Product differentiation by analysis of DNA melting curves during the polymerase chain reaction
Product differentiation by analysis of DNA melting curves during the polymerase chain reaction
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DOI：
10.1006/abio.1996.9916
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发表时间：
1997-02-15
影响因子：
2.9
通讯作者：
Wittwer, CT
中科院分区：
文献类型：
--
作者：
Ririe, KM;Rasmussen, RP;Wittwer, CT
与热循环仪集成的微量荧光计用于通过双链 DNA 特异性染料 SYBR Green I 的荧光监测来获取聚合酶链式反应过程中的 DNA 熔解曲线。当热循环仪加热到产物的解离温度时，将荧光绘制为温度的函数，从而得出 DNA 熔解曲线。该 DNA 熔解曲线的形状和位置是 GC/AT 比率、长度和序列的函数，可用于区分熔解温度相差小于 2°C 的扩增产物。可以将所需产物与不需要的产物区分开来，在许多情况下无需凝胶电泳。当用双链 DNA 特异性染料监测扩增时，熔解曲线分析可以扩展初始模板定量的动态范围。产物的完整扩增和分析可在 15 分钟内完成。 (C) 1997 年学术出版社。
A microvolume fluorometer integrated with a thermal cycler was used to acquire DNA melting curves during polymerase chain reaction by fluorescence monitoring of the double-stranded DNA specific dye SYBR Green I. Plotting fluorescence as a function of temperature as the thermal cycler heats through the dissociation temperature of the product gives a DNA melting curve. The shape and position of this DNA melting curve are functions of the GC/AT ratio, length, and sequence and can be used to differentiate amplification products separated by less than 2 degrees C in melting temperature. Desired products can be distinguished from undesired products, in many cases eliminating the need for gel electrophoresis. Analysis of melting curves can extend the dynamic range of initial template quantification when amplification is monitored with double-stranded DNA specific dyes. Complete amplification and analysis of products can be performed in less than 15 min. (C) 1997 Academic Press.