質體 DNA 構形對即時定量聚合酶鏈反應定量偏差之探討

即時定量聚合酶鏈反應 (real-time quantitative polymerase chain reaction，real-time qPCR) 為目前檢測與定量基因改造生物的主要方法，現行較普遍的方法是以質體 DNA 建構標準曲線進而推算基改成份含量的絕對定量法。近期有研究指出環狀 DNA 的構形會影響 PCR 產物的增幅，因此本研究探討超螺旋狀、線狀、缺口性環狀及封閉環狀四種不同構形的質體，在 real-time qPCR 以及一般 DNA 定量上所造成的偏差，並實際進行基因改造玉米 NK603 的定量檢測。本研究選用四種 DNA 定量方法，分別是 O.D. 260 nm 定量法、Hoechst 33258 螢光染劑定量法、Quant-iT 定量法及酵素反應之 HPLC 定量法，結果顯示 O.D. 260 nm 定量法得到較高的定量結果，而質體構形對於 Quant-iT 定量法影響最大。超螺旋狀質體與其他構形的質體，在定量的差異可達 1.62 至 1.88 倍。在 real-time qPCR 的部份，選用四種系統，分別是 SYBR Green I、TaqMan、TaqMan MGB 及 LNA TaqMan，結果顯示無論那一個系統，當質體構形為線狀或缺口性環狀時，real-time qPCR 的測定結果較高，為超螺旋狀質體的 1.9 至 5.4 倍不等，可能是因為質體構形影響前幾個循環的 PCR 增幅效率而造成的。本研究並推論一數學模型討論計算之，顯示超螺旋狀構形的 PCR 增幅效率與缺口性環狀構形相比，可低至 20%，因而造成增幅曲線及標準曲線的差異。若加上先前 DNA 定量的偏差，則最壞情況會產生 10.2 倍的差異。在基因改造成分檢測方面，由於目標基因與內源性基因都是經由質體標準曲線推算，因此定量上的偏差會互相抵消，對於定量基因改造成分方面，質體構形不會造成顯著的影響。但值得注意的是，線狀質體建立的標準曲線可能不足以涵蓋低含量的樣品。本研究結果顯示在使用以質體作為標準曲線進行 real-time qPCR 之絕對定量法時，參考質體的製備與定量方法必須嚴謹，加以標準化，以避免定量結果的不確定性。

關鍵字

即時定量聚合酶鏈反應；基因改造生物；質體構形； PCR 增幅效率； DNA 定量；絕對定量

並列摘要

Real-time quantitative PCR (qPCR) is the “gold standard” method of accurate quantification of genetically modified organisms (GMO). Precise quantification of target gene could be achieved by absolute quantification method based on a standard curve constructed by plasmid DNA. A recent study showed that supercoiled plasmid may suppress PCR amplification in real-time qPCR assay. In this study, we investigate the bias of DNA quantification caused by plasmid DNA conformations (supercoiled, linear, nicked circular and closed circular) both in DNA quantification methods and qPCR. Three DNA quantitative methods we used are UV absorbance at 260 nm, Hoechst 33258 method, Quant-iT assay and HPLC method. The DNA amount measured by UV absorbance is significantly higher than other methods. The effect of plasmid DNA conformations is most significantly in Quant-iT assay, and the differences among plasmid DNA conformations were between 1.62 to 1.88 fold. The qPCR systems we tested were SYBR Green I, TaqMan, TaqMan MGB and LNA TaqMan. The results showed that the standard curves which constructed with linear or nicked circular plasmid have higher relative amplifications which approximately 1.9 to 5.4 fold. The difference observed in this study may due to the difference of amplification efficiency caused by plasmid DNA conformations in the early stage of PCR. We constructed a theoretical model to elaborate possible mechanism underneath this phenomenon. The difference of PCR efficiency between supercoiled and nicked circular plasmids could be nearly 80%. In a worst-case scenario, the bias caused by DNA quantification and qPCR will be 10.2-fold. The bias of quantification which caused by plasmid conformations in the real-time qPCR assay of GM NK603 maize will neutralized due to the same bias tendency of target and reference gene standard curves. However, the plasmid conformations still have significant effect on the limit of detection of real-time qPCR assay.

並列關鍵字

real-time qPCR ； genetically modified organism (GMO) ； plasmid conformation ； PCR amplification efficieny ； DNA quantification ； absolute quantification

參考文獻

Ahmed, F.E. 2002. Detection of genetically modified organisms in foods. Trends Biotechnol 20: 215-223.

Andersen, C.B., Holst-Jensen, A., Berdal, K.G., Thorstensen, T. and Tengs, T. 2006. Equal performance of TaqMan, MGB, molecular beacon, and SYBR green-based detection assays in detection and quantification of Roundup Ready soybean. J Agric Food Chem 54: 9658-9663.

Andrus, A. and Kuimelis, R.G. 2001a. Analysis and purification of synthetic nucleic acids using HPLC. Curr Protoc Nucleic Acid Chem Chapter 10: Unit 10 15.

Andrus, A. and Kuimelis, R.G. 2001b. Base composition analysis of nucleosides using HPLC. Curr Protoc Nucleic Acid Chem Chapter 10: Unit 10 16.

Arumuganathan, K. and Earle, E.D. 1991. Nuclear DNA content of some important plant species. Plant Mol Biol Report 9: 208-218

國際替代計量

質體 DNA 構形對即時定量聚合酶鏈反應定量偏差之探討

全文下載

主題瀏覽