.qRTPCR生物分析检测技术.ppt

第二节 实时荧光定量PCR SYBR Green I 定量原理 确定初始模板的浓度: 初始DNA量越多, 荧光达到某一值（域值）时所需要的循环数越少Log浓度与循环数呈线性关系：根据样品扩增达到域值的循环数就可计算出样品中所含的模板量 PRIMER AND PROBE DESIGN Primers and probes must be carefully designed because of the costs associated with producing probes with different dyes at 5 and 3 ends. PE/ABD Primer Express software, which is specifically designed to select the primers and probes. The required parameters for well-designed primers and probe have been well built into the program. These parameters include a Tm for the probe that is 10°C higher than the primers, primer Tm is between 58°C and 60°C, amplicon size between 50 and 150 bases, absence of 5 Gs. Primer and probe design is different for the allelic discrimination assays. The probes designed for each allele should be centered over the mismatched base, and the probes only have to differ by that base. Another major difference is that the probe for these assays has a lower Tm requirement than the TaqMan PCR assays. Prescreening assay SYBR Green can be used as a probeless alternative to the TaqMan system. Because it binds to all double-stranded DNA, it is imperative to ensure that the PCR product being quantified is a clean, single product, because any primer-dimers or background smears are detected. It can be used as a prescreening assay before ordering a probe. If the results are satisfactory, the probe can be ordered and TaqMan reactions run with higher specificity. 11. 内标技术介绍 三、 实时荧光定量PCR技术的应用介绍 荧光定量PCR技术的应用 对DNA、RNA样品进行定量和定性分析 定量分析包括绝对定量分析和相对定量分析。 前者可以得到某个样本中基因的拷贝数和浓度； 后者可以对不同方式处理的两个样本中的基因表达水平进行比较。 可以不定期对PCR产物或样品进行定性分析如： 利用熔解曲线分析识别扩增产物和引物二聚体，以区分非特异扩增； 利用特异性探针进行基因型分析及SNP检测等。 目前实时荧光PCR技术已经被广泛应用于基础科学研究、临床诊断、疾病研究及药物研发等领域。其中最主要的应用集中在以下几个方面： 比较经过不同处理样本之间特定基因的表达差异（如药物处理、物理处理、化学处理等），及特定基因在不同相的表达差异； 比较正常组织与病理组织中各种mRNA 表达量差异； 验证基因芯片实验结果； 验证RNA干扰实验结果。 分子 Beacons是一种在靶DNA不存在时形成茎环结构的双标记寡核苷酸探针。 在此发夹结构中：位于分子一端的荧光基团与分子另一端的淬灭基团紧紧靠近。 在此结构中，荧光基团被激发后不是产生光子，而是将能量传递给淬灭剂，这一过程称为荧光谐振能量传