定量PCR限制

nullAdvantages and limitations of quantitative PCR (Q-PCR)-based approaches in microbial ecologyAdvantages and limitations of quantitative PCR (Q-PCR)-based approaches in microbial ecologyLvyi Cindy J.Smith&A.Mark Osborn Department of Animal and Plant Sciences,University of Sheffield,Western Bank, Sheffield ,UKnullAbstract Introduction Advantages of Q-PCR over traditional endpoint PCR Fluorescence detection chemistries used to detect template amplification during Q-PCR Target quantification using the cycle threshold (Ct) method Biological and methodological factors affecting quantification of genes and transcripts from environmental samples Quantifying gene expression by RT-Q-PCR nullPracticalities of (RT)-Q-PCR protocols Application of Q-PCR for investigating the microbial genetic potential within the environment Quantifying gene expression in environmental samples using RT-Q-PCR : a step closer to determining the functioning of target genes in the environment Combining (RT)-Q-PCR with other approaches to provide greater insight into community function and dynamics ConclusionsAbstractAbstractQuantitative PCR(Q-PCR orreal-timePCR) approaches are now widely applied In microbial ecology to quantify the abundance and expression of taxonomic and functional gene markers within the environment.null Firstly,this review addresses the theoretical and practical implementation of Q-PCR and RT-Q-PCR protocols in microbial ecology Secondly, review the applications of (RT)-Q-PCR analyses in environmental microbiology and evaluate the contribution and advances gained from such approaches. Finally, furthering understandingIntroductionIntroductionQuantitative -PCR定义： 在PCR反应体系中加入荧光基团，利用荧 光信号累积实现了实时监测整个PCR进程， 对起始模板进行定量的方法。Quantitative-PCR的应用Quantitative-PCR的应用①Quantitatively track phylogenetic and functional gene changes across temporal and spatial scales under varying environmental or experimental conditions. ②the quantitative data generated can be used to relate variation in gene abundances and/or levels of gene expression (in terms of transcript numbers) in comparison with variation in abiotic or biotic factor and/or biological activities and process rates.null③complement other quantitative environmental data sets ④reverse transcription (RT)analyses are now increasingly combined with Q-PCR Methods in RT-Q-PCR assays, offering a powerful tool for Quantifying gene expression (in terms of numbers of rRNA and mRNA transcripts) and relating biological activity to Ecological functionAdvantages of Q-PCR over traditional endpoint PCR Advantages of Q-PCR over traditional endpoint PCR 普通PCR技术：end- point PCR 在PCR结束后对终点产物进行定量分析。(反应不出模板初始量的不同) 实时定量PCR技术： 实时检测PCR扩增，在扩增的指数期对起始模板进行定量。nullTarget quantification using the cycle threshold (C t) methodTarget quantification using the cycle threshold (C t) methodThe Q-PCR amplification curve can be subdivided into four stages: Background noise Exponential amplification(指数期) Linear amplification（线性期） A plateau stage（平台期） nullnull荧光阈值：在荧光扩增曲线上人为设定的一个值，它可以设定在荧光信号指数扩增阶段任意位置上，但一般我们计算机将荧光域值的默认设置是 3-15 个循环的荧光信号的偏差的 10 倍。 Ct 值：每个反应管内的荧光信号到达设定的域值时所经历的循环数（cycle threshold ）nullnullnullBy detection of amplicons during the early exponential phase of the PCR,this enables the quantification of gene (or transcript) numbers when these are proportional to the starting template concentration. Log X0与Ct呈线性关系nullX0：初始模板量 Ex：扩增效率 XCt:荧光扩增信号达到阈值强度时扩增产物的量在阈值线设定以后,它是一个常数,我们设为M log X0= -log(1+Ex) *Ct+ log M CT 值与起始模板的关系研究明，每个模板的 CT 值与该模板的起始拷贝数的对数存在线性关系，起始拷贝数越多， CT 值越小。