- 學位論文
毛細管熱對流聚合酶鍊鎖反應產物專一性檢測機制
Development of a specificity detection mechanism of amplicons by capillary convective Polymerase Chain Reaction
摘要
解鏈曲線分析(Melting Curve Analysis , MCA)是利用DNA的雙股解鏈現象與螢光染劑亮度之關係，吾人可透過螢光訊號隨溫度變化得知該DNA之解鏈溫度，由於不同DNA之解鏈溫度不同，因此MCA可用來檢測檢體專一性。 市面上販售的商用MCA machine主要是以溫控系統對DNA檢體進行PCR擴增，擴增過程結束後再對DNA檢體進行MCA，但由於此種升溫系統十分精密，因此這些機台普遍有造價昂貴的缺點。 2011年Chou團隊開發出毛細管熱對流PCR[1] (Capillary Convection PCR , CCPCR)，這種以單一溫控即可完成核酸擴增的方法，改善了傳統PCR機台溫控系統所需的昂貴成本，但CCPCR目前仍缺與MCA整合的系統。 本研究嘗試將CCPCR與空間式的MCA結合起來，將DNA檢體進行CCPCR後直接進行空間式MCA，在兩個過程中都是使用同一容器，以達到像Roche LightCycler2.0機台擁有核酸擴增與基因型分析的整合功能。 實驗先建立螢光偵測與加熱平台，然後決定毛細管內的溫度梯度，為了穩定毛細管內溫度場，因此本實驗使用紅銅鰭片來穩定溫度梯度。 最後針對三種DNA檢體進行MCA，將進行CCPCR後的試劑放置在螢光偵測平台，將拍出的螢光梯度對距離做一階微分取峰值，再對應到溫度梯度以決定檢體的Tm值，然後將結果與Roche LightCycler 2.0 和預測值比較，比較的結果顯示本研究與商用機台之結果之趨勢相符。
並列摘要
The principle of Melting Curve Analysis (MCA) is to determine the specificity of amplicons through the relationship between intensity of fluorescence dye and the temperature of reagent in which the amplicons are produced. Commercial MCA machines are typically combined with a Polymerase Chain Reaction (PCR) process. After DNA templates have been amplified by PCR to the enough amount which can be detected, then the MCA process is turned on to determine the specificity of amplicons. Since a commercial MCA machine has very high accuracy of temperature control, the cost of machine is expensive. Fortunately, Chen’s research group published and developed a Capillary Convection PCR (CCPCR) process to produce amplicons in 2011 [1]. This technique requires a single temperature control and can reduce the cost of PCR machine dramatically. However, their CCPCR machine is not equipped with MCA capability. In the past few years, some researchers used spatial temperature gradient to perform MCA, the advantage of which is the low cost of temperature control system and the short detection time. This thesis attempts to combine CCPCR with spatial- temperature- gradient MCA solution. DNA templates are amplified by a CCPCR platform in which the specificity of amplicons can be determined by the MCA approach. Then, such a CCPCR platform can have the same capability as those of Roche LightCycler, a commercial PCR machine integrate with MCA solution. This thesis first established a melting curve detection platform, including heating surface and optical system. The temperature gradient of reagent in the capillary tube can be established by heating the bottom surface of capillary tube and by cooling the capillary through the surrounding air. For the stability of temperature gradient, this work introduces a trapezoidal copper fin to cover the capillary tube to obtain stable temperature gradient along the tube axis. Lastly, three different DNA samples were tested. The measured melting temperatures of three tested samples are compared with those obtained by the commercial machine Roche LightCycler 2.0, and predicted values by well-known formula. The comparison shows a good agreement between the present measured results and those by the commercial machine.