[医药卫生]pyrosequencing焦磷酸测序.pptVIP

焦磷酸测序法的原理及应用 （Pyrosequencing遗传分析技术） Detection of the light Pyrosequencing 系统平台 Pyrosequencing 系统平台 1. Assay Design 2. PCR扩增 一对扩增引物中的一条带有生物素标记，另一条不带标记。 3. Single Strand Template Preparation 真实的序列：遗传检测的黄金标准 快速的实时输出：1 碱基/分钟 96个样本平行处理能力 新一代的Pyro Gold试剂：表现大为提升 5. Pyrogram Analysis Pyrosequencing技术的特点 得到定量序列结果的技术 极高的准确性 Built-In QC 重现性极佳 通用型技术平台 功能多样，应用领域极广 可以用于任何遗传多态性的分析 甲基化研究的平台 高通量、低成本的快速临床微生物诊断 实验设计灵活 自动化程度高，操作简单 Why Pyrosequencing is quantitative？ Pyrosequencing：Accurate Quantifying What are ”Inbuilt Controls”? Benefits of Built-in Quality Control QC基于周边的序列信息 鉴定的黄金标准 从primer后的第一个碱基开始——便于数据alignment QC 验证了实验的成功与否 具体到每一轮的每一个样品 万一实验失败，可以帮助分析原因，解决问题 Integrated Software Package Pyrosequencing的应用 已发表的文章 Relative peak height = = peak1 height peak1 height + peak2 height Allele frequency = = relative peak height x 100 (%) Relative peak height Allele frequency (%) 0,000 0,100 0,200 0,300 0,400 0,500 0,600 0,700 0,800 0,900 1,000 0 10 20 30 40 50 60 70 80 90 100 15 % 30 % 75 % E S G C T A G T A G A G C/C E S G C T A G T A G A G T/T E S G C T A G T A G A G C/T Since Pyrosequencing shows polymorphisms with sequence context, you can always trust the validity of the data. Pyrosequencing焦磷酸测序系统的卓越表现： 99.998% accurate (based on analysis of 100,000 wells with known genotype) 整合且不断扩展的软件包 SNP - SNP and mutation analysis AQ - Included in SNP software - Easy statistics function SQA - Base calling - Sequence alignment All three with quality assessment Running Analyzing Planning Documenting Sequence analysis Sequence identification Microbial typing Cloned DNA re-sequencing mtDNA/Forensics Transgenetics Expression profiling Oligo ID Microsatellites Known positions Unknown positions within hot  spots Point mutations Insertions/