荧光
分析化学(期刊)
温度循环
热电偶
磺酰罗丹明B细胞培养试剂染料
熔化曲线分析
光漂白
温度测量
荧光计
材料科学
化学
热的
色谱法
热力学
实时聚合酶链反应
光学
复合材料
物理
细胞毒性
基因
体外
生物化学
作者
Lindsay N. Sanford,Carl T. Wittwer
标识
DOI:10.1016/j.ab.2012.10.037
摘要
Accurate control of the sample temperature during thermal cycling is critical for successful polymerase chain reaction (PCR). Direct sensor contact with the reaction is problematic, forcing measurements external to the sample and compromising accuracy during rapid temperature transitions. The widespread use of fluorescence in real-time PCR and melting analysis suggests another measure of temperature, the intrinsic fluorescence of temperature-sensitive passive dyes. Calibration curves correlating sulforhodamine B fluorescence to temperature on nine real-time PCR instruments were obtained by heating at 0.018–0.1 °C/s between 50 and 95 °C, with a twofold change in fluorescence. After instrument stabilization for 20 min, no dye photobleaching was observed and thermal degradation was 2.2%/h at 80 °C. During cycling, solution temperatures derived from fluorescence were well matched to thermocouples placed within samples, but not to temperatures recorded by the instrument. Solution temperatures lagged instrument temperatures by up to 8 °C during cycling, often requiring 5–10 s at target temperatures for equilibration. Melting curves were displaced by 0.2–1.1 °C. Temperature inaccuracies were dependent on the instrument, the ramp rate, and the sample volume. The fluorescence of passive dyes can be used to accurately assess solution temperatures during PCR and should be particularly useful at fast cycling speeds.
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