Continuous fluorescence monitoring of rapid cycle DNA amplification
Continuous fluorescence monitoring of rapid cycle DNA amplification
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DOI：
10.2144/97221bi01
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发表时间：
1997-01-01
期刊：
影响因子：
2.7
通讯作者：
Rasmussen, RP
中科院分区：
文献类型：
--
作者：
Wittwer, CT;Herrmann, MG;Rasmussen, RP
通过三种不同的荧光技术对快速循环DNA扩增进行连续监测。荧光监测通过以下方式进行：（i）双链特异性染料SYBR® Green I；（ii）在双标记水解探针经核酸外切酶切割后，罗丹明对荧光素的猝灭作用降低；（iii）通过相邻杂交探针对荧光素到Cy5™的共振能量转移。每个循环采集一次荧光数据，可对初始模板拷贝数进行快速绝对定量。SYBR Green I检测的灵敏度受非特异性产物形成的限制。使用单个核酸外切酶水解探针或两个相邻杂交探针可提高特异性水平。与每个循环测量一次荧光不同，在每个循环中持续监测可监测荧光的温度依赖性。水解探针的累积、不可逆信号可很容易地与杂交探针的温度依赖性、可逆信号区分开来。通过使用SYBR Green I，可以在每个循环内跟踪产物变性、退火和延伸。在后期扩增循环中发生大量产物与产物的退火，这表明产物退火是平台期效应的一个主要原因。循环内的持续监测可快速优化扩增条件，在开发新的标准化临床检测方法中应该特别有用。
Rapid cycle DNA amplification was continuously monitored by three different fluorescence techniques. Fluorescence was monitored by (i) the double-strand-specific dye SYBR(R) Green I, (ii) a decrease in fluorescein quenching by rhodamine after exonuclease cleavage of a dual-labeled hydrolysis probe and (iii) resonance energy transfer of fluorescein to Cy5(TM) by adjacent hybridization probes. Fluorescence data acquired once per cycle provides rapid absolute quantification of initial template copy number. The sensitivity of SYBR Green I detection is limited by nonspecific product formation. Use of a single exonuclease hydrolysis probe or two adjacent hybridization probes offers increasing levels of specificity. In contrast to fluorescence measurement once per cycle, continuous monitoring throughout each cycle monitors the temperature dependence of fluorescence. The cumulative, irreversible signal of hydrolysis probes can be distinguished easily from the temperature-dependent, reversible signal of hybridization probes. By using SYBR Green I, product denaturation, annealing and extension can be followed within each cycle. Substantial product-to-product annealing occurs during later amplification cycles, suggesting that product annealing is a major cause of the plateau effect. Continuous within-cycle monitoring allows rapid optimization of amplification conditions and should be particularly useful in developing new, standardized clinical assays.