KINETIC PCR ANALYSIS - REAL-TIME MONITORING OF DNA AMPLIFICATION REACTIONS
KINETIC PCR ANALYSIS - REAL-TIME MONITORING OF DNA AMPLIFICATION REACTIONS
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DOI：
10.1038/nbt0993-1026
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发表时间：
1993-09-01
期刊：
影响因子：
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通讯作者：
WATSON, R
中科院分区：
文献类型：
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作者：
HIGUCHI, R;FOCKLER, C;WATSON, R
我们描述了一个简单的，定量分析任何可扩增的DNA序列，使用摄像机监测多个聚合酶链反应（pcr）同时在热循环过程中。摄像机利用结合双链DNA的溴化乙啶（EtBr）荧光的增加来检测每个PCR中双链DNA （dsDNA）的积累。热循环过程中荧光积累的动力学与DNA拷贝起始数直接相关。产生可检测荧光所需的周期越少，目标序列的数量就越多。该方法的结果表明，PCR分析的动力学方法可以在很大的动态范围内灵敏、选择性地定量DNA。这种方法还提供了一种确定不同反应条件对扩增效果的影响的方法，因此可以深入了解基本的PCR过程。
We describe a simple, quantitative assay for any amplifiable DNA sequence that uses a video camera to monitor multiple polymerase chain reactions (PCRs) simultaneously over the course of thermocycling. The video camera detects the accumulation of double-stranded DNA (dsDNA) in each PCR using the increase in the fluorescence of ethidium bromide (EtBr) that results from its binding duplex DNA. The kinetics of fluorescence accumulation during thermocycling are directly related to the starting number of DNA copies. The fewer cycles necessary to produce a detectable fluorescence, the greater the number of target sequences. Results obtained with this approach indicate that a kinetic approach to PCR analysis can quantitate DNA sensitively, selectively and over a large dynamic range. This approach also provides a means of determining the effect of different reaction conditions on the efficacy of the amplification and so can provide insight into fundamental PCR processes.