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1 实时荧光定量PCR(real-time PCR)的工作原理和方法1.1 原理 1991年Holland首先提出了Taqman探针的原理,该法利用TaqDNA酶的5’→3’核酸外切酶活性水解结合在靶扩增子上的杂交探针。该探针的5’末端有一个报告基团为6-羧基荧光素(6-carboxy-fluoroscein,FAM),发射峰值在518nm;3’末端的猝灭基团为6-羧基四甲基若丹明(6-car-boxy-tetramethyl-rhodamine,TAMRA),发射峰值在582nm。3’末端被磷酸化,以防止探针在PCR过程中被延伸。当探
1 Real-time PCR (real-time PCR) working principle and method 1.1 Principle 1991 Holland first proposed the principle of Taqman probe, the method of Taq DNA enzyme 5 ’→ 3’ exonuclease activity in the target hydrolysis Hybridization probe on amplicon. The reporter probe has a reporter group at the 5 ’end of 6-carboxy-fluoroscein (FAM) with an emission peak at 518 nm; the quenching group at the 3’ end is 6-carboxytetramethylorrhodamine (6-car-boxy-tetramethyl-rhodamine, TAMRA) emission peak at 582nm. The 3 ’end is phosphorylated to prevent the probe from being extended during PCR. When exploration