null利用已知起始拷贝数的标准品可作出标准曲线，因此，只要获得未知样品的 Ct 值，即可从标准曲线上计算出该样品的起始拷贝数。 Ct值logX0Absolute quantification VS. relative quantificationAbsolute quantification VS. relative quantification绝对定量:检测起始模板数的精确拷贝数， ----------标准曲线法 ‰相对定量:确定经过不同处理的样本之间基因的表达差异（研究真核较多，不适用于原核基因） ------------双标准曲线法 绝对定量的标准样品绝对定量的标准样品绝对定量的标准样品： 已知拷贝数的质粒DNA，做系列稀释。 标准样品的种类： •含有和待测样品相同扩增片段的克隆质粒（不可培养微生物定量） •含有和待测样品相同扩增片段的cDNA •PCR的产物 绝对定量代表的是与标曲比较的目的gene的量，而不是环境样品中的量。 Minor differences in Ct values and standard curves result in large Differences in gene copy numbers. Fluorescence detection chemistries used to detect template amplification during Q-PCRFluorescence detection chemistries used to detect template amplification during Q-PCR实时荧光定量 PCR 的化学原理包括 探针类、非探针类两种 探针类：利用与靶序列特异杂交的探针来指示扩增产物的增加（特异性更高） 非探针类：利用荧光染料或者特殊的引物来指示扩增的增加 （简便易行 ）（1）probe------TaqMan（1）probe------TaqMan™ 5′端标记有基团(Reporter, R) ™ 3′端标记有荧光淬灭基团(Quencher, Q) 探针完整，R所发射的荧光能量被Q基团吸收，无荧光，R与Q分开，发荧光 ™( Taq酶有5′→3′外切核酸酶活性，可水解探针)nullnull每扩增一条DNA分子，释放一个荧光信号Protocols of TaqmanProtocols of Taqman1、引物、探针的设计： 探针Tm为68-70℃，<30 bp, 5’不能有G，G可能会淬灭荧光素， 探针尽量靠近F引物，扩增片段<400 bp，引物Tm为59-60℃ 2、反应参数的确定： 一般为：95 ℃,3min 94 ℃，15S 40cycles 60 ℃，60S （Taq酶5′→3′外切酶活性在60℃最高也可通过温度梯度优化退火温度） 3、优化引物和探针浓度：获得最小Ct值，最大信号/背景比值 引物浓度：50-900nM 探针浓度：50-250nM 4、其他与常规PCR相同TaqMan法优缺点TaqMan法优缺点‰优点 对目标序列的高特异性--阴性结果确定 ‰引物设计相对简单 ‰重复性比较好 ‰缺点 只适合一个特定的目标 ‰委托公司标记，价格较高 (1)probe--------分子信标（molecular beacon） (1)probe--------分子信标（molecular beacon） 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。 分子信标的茎环结构中，环一般为 15-30 个核苷酸长，并与目标序列互补 设计要非常仔细，要保证：在复性温度下，模板不存在时形成茎环结构，模板存在时则与模板配对。与模板配对后，分子信标的构象改变使得荧光基团与淬灭剂分开。当荧光基团被激发时，它发出自身波长的光子 null变性时退火时延伸（2）no probe------SYBR Green（2）no probe------SYBR Green一种只结合于双链 DNA小沟中的染料。与双链 DNA 结合后，其荧光大大增强。 ‰变性时，DNA双链分开，无荧光 ‰在延伸结束阶段采集荧光信号。 ‰SYBR Green也能和非特异的双链DNA结合发光，所以必须在反应结束时做熔解曲线分析。null延伸结束阶段null优化PCR体系优化PCR体系SYBR Green 使用浓度:太高抑制Taq酶活性，太低，荧光信号太弱，不易检测 Primer：引物的特异性高，否则扩增有杂带，定量不准SYBR Green法优缺点SYBR Green法优缺点 优点 对DNA模板没有选择性--适用于任何DNA 使用方便--不必设计复杂探针 非常灵敏 便宜 缺点 容易与非特异性双链DNA结合，由引物二聚体、单链二级结构以及错误的扩增产物引起的假阳性会影响定量的精确性，但可以通过融解曲线的分析，优化反应条件 ‰ 对引物特异性要求较高（2）no probe-------LUX Primers （2）no probe-------LUX Primers LUX (light upon extention) 引物 目标特异的引物对中的一个引物 3’ 端用荧光报告基团标记 , 在没有单链模板的情况下，该引物自身配对，形成发夹结构，使荧光淬灭 . 有目标片断的时候，引物与模板配对，发夹结构打开，产生特异的荧光信号 LUX 引物通过二级结构实现淬灭，不需要荧光淬灭基团，也不需要设计特异的探针序列 (NEW!!!!!)nullBiological and methodological factors affecting quantification of genes and transcripts from environmental samplesBiological and methodological factors affecting quantification of genes and transcripts from environmental samplesFirstly, the choice of method used for nucleicacid extraction, extraction efficiencies vary considerably between different methods and the final nucleicacid yield is dependent on both the method used and the type of environmental sample being studied. Moreover, different extraction protocols, different laboratories.所得到的绝对定量就没有可比性，标曲的数量级设置不同也没有可比性。nullEnsure that the environmental template and the standard curve target gene have equivalent amplification efficiencies.相同的扩增效率（正常环境样品会受抑制剂的影响） A direct comparison should be used only for gene numbers determined within a single Q-PCR assay and using the same standard curve.Quantifying gene expression by RT-Q-PCRQuantifying gene expression by RT-Q-PCRCombining Q-PCR with an initial RT reaction facilitates the quantification of RNA transcripts (rRNA or mRNA), enabling quantitative estimates of the activity of specific taxa or functional guilds within a microbial community. As with DNA quantification, the first step towards accurate RNA quantification lies in the preparation of a high-quality template, free from inhibitors.