研究生: |
王韋凱 Wang, Wei-Kai |
---|---|
論文名稱: |
利用焦磷酸定序技術和選擇性富集胎兒游離DNA開發非侵入性產前檢測地中海貧血 Non-invasive prenatal testing of thalassemia by pyrosequencing technique and selective enrichment of cell-free fetal DNA |
指導教授: |
黃則達
Huang, Tze-Ta 黃振勳 Huang, Jehn-Shyun |
學位類別: |
碩士 Master |
系所名稱: |
醫學院 - 口腔醫學研究所 Institute of Oral Medicine |
論文出版年: | 2020 |
畢業學年度: | 108 |
語文別: | 中文 |
論文頁數: | 74 |
中文關鍵詞: | 地中海貧血 、游離DNA 、次世代定序 、非侵入產前檢測 、焦磷酸定序 、選擇性富集 |
外文關鍵詞: | Thalassemia, Cell-free DNA, Non-invasive prenatal testing, Pyrosequencing, Selective enrichment |
相關次數: | 點閱:97 下載:0 |
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地中海貧血是一種體染色體隱性遺傳疾病,為世界上常見的單基因疾病。在台灣,地中海貧血的帶因者約為5-8%,其中α-地中海貧血約為3-5%,β-地中海貧血約為1-3%。產前遺傳診斷取得胎兒組織有三種方法,羊膜穿刺術、絨毛膜取樣術、臍帶血採樣術。羊膜穿刺有0.3%機率導致流產,另外兩種有1%機率導致流產。在血液中存在游離DNA(cfDNA),孕婦的血漿中除了自身的游離DNA也存在胎兒的游離DNA。非侵入式產前檢測便利用這個特性,可以對胎兒進行染色體檢測,但現在能診斷出來的大約是5 MB以上的基因缺失或增加。孕婦和胎兒游離DNA的大小大致重疊但有些許差異,在150 bp以下時胎兒的游離DNA佔的比例較高。我們將利用DNA大小選擇性的方法使胎兒游離DNA的比例提高,再利用焦磷酸定序技術,測定α和β-地中海貧血中,突變或缺失的比例是否有改變。我們預期帶因者是正常的序列和突變或缺失比例是一半一半,當有夫妻為同型帶因者所懷重型地中海貧血胎兒時,母親孕期血漿中游離DNA其一半一半的比例將會改變。實驗發現焦磷酸定序本身會有5%的誤差,胎兒cfDNA佔比要大於10%才能檢測出來。並利用PCR產物和cfDNA類似物模擬不同比例的突變和正常基因,證實焦磷酸定序的可以觀察到比例的變化,而cfDNA類似物正常和突變比例在2:1以下時才有DNA大小選擇性富集的效果。我們使用焦磷酸定序Codon 26孕婦樣品,正常和突變的比例51:49,推斷胎兒和媽媽的基因型一樣。經過富集後,我們再次使用焦磷酸定序,正常和突變接近50:50,所以我們推斷胎兒為codon 26帶因者應該是正確的。
Thalassemia is a somatic recessive genetic disease, and a common single-gene disease in the world. If the parents are the same type heterozygous, the offspring have a quarter chance to become a thalassemia patient. There is cell-free DNA (cfDNA) in the blood, and in pregnant women, there are not only pregnant women cfDNA but also fetus cfDNA. The feature is used by Non-invasive Prenatal Testing (NIPT) to perform chromosomal testing of the fetus, but the size of genes deletion or duplication diagnosed are needed to be more than 5 MB. The size of cfDNA in pregnant women and fetuses overlaps, but there is a slight difference in the proportion of cfDNA that the fetus is below 150 bp. We first used the size-selective enrichment to increase the proportion of fetal cell-free DNA, then used pyrosequencing technology to determine whether the proportion of mutations or deletions in α & β-thalassemia has changed. We expect that normal and mutation or deletion sequence ratio of carrier is half and half. When there is homozygous fetal cfDNA added, the ratio will change. We found that pyrosequencing has a 5% error. So, when fetal fraction is greater than 10%, it will be detected. We used PCR product and cfDNA mimic to simulate different proportion of mutant and normal gene, it was confirmed that pyrosequencing can observe the ration change. The proportion of cfDNA mimic normal and mutant were less than 2:1, then DNA size selective enrichment has effect. We detected the codon 26 pregnant women ratio of normal to mutation 51:49 by pyrosequencing. It is inferred that the fetus and mother have the same genotype.
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