Crockett A O, Wittwer C T
Department of Pathology, University of Utah Medical School, Salt Lake City, Utah 84132, USA.
Anal Biochem. 2001 Mar 1;290(1):89-97. doi: 10.1006/abio.2000.4957.
Fluorescein-labeled oligonucleotide probes can be used to continuously monitor the polymerase chain reaction. Depending on the sequence, the fluorescence intensity of the probe is either increased or decreased by hybridization. The greatest effect is probe quenching by hybridization to amplicons containing deoxyguanosine nucleotides (Gs), giving a sequence-specific decrease in fluorescence as product accumulates. Quenching of the probes by Gs is position dependent. A 25% decrease in fluorescence of 5'-labeled probes was observed with a G at the first position of the 3'-dangling end. Additional Gs can increase quenching to about 40%. This change in fluorescence with hybridization allows real-time quantification and mutation detection with a simple single labeled probe. Quantification of the initial template copy number is possible by monitoring fluorescence at each cycle at a constant temperature. Mutation detection by Tm estimates from melting curve analysis for factor V Leiden, hemoglobin C, hemoglobin S, the thermolabile mutation of methylenetetrahydrofolate reductase, and the cystic fibrosis-associated deletion F508del is demonstrated. By using the inherent quenching of deoxyguanosine nucleotides in the amplicon, complicated probe designs involving internal quenching can be avoided.
荧光素标记的寡核苷酸探针可用于连续监测聚合酶链反应。根据序列的不同，探针的荧光强度会因杂交而增强或减弱。最大的影响是探针与含有脱氧鸟苷核苷酸（G）的扩增子杂交导致淬灭，随着产物积累，荧光会出现序列特异性降低。G对探针的淬灭作用取决于位置。当3'端悬垂末端的第一个位置为G时，观察到5'端标记探针的荧光降低了25%。额外的G可使淬灭增加至约40%。这种杂交时荧光的变化允许使用简单的单标记探针进行实时定量和突变检测。通过在恒定温度下监测每个循环的荧光，可以对初始模板拷贝数进行定量。通过熔解曲线分析对因子V莱顿、血红蛋白C、血红蛋白S、亚甲基四氢叶酸还原酶的热不稳定突变以及囊性纤维化相关缺失F508del进行熔解温度（Tm）估计来检测突变。通过利用扩增子中脱氧鸟苷核苷酸的固有淬灭作用，可以避免涉及内部淬灭的复杂探针设计。