但通过稀释来降低抑制物的浓度不可取，因为少量的RNA的量的变化会使RT体系改变，会导致转录物数量上的log变化。nullRNA template must be free from contaminating DNA that could contribute to the final amplification signal. Absolute numbers of RNA transcripts should be determined from standard curves constructed from cDNA. The efficiency of the initial RT step is critical for sensitive and accurate quantification.nullRT-Q-PCR amplifications can be conducted using either a one-step or a two-step reaction. one-step RT-Q-PCR Advantage: both the RT reaction and the Q-PCR are carried out Consecutively in a single tube.RNA is first reverse transcribed, with all resultant cDNA serving as templates in the Subsequent Q-PCR amplification. In addition to the reduced risk of contamination and the convenience of Setting up only a single reaction,a further advantage of this Method is that all the resulting cDNA produced is used to Quantify the target RNA sequence.one-step RT-Q-PCR disadvantageone-step RT-Q-PCR disadvantageFor the study of eukaryotes,one-step RT-Q-PCR reactions have been reported to have reduced sensitivity, as reaction conditions are compromised to accommodate the two Different enzymes required within a single reaction.（反转酶，合成酶）two-step RT-Q-PCR advantagetwo-step RT-Q-PCR advantageThe RT reaction and the Subsequent Q-PCR are carried out separately. Firstly,cDNA is generated in an independent RT reaction and subsequently an aliquot of this cDNA is used as a template for the Q-PCR. cDNA generated in the RT reaction can be used as a template for a number of different Q-PCR reactions. More economicallynullRandom primers can maximize the number of different cDNA templates generated, gene-specific reverse primers can increase the Sensitivity and specificity of the cDNA created and at the Same time reduce the amount of unspecific background cDNAApplication of Q-PCR for investigating the microbial genetic potential within the environmentApplication of Q-PCR for investigating the microbial genetic potential within the environmentBy targeting highly conserved regions of the 16SrRNA gene, Q-PCR assays have been designed to quantify‘total’ bacterial (and/orarchaeal) numbers while targeting of taxa specific Sequences within hypervariable regions within the gene enables quantification of sequences from phylum to species levels,provided that there are sequenced at a available that enable the design of primers and probes.nullnullnullQuantifying gene expression in environmental samples using RT-Q-PCR : a step closer to determining the functioning of target genes in the environment Quantifying gene expression in environmental samples using RT-Q-PCR : a step closer to determining the functioning of target genes in the environment While the Quantification of both rRNA genes and/or functional genes from the environment can be used as an indicator of the genetic potential within an environment and is suggestive of potential functional activity within a community. 直接检测mRNA比DNA更能直接表现功能基因的表达。Combining (RT)-Q-PCR with other approaches to provide greater insight into community function and dynamicsCombining (RT)-Q-PCR with other approaches to provide greater insight into community function and dynamicsSIP: Microarrays:One major disadvantageOne major disadvantage Require for prior sequence data of the specific target gene of interest.