新型核酸解鏈曲線之技術開發與應用

Abstract

本論文提出一種創新的Melting Curve Analysis方法，主要以空間距離取代升溫速率為檢測系統參數，使檢體由於線性溫度梯度而受熱發生Melting現象，藉由分析特定位置之溫度對應檢體的Melting Curve，判斷Tm值結果來達成基因型分析。 本研究實驗第一部分為系統參數測試，包含加熱平台溫度分布、影像系統參數、玻璃毛細管螢光反應時間測試、平台管道位置變異分析實驗等。由實驗結果發現，新型MCA平台系統特性為：(1)兩端定溫架構可維持一穩定線性溫度梯度；(2)光學系統之曝光時間與增益值參數將影響影像品質，適當的調整曝光時間可提高影像靈敏度，而增益值則愈低愈好；(3)玻璃毛細管因材料與幾何形狀將使檢體螢光產生延遲，但可於短時間內達成熱平衡；(4)分析結果不因加熱平台上不同管道位置所影響。 第二部分為新型MCA實驗測試，以多種基因型檢體於新型MCA平台與商用儀器之Tm值量測結果進行比較，實驗結果顯示兩套系統分析之Tm值非常接近，可成功印證新型MCA之可行性。其次，進一步以混合樣本測試實驗，可達與商用儀器相同的檢測效能，檢測出樣本中含有兩種Tm值不同的DNA。 本研究提出之一維空間性質線性溫度梯度的新型MCA，以簡單之兩端定溫的機制，可大幅減少系統硬體成本；非時間性質的檢測亦具有自動化平行大量檢測分析的潛力。以灰階值分析的方式適合發展高解析度的SNP檢測，藉由像素與影像分析提高檢測的精度與效能。未來可與前端聚合酶鏈鎖反應 ( PCR ) 技術整合，完成核酸擴增與產物基因型辨識的一體化檢測流程。

This work proposes a new method of melting curve analysis. The fluorescence would change in temperature as a function of distance. The samples in glass capillary were melted by a heating surface with temperature gradient. The fluorescence intensity was decreased along with the distance. The melting temperature can be calculated by analyzing temperature versus position with the change in sample fluorescence. The first main part of experiment is system testing, including the temperature distribution of heating surface, the exposure time and gain of the optical device, the thermal-induced reaction time of fluorescence, and the variation analysis within different channels. The experimental results indicate that the characteristic of new MCA platform are described as follow. (1) By using two ends of heating/cooling system, a steady temperature gradient can be induced. (2) The value of exposure time and gain will affect the noise signal. The image intensity and sensitivity can be enhanced by increasing exposure time appropriately. But gain should be kept short. (3) The transient effects associated with the material and wall thickness of the sample container should be considered. (4) The variation in analytic results of different channels can be neglect. The second main part is the experiment of new melting curve analysis. For proof-of-concept testing, several DNA sample has been investigated. The results from new melting curve analysis were compared to the commercial instrument. Further, we also successfully distinguished the genotyping of 1:1 mixing sample. This work demonstrated the platform of the distance-dependent melting curve analysis by simple two-ends heating/cooling devices. The cost of system can be reduced significantly. The time-independent method has the potential advantages in automation and parallel detection. One application could be in the field of SNP detection. Combine the new melting curve analysis with PCR systems has a promising future and it offers much the same functionality.