real-time PCR相关问题

宝生物工程（大连）有限公司TaKaRaBiotechnology（Dalian）Co.,Ltd.Add:辽宁省大连经济技术开发区东北二街19号邮编：116600No.19Dongbei2ndStreet,DevelopmentZone,Dalian(116600),ChinaTEL:0411-87641685，87641686；FAX：[phone]；E-mail:[e-mail]大连TaKaRa产品问题集F定量PCR相关制品问题集目录Q-1：荧光定量PCR（RealTimePCR）基础知识?······························································3Q-2：荧光定量检测方法如何选择?······················································································4Q-3：TaqMan探针法进行荧光定量，探针如何选择·······························································4Q-4：RealTimeRT-PCR如何选择TwoStepRT-PCR和OneStepRT-PCR?·······················5Q-5：绝对定量与相对定量有哪些差别?················································································5Q-6：荧光定量实验的成功因素有哪些?················································································5Q-7：进行RealTimePCR实验时，需要做哪些确认工作?····················································8Q-8：如何确认模板中是否含有阻害物质?·············································································8Q-9：扩增曲线的荧光信号值低怎么办?················································································8Q-10：出现扩增曲线朝右下方下落现象的原因是什么?···························································9Q-11：PCR扩增效率低的原因有哪些?················································································9Q-12：PCR扩增效率过高的原因是什么?···········································································10Q-13：融解曲线分析有复数峰产生的原因有哪些?·······························································10Q-14：TaKaRa有哪些「PerfectRealTime」产品?··························································10Q-15：目前宝生物提供不同特点的SYBRGreenI检出用试剂，分别为DRR041、DRR081和DRR091，应如何选择?·························································································12Q-16：TaKaRa「PerfectRealTime」制品应如何保存?····················································12Q-17：TaKaRa「PerfectRealTime」系列产品使用了HotStartDNA聚合酶，反应前是否需要进行酶的活性化步骤?···························································································12Q-18：进行二步法RealTimeRT-PCR反应时，RT反应后，以cDNA（RT反应液）为模板加入到PCR反应体系中的量是多少?·············································································13Q-19：使用TaKaRa「PerfectRealTime」系列产品，是否需要对Mg2+浓度进行研讨?···········13Q-20：通常PCR反应按三步法进行，为何使用TaKaRa「PerfectRealTime」系列产品时推荐的PCR程序多为两步法?························································································131Q-21：使用TaKaRa「PerfectRealTime」系列产品对引物有何要求?································13Q-22：TaKaRa「PerfectRealTime」系列产品中附带的ROXReferenceDye和ROXReferenceDyeII如何使用?·································································································13Q-23：使用TaKaRa「PerfectRealTime」系列产品应用ABIPRISM仪器进行RealTimePCR反应时实验结果不好，融解曲线无主峰，为什么?·····················································14Q-24：使用TaKaRa「PerfectRealTime」系列产品应用ABIPRISM仪器进行RealTimePCR反应时，是否需要进行了95℃、10分钟的变性步骤?···············································14Q-25：和某些公司制品相比，SYBRPremixExTaq和SYBRPremixExTaqII制品的颜色较淡，为什么?···············································································································14Q-26：PremixExTaq「PerfectRealTime」融解时，有絮状沉淀物，怎么办?····················14Q-27：如何利用CycleavePCR法进行SNP分型解析实验?················································14Q-28：CycleavePCR法有哪些应用?···············································································16Q-29：为什么在ROCHE的LightCycler2.0荧光定量PCR仪上检测不到5’CY5-3’BHQ3探针中CY5信号?···········································································································16Q-30：FAM染料的荧光强度是否随溶液pH的变化而改变?···················································16Q-31：在荧光定量实验中是否可以使用未经纯化的脱盐引物?················································17Q-32：ECLIPSE标记的探针在进行质谱检测时，为什么会出现比目的峰分子量小170.5Da的峰和与目的分子量有±15Da差别的峰出现?·································································172Q-1：荧光定量PCR（RealTimePCR）基础知识?A-1：技术原理：实时荧光定量PCR，利用荧光信号积累实时监测整个PCR进程，最后通过标准曲线对未知模板进行定量分析的方法。PCR反应过程中产生的DNA拷贝数是呈指数方式增加的，随着反应循环数的增加，最终PCR反应不再以指数方式生成模板，从而进入平台期。将已知浓度的标准品梯度稀释进行RealTimePCR，按照起始DNA量由多到少的顺序等间隔得到一系列扩增曲线。Ct值与起始模板数的对数值之间存在线性关系，可以制作标准曲线。未知浓度的样品也得到Ct值，代入标准曲线，就可以求出未知样品的起始模板量。RealTimePCR的定量原理图应用进展：RealTimePCR是具有划时代意义的技术，具有操作简单、快速方便、灵敏度高、重复性好、污染率低等优点，不仅应用于基因表达解析，还广泛应用于SNP分型解析、物种鉴定、病毒和病原菌的检测、转基因食品的定量分析、导入基因拷贝数的解析等诸多领域。阈值（threshold）：在扩增曲线的指数增长区域内适当位置上设定的荧光检出界限。Ct值（Cyclethreshold）：表示每个PCR反应管内荧光信号到达设定的域值时所经历的循环数。经数学理论证明，Ct值与起始模板数的对数值成反比线性关系。阈值和Ct值的相互关系图相关系数（rSquared）反映标准曲线的直线性，理想值应大于0.98，越接近于1说明直线性越好，定量越准确。斜率（slope）反映PCR扩增效率，-0.25～-0.33（根据不同装置数值不同）。扩增效率（E）理想扩增效率应在0.8﹤E﹤1.2。3标准曲线的评价指标图例Q-2：荧光定量检测方法如何选择?A-2：必须根据具体的实验用途进行选择。如果用于区别同源性高的序列以及进行SNP分型解析等多重PCR检测，荧光探针法无可替代，而进行除此之外的RealTimePCR实验，我们推荐使用简单易行、成本低的SYBRGreenI荧光嵌合法。可以参考如下信息：SYBRGreenI嵌合荧光法探针法优点简单易行，成本较低，无需合成特异性探针。特异性强，能进行多重PCR。缺点对扩增的特异性要求高；不能进行多重PCR。需要设计特异性探针，成本较高；有时探针设计困难。Q-3：TaqMan探针法进行荧光定量，探针如何选择?A-3：TaqMan探针法是使用5′端带有荧光物质（如：FAM等），3′端带有淬灭物质（如：TAMRA、Eclipse、BHQ等）的TaqMan探针进行荧光检测的方法。当探针完整时，5′端的荧光物质受到3′端的淬灭物质的制约，不能检出荧光。探针的报告基团的发射波长要在淬灭基团的吸收波长范围内。淬灭基团种类淬灭基团性质使用的报告基团种类TAMRA荧光物质FAM,HEX,TET,JOE等ECLIPSE非荧光物质FAM,HEX,TET,JOE,TAMRA,ROX等BHQ0非荧光物质BODIPY493/503,BODIPYFL等BHQ1非荧光物质FAM,HEX,TET,JOE等BHQ2非荧