臺灣博碩士論文加值系統

胚胎著床前基因診斷（PGD）為一種替代性的產前診斷，在具有遺傳疾病的家族中，胚胎著床前基因診斷提供一個有正常小孩的機會，並且不用面臨最壞的情況—終止妊娠。胚胎切片技術，目前有三種可能來源的遺傳物質進行檢測，分別為極體切片、卵裂期的胚葉細胞切片、及囊胚期的滋養層切片，不論是何種來源的胚胎切片，由如此少量的遺傳物質進行以聚合酶連鎖反應為基礎，來進行胚胎著床前基因診斷的分析是非常困難的。而全基因組放大技術可以將如此少量的遺傳物質放大再進行診斷，可獲得較準確的結果，並且擴大了更多可使用的檢測平台，當然有其主要的問題之一為allele drop out (ADO)，而ADO的情形造成了誤診，及降低胚胎著床前基因診斷的準確性，因此本論文中使用不同的方式來改善ADO的情況。
我們比較了兩種不同的胚胎切片技術的來源細胞所進行的胚胎著床前基因診斷，發現囊胚期切片細胞來源變多，可降低ADO的情況，並且提高胚胎著床前基因診斷的準確性。接著使用了新的全基因組放大技術Rubicon PicoPlex WGA kit，雖然可以降低ADO的情況，但由於新的全基因組放大技術的局限，對於胚胎著床前基因診斷的實用性可能還須評估，而目前Rubicon PicoPlex WGA kit使用於胚胎著床前篩檢（PGS）上。另外在已造成ADO的情況下，我們使用PCR clamping技術合併鎖核酸探針（LNA probe），的確可成功的改變已造成ADO的檢測結果。
最後，不論從來源、過程及檢測中，使用不同的方式來提高準確性，但ADO的情況還是存在，因此一個高準確性胚胎著床前基因診斷技術平台的建立，經由囊胚期切片技術，進行全基因組放大技術，直接及間接檢測法之雙平台的檢測，建立一個高優質的胚胎著床前基因診斷系統，給予接受胚胎著床前基因診斷的病人更加的安心。

Preimplantation genetic diagnosis (PGD) is an alternative for prenatal diagnosis. For families with genetic diseases, PGD offers a chance to have an unaffected child, without facing termination of pregnancy. There are three stages of biopsy: polar bodies, one or two blastomeres from the cleavage-stage embryos, and trophectoderm cells from the blastocyst-stage embryos. Validation of polymerase chain reaction (PCR)-based assays are challenging because only limited genetic material can be obtained for PGD. The whole genome amplification (WGA) can amplify the limited DNA to process diagnosis. By using WGA, we can improve the accuracy of diagnosis and expand other diagnosis methods for PGD. The main problem of WGA is allele drop out (ADO), which is critical for misdiagnosis and low accuracy. Therefore, we used a different approach to improve the ADO.
We compare amplification rate and efficiency in blastomere and trophectoderm biopsy. We can retrieve more cells in the trophectoderm than blastomere, and improved accuracy for PGD. Furthermore, we apply the Rubicon PicoPlex WGA kit for whole genome amplification. Although the ADO is thus reduced, the practicality of Rubicon PicoPlex WGA kit for PGD need to be further evaluated due to the limitation of the new WGA technique. In addition, we successfully improve the ADO by using the LNA probe and PCR clamping.
In the end, no matter how we try to improve the examination process, the ADO is still inevitable. Therefore, to establish a high-accuracy method, through the biopsy of blastocyst stage, whole genome amplification, and the double confirmatory platform, are crucial for a high-quality PGD system. Consequently, we could provide a more credible and reliable system for the patients.

目 錄
口試委員會審定書…………………………………………………………………Ⅰ
誌謝…………………………………………………………………………………Ⅱ
中文摘要……………………………………………………………………………Ⅲ
英文摘要……………………………………………………………………………Ⅳ
目錄…………………………………………………………………………………Ⅴ
圖目錄………………………………………………………………………………Ⅶ
表目錄………………………………………………………………………………Ⅷ
第一章 緒論…………………………………………………………………………1
1.1 研究背景與動機…………………………………………………………………1
1.2 胚胎著床前基因診斷（PGD）之胚胎切片技術………………………………4
1.3 胚胎著床前基因診斷（PGD）之分子檢測技術………………………………5
1.4 PCR clamping……………………………………………………………………8
1.5 研究目的…………………………………………………………………………9
第二章 研究方法……………………………………………………………………10
2.1 全基因組放大技術合併聚合酶連鎖反應實驗………………………………10
2.2 Real-time PCR合併PCR clamping實驗………………………………………12
第三章 結果…………………………………………………………………………14
3.1 比較單一淋巴細胞與五顆淋巴細胞之ADO rate及成功放大率……………14
3.2 比較於卵裂期及囊胚期的胚胎切片所進行的胚胎著床前基因診斷（PGD）
………………………………………………………………………………………14
3.3 比較不同全基因組放大技術組（WGA kit）之ADO rate及成功放大率…...15
3.4 利用LNA probe改變已造成的ADO的情形…………………………………15
第四章 討論…………………………………………………………………………17
4.1 胚胎切片技術之探討…………………………………………………………17
4.2 全基因組放大技術之探討……………………………………………………18
4.3 胚胎著床前基因診斷（PGD）之分子檢測探討……………………………19
第五章 胚胎著床前基因診斷（PGD）之遺傳諮詢與倫理議題…………………21
第六章 胚胎著床前篩檢（preimplantation genetic screening）…………………24
第七章 參考文獻……………………………………………………………………27

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