BIORAD荧光定量PCR原理和方法介绍讲课文档.pptVIP

引物和探针的设计 现在六十二页，总共六十六页。 查找文献 确定目的基因 查找基因序列 序列分析 引物与探针的设计 验证引物与探针的特异性 现在六十三页，总共六十六页。 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 现在六十四页，总共六十六页。 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 现在六十五页，总共六十六页。 Primer Express Beacon Designer 引物和探针的设计 现在六十六页，总共六十六页。 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. SNP 检测，较Taqman探针有更大的温度选择性 多重PCR 难设计和合成 产生的荧光信号较低 价格昂贵 现在三十页，总共六十六页。 5’ 5’ 3’ 3’ d.NTPs Taq酶 Primers 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ 反应组分 FRET 探针 变 性 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ Taq 5’ 3’ R Q D R 现在三十一页，总共六十六页。 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