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荧光定量PCR技术及其应 用 FieldApplicationScientist Molecular&CellularBiology 8008208982/CNMCBSupport@lifetech.com Agenda 荧光定量PCR技术的工作原理 荧光定量PCR技术的应用 2|LifeTechnologiesProprietary&Confidential|2/20/2012 荧光定量PCR的工作原理 化学原理 数学原理 检测原理 3|LifeTechnologiesProprietary&Confidential|2/20/2012 •与传统PCR一样用同样的基本成分(双链DNA,引物,dNTPs, PCRbuffer,Taq酶等) TGA T AG C GGAC TGCAAACAGATTAGACATAGATAGACAGATTAGATAGACTTAGATGTTTGGCA ACGTTTGTCTAATCTGTATCTATCTGTCTAATCTATCTGAATCTACAAACCGT Mg2+Mg2+ Mg2+ Mg2+ Mg2+Mg2+ 4|LifeTechnologiesProprietary&Confidential|2/20/2012 •PCR反应液加入各种类型的荧光染料 5|LifeTechnologiesProprietary&Confidential|2/20/2012 两种化学方法 TaqMan探针法染料法 6|LifeTechnologiesProprietary&Confidential|2/20/2012 TaqMan水解探针作用机理 一对PCR引物 7|LifeTechnologiesProprietary&Confidential|2/20/2012 TaqMan水解探针作用机理 引物之间一条寡核苷酸,称作探针 8|LifeTechnologiesProprietary&Confidential|2/20/2012 TaqMan水解探针作用机理 报告基团淬灭基团 9|LifeTechnologiesProprietary&Confidential|2/20/2012 TaqMan水解探针作用机理 完整的探针:5′端荧光基团吸收能量后将能量转移给临近的3′ 端荧光淬灭基团(发生荧光共振能量转移,FRET) 激发光 能量无荧光信号产生 10|LifeTechnologiesProprietary&Confidential|2/20/2012 TaqMan水解探针作用机理 Lightenergy 11|LifeTechnologiesProprietary&Confidential|2/20/2012 变性(9595ºCºC) 12|LifeTechnologiesProprietary&Confidential|2/20/2012 退火(60(60ºC)ºC) 13|LifeTechnologiesProprietary&Confidential|2/20/2012 14|LifeTechnologiesProprietary&Confidential|2/20/2012 15|LifeTechnologiesProprietary&Confidential|2/20/2012 16|LifeTechnologiesProprietary&Confidential|2/20/2012 17|LifeTechnologiesProprietary&Confidential|2/20/2012 18|LifeTechnologiesProprietary&Confidential|2/20/2012 ? 19|LifeTechnologiesProprietary&Confidential|2/20/2012 Taqisspecial... •5′-3′外切酶活性 •将探针5′端连接的荧光基团从探针上 切割下来 20|LifeTechnologiesProprietary&Confidential|2/20/2012 21|LifeTechnologiesProprietary&Confidential|2/20/2012 22|LifeTechnologiesProprietary&Confidential|2/20/2012 23|LifeTechnologiesProprietary&Confidential|2/20/2012 24|LifeTechnologiesProprietary&Confidential|2/20/2012 25|Lif