BIO-RAD荧光定量PCR原理和方法介绍PPT.pptVIP

引物设计原则 长度：20-27nt G+C含量：45-55% 碱基的随机分布：3’端不应超过3个连续的G或C 引物自身：不应存在互补序列 引物之间：不应有互补性，尤应避免3’端的互补重叠 引物的3’端:3’也不能有形成任何二级结构的可能 引物的特异性 探针设计原则 序列保守 G+C含量比较高 Tm值：高10℃ 5’端不能是G Primer Express Beacon Designer 引物和探针的设计 谢谢! Self explanatory Practice with the animation so you understand the timing of this slide (if you haven’t used it before) - ask the audience to participate in this, it is a good way for you to determine the different comfort levels your audience has with the Ct concept. These data are a 5-fold serial dilution of cDNA from Human total RNA, but this is not the main focus of the slide. The standard curve is made from a known sample. The log of the copy number (x-axis) is plotted versus the threshold cycle (y-axis) to generate this graph. This slide explains the relative importance of r (correlation coefficient) and m (slope) from the standard curve equation. In effect, the r value reflects reproducibility - as in replicates. And more importantly the “linearity” of the curve. The efficiency can give you valuable information about the chemistry of your reaction. Efficiency = [10(-1/slope) ] - 1. For example: a slope of 3.322 ~ 100% , 3.5 ~ 95%. Same basic format as with standard PCR. However, the Intercalation Dye is also included. Very low background when not bound to dsDNA. Detection is performed at the annealing step due to the opening of the probe. The binding and unfolding of the probe is because it is energetically more favorable to bind the target sequence than to remain in the folded conformation. Same basic format as standard PCR. FRET 探针 5’ 3’ 5’ 3’ l 3’ D R 5’ 退 火 5’ 3’ 链替换,荧光熄灭 5’ 3’ 3’ 5’ Taq 3’ D R 1-5 bases D R Taq 5’ R 5’ 特异性好 SNP 检测 难设计 费用昂贵 Texas Red, ROX CY3.5, Redmond Red 常用的荧光基团 FAM TET, VIC CY5 CY5.5, LC705 LC640 HEX JOE CY3 TAMRA 应 用 相对定量 绝对定量 突变检测 等位基因检测 相对定量 参考基因：Actin, GAPDH, 18sRNA 靶基因A GAPDH ΔCt 对照组 25.2 19.5 5.7 实验组 22.1 20.8 1.3 1．计算ΔCt：ΔCt=Ct 靶基因－Ct GAPDH，分别计算实验组和对照组的ΔCt。 2．计算ΔΔCt：ΔΔCt=ΔCt实验组－ΔCt对照组，本例中ΔΔCt= －4.4