实时荧光定量PCR在临床医学中的应用.pptVIP

图示为杂合子样本，PCR后经过完全变性和退火，形成完全匹配的两种双链和有一个错配的两种双链，由此进行HRM会出现两个峰 如果是纯合子样本（野生或突变），则HRM相似，均为完全匹配 两种纯合子样本的差异靠突变位点的A-T, G-C的Tm区分 To perform High Resolution Melting analysis, the target sequence is amplified by PCR in the presence of a fluorescent dye that binds only to dsDNA. The dye does not interact with ssDNA, but fluoresces strongly in the presence of dsDNA. This change in fluorescence can be used both to measure the increase in DNA concentration during PCR and then to directly measure temperature-induced DNA dissociation during High Resolution Melting. After PCR, amplicons are shortly denatured