜膠電泳確認整體染色體放大（WGA）：MDA 方法的結果……..............................................…..31
圖八、洋菜膠電泳確認整體染色體放大（WGA）：Linker adaptor 方法的結果..............................................32
圖九、洋菜膠電泳確認聚合酶連鎖反應：MDA 方法的結果………33
圖十、洋菜膠電泳確認聚合酶連鎖反應：Linker adaptor 方法的結果……….............................................….34
圖十一、洋菜膠電泳確認聚合酶連鎖反應：MDA 方法的結果……………........................................……35
圖十二、使用不同濃度DNA 量以洋菜膠電泳確認聚合酶連鎖反應……………............................................36
圖十三、C.9 C>T 直接序列分析的各種結果………..…………..37
圖十四、C.316-185 C>T 直接序列分析的各種結果………………38

表目錄
表一、各種單一細胞倍增方法之比較……………….…………….39
表二、Beta thalassemia 常見SNP 的點位………….……………40
表三、聚合酶連鎖反應所需引子………………….……………….42
表四、PCR反應試劑的濃度與總體積……………………………….43
表五、PCR熱循環參數……………………………………………….44
表六、MDA方法Long primer (423 bp)統計結果……..………….45
表七、Linker adaptor與MDA兩種方法的比較……….………....46
表八、MDA 方法：使用五個不同Sample 利用相同primer 的PCR 方法amplified 3 次並送Sequence 的結果…….................47
表九、Linker adaptor 方法：使用五個不同Sample 利用相同primer 的PCR 方法 amplified 3 次並送Sequence 的結果.....48
表十、MDA 方法相同的檢體以不同的引子做PCR 並序列分析的結果……...............................................….49

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