即時聚合酶鏈鎖反應(qPCR)被廣泛應用於分子研究與診斷上，尤其是核酸分子的定量與檢測。qPCR結合PCR放大及偵測每個循環的螢光訊號，來達到即時定量的目的，由於不需要使用電泳來檢測PCR產物，因此能夠更精確的定量與分析實驗結果。 然而，研究發現許多疾病的成因是緣於單一核苷酸的突變，並且難以被一般DNA引子或探針辨識，而錯誤的診斷常造成治療的延誤。為了使疾病的診斷更為準確，以現有分子檢測為主的PCR或qPCR而言，只能從核酸放大及標定所需的primer與targeting probe來增加專一性。本研究利用一種核酸類似物，促使兩股DNA間的靜電排斥力下降，以便在進行互補股序列雜交時可以形成更穩定的雙股螺旋結構。期望在特定的PCR操作條件及核酸類似物的設計下，使perfect match之Tm上升或是mismatch之Tm下降，以獲得更大的ΔTm，並藉由調整PCR實驗過程中引子與模板進行結合時的黏合溫度(Annealing temperature)來達到專一性的提升。 在本研究中，針對PCR及qPCR檢測平台進行引子與探針的修飾，利用修飾核酸類似物來增加其專一性。從實驗結果可以得知，經過核酸類似物修飾的引子在辨識單一鹼基錯誤配對上的能力是明顯優於一般DNA引子，此外，在特定的PCR條件下，提升黏合溫度能夠使改質後的引子的辨識效果更加專一。在SNP基因分型的實驗中，將1-2個核酸類似物修飾在帶有不同螢光分子的TaqMan probe上，並與一般TaqMan probe和LNA probe進行比較，實驗結果顯示，帶有核酸類似物的探針不僅具有相似的分型表現，隨著黏合溫度的調整，還可以增加實驗的專一性。期望未來能夠運用此種核酸類似物的特性，建立相關之最佳化操作條件與核酸類似物的設計，以便能提供更高精確度之生物分子檢測平台。 ;Quantitative polymerase chain reaction (qPCR) is one of the powerful techniques for detection and quantification of nucleic acids and has enormous utilization in molecular research and diagnostics. qPCR combines PCR amplification and detection into a single step. This eliminates the need to detect products using gel electrophoresis, and provides a more precise quantification. Nevertheless, some diseases which are caused by single nucleotide mutation are difficult to identify when using native DNA primers and probes. A false diagnosis may lead to incorrect treatment or delayed treatment. In order to achieve more accurate detection and diagnosis of the diseases, we need to develop a more precise method. For the existing molecular detection methods, PCR or qPCR, can only increase the specificity from the nucleic acid amplification of the desired primers and targeting probes. With the aim of enhancing the specificity, we use neutralized DNA (nDNA) which is a DNA analogue with the backbone phosphate groups replaced by phosphate methylated groups to modify DNA primers and probes. Because of the reduction of electrostatic repulsion, the complementary nDNA/DNA duplex shows stronger hybridization affinity compared to the DNA/DNA duplex. It is expected to obtain greater ΔTm and the enhancement of specifity through the adjustment of PCR operating conditions and the desing of nDNA. In this study, we focus on the specificity enhancement of PCR and qPCR assay with primers and probes containing one and/or two nDNA base. According to the experimental results, we observe that the nDNA-modified primers have better discrimination ability between perfect match oligonucleotide and mismatch oligonucleotide than the native DNA primers and the intensity ratio can achieve 3 times higher with increasing temperature under certain PCR conditions. In SNP (Single Nucleotide Polymorphism) genotyping experiments, the results demonstrate that nDNA-taqMan probes can improve the specificity with increasing annealing temperature and perform as well as taqMan probes and LNA-TaqMan probes. Consequently, nDNA is a promising DNA analogue applied in primers and probes to enhance the specificity, and provide a more precise detection. It is expected to use the properties of nDNA in the future to establish the optimal PCR operating conditions and nDNA design to provide a higher accuracy biomolecular detection platform.
