利用SPRi探討中性DNA探針相較於一般DNA探針在低鹽雜交環境下之優勢

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林仲恩(Zhong-En Lin) 查詢紙本館藏

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化學工程與材料工程學系

論文名稱

利用SPRi探討中性DNA探針相較於一般DNA探針在低鹽雜交環境下之優勢
(Advantages of the neutralized DNA (nDNA) probe compare with that of the regular DNA probe under the low ionic strength hybridization buffer by using SPRi)

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摘要(中)

自2003年科學家把人類基因組解讀完成後，科學家發現人類的DNA序列中平均每1000個鹼基就會出現1至4個差異位點，稱之為單一核苷酸多型性(Single Nucleotide Polymorphism；SNP)。然而，這SNP的存在已經被科學家們發現其對人類常見疾病是有相互關係的，在本研究中會使用表面電漿共振影像儀(Surface plasmon resonance imaging ; SPRi)作為生物感測器去觀測探針對於SNP的辨識能力，其感測方式是利用待測物在生物晶片上的吸附行為而造成晶片表面的折射率變化來得知我們所要的結果，SPRi的優勢在於不用在待測物上標定任何東西，進而減少檢測成本，可以即時的看到檢測成果，進而減少檢測上的時間等等。生物感測器中還有另一項重要元素，即在生物晶片上的生物辨識探針，一個良好的探針可以提高檢測的精確度。在本研究中，使用本實驗室開發的一種DNA類似物，中性DNA(nDNA)作為生物辨識探針，其結構上，DNA磷酸骨幹的負電由甲基化所遮蔽，使核酸分子變成不帶電的DNA類似物，特點在於進行兩條DNA雜交時，由於靜電排斥力的減少，使得在低鹽環境下，可以形成穩定雙股螺旋結構。而在本研究中，會探討nDNA探針相對於DNA探針的優勢，以本研究得到的實驗結果中，nDNA探針在低鹽環境下，互補股DNA的雜交量比一般DNA探針來的多，以此證實前述對於 nDNA探針在低鹽雜交環境下可以形成穩定的雙股螺旋結構的理論特性是正確的。再來，探針對於檢測的精確度實驗中，於低鹽環境下，nDNA探針辨識一個鹼基錯誤配對序列的能力是優於一般DNA探針(約4.37倍)，顯示nDNA探針在此環境下有很好的專一性。當進一步提高雜交環境的溫度至nDNA與帶有一個鹼基錯誤配對之雙股Tm值附近，nDNA探針辨識一個鹼基錯誤配對序列的能力又可以再次提升(約一般DNA探針的9.38倍)，讓基因檢測的精確度有效提高。

摘要(英)

The development of DNA sensors has gained popularity over the past few years, for its potential interest in applications for DNA sequencing in clinical diagnostics such as the detection of single nucleotide polymorphisms (SNPs). Surface plasmon resonance imaging (SPRi) is a surface analysis technique that measures refractive index changes induced by molecular adsorption on a noble metal film. In this study, we take neutralized DNA (nDNA) as a detection probe on the sensor surface. The nDNA is an uncharged DNA analogue due to the backbone phosphate groups changed by methylposphate groups, which would make no electrostatic repulsion during the hybridization between nDNA and regular DNA. By the experimental results, target DNA of the SPR response was linear in the concentration range 1-20μM. We find out the nDNA probe can catch more nucleic acids than the regular probe at the low ionic strength buffer condition because of the reduction of the electrostatic repulsion. In addition, we use nDNA probe to discriminate between the perfect matched sequences and　single-mismatched sequences. We also make the improvement of discrimination by controlling the salt concentration of the sample solutions and the experimental temperature. At the low ionic strength buffer, the discriminating capability of nDNA probe is 4.37 times greater than that of regular DNA probe. After raising the experimental temperature, the discriminating capability of nDNA probe is 9.38 times greater than that of regular DNA probe. Based on these results, we are sure that nDNA probe can demonstrate the well discrimination at the particular conditions. These features of nDNA probe can improve the accuracy of the DNA detection.

關鍵字(中)

★ 表面電漿共振影像儀
★ 不帶電探針
★ 低離子強度
★ 不帶電探針專一性

關鍵字(英)

★ SPRi
★ nDNA probe
★ low ionic strength
★ SNP

論文目次

中文摘要 i
Abstract iii
誌謝 v
目錄 vii
圖目錄 x
表目錄 xiv
第一章緒論 1
第二章文獻回顧 3
2.1 基因檢測 3
2.1.1 基因檢測技術 4
2.2 表面電漿共振 5
2.2.1 表面電漿共振原理 6
2.2.2 表面電漿共振儀分類 8
2.2.3 表面電漿共振儀 11
2.3 晶片改質 14
2.3.1 自組裝單分子層膜技術 15
2.3.2 表面分子之固定化 22
2.4 核酸類似物探針 24
2.4.1 肽核酸 24
2.4.2 鎖核酸 26
2.4.3 磷酸根甲基化去氧核醣核酸 28
第三章實驗藥品、儀器設備與方法 31
3.1 實驗藥品 31
3.2 儀器設備 33
3.3 實驗方法 34
3.3.1 Circular dichroism (CD)實驗 34
3.3.2 SPR晶片製備 34
3.3.3 SPR DNA晶片之表面改質 35
3.3.4 緩衝溶液配置 36
3.3.5 SPRi DNA雜交實驗 36
3.3.6 SPRi辨識SNP實驗 37
3.3.7 雙股DNA之Tm量測 37
第四章結果與討論 39
4.1 nDNA 專一性雜交之鑑定 39
4.2 晶片表面探針固定化之鑑定 42
4.3 利用SPRi探討nDNA探針相較於一般DNA探針之優勢 45
4.3.1 一般DNA探針之雜交實驗 45
4.3.2 nDNA探針之雜交實驗 48
4.3.3 一般DNA探針與nDNA探針於低鹽環境下的雜交實驗比較 51
4.4 探針於辨識SNP的能力探討 55
4.4.1 一般DNA探針辨識SNP能力探討 56
4.4.2 nDNA探針辨識SNP能力探討 58
4.4.3 於低鹽環境下nDNA/DNA探針辨識SNP之探討 60
4.4.4 提升雜交環境溫度以提高nDNA探針的辨識效果 64
第五章結論 67
第六章參考文獻 69

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指導教授

陳文逸(Wen-Yih Chen)

審核日期

2016-7-22

推文