《实验四PCR技术在动物传染病诊断中的作用》由会员分享，可在线阅读，更多相关《实验四PCR技术在动物传染病诊断中的作用（47页珍藏版）》请在金锄头文库上搜索。
1、实验四Evaluation only.Evaluation only. Created with Aspose.Slides for .NET 3.5 Client Profile 5.2.0.0.Created with Aspose.Slides for .NET 3.5 Client Profile 5.2.0.0. Copyright 2004-2011 Aspose Pty Ltd.Copyright 2004-2011 Aspose Pty Ltd.Evaluation only.Evaluation only. Created with Aspose.Slides for .NE
2、T 3.5 Client Profile 5.2.0.0.Created with Aspose.Slides for .NET 3.5 Client Profile 5.2.0.0. Copyright 2004-2011 Aspose Pty Ltd.Copyright 2004-2011 Aspose Pty Ltd.聚合酶链反应(Polymerase chain reaction)简称PCR，是一种选择 性体外扩增DNA或RNA的方法。PCR又称为无细胞克隆系统或特异性DNA序列体外引物定向酶促 扩增法，可将极微量的靶DNA特异地扩增上百万倍。Evaluation only.Evalu
3、ation only. Created with Aspose.Slides for .NET 3.5 Client Profile 5.2.0.0.Created with Aspose.Slides for .NET 3.5 Client Profile 5.2.0.0. Copyright 2004-2011 Aspose Pty Ltd.Copyright 2004-2011 Aspose Pty Ltd.n 模板DNA或RNAn 特异性引物: sense和 antisensen Taq酶：耐热DNA聚合酶n dNTPsn Mg2+ n PCR缓冲液： 10mmol/LTris-HCl
4、 pH8.450mmol/L KClMg2+0.1mg/mL乙酰BSAPCR体系基本组成成分Evaluation only.Evaluation only. Created with Aspose.Slides for .NET 3.5 Client Profile 5.2.0.0.Created with Aspose.Slides for .NET 3.5 Client Profile 5.2.0.0. Copyright 2004-2011 Aspose Pty Ltd.Copyright 2004-2011 Aspose Pty Ltd.模板DNAMg2+dNTPTaq酶引物Evalu
5、ation only.Evaluation only. Created with Aspose.Slides for .NET 3.5 Client Profile 5.2.0.0.Created with Aspose.Slides for .NET 3.5 Client Profile 5.2.0.0. Copyright 2004-2011 Aspose Pty Ltd.Copyright 2004-2011 Aspose Pty Ltd.引物设计注意事项引物的长度一般为15-30bp，常用的是18-27 bp，但不应大于38bp，因为过长会导致其延伸温度大于74，不适于TaqDNA聚合
6、酶进行反应。 引物3端的末位碱基对Taq酶的DNA合成效率有较大的影响。末位碱基为A的错配效率明显高于其他3个碱基，因此应当避免在引物的3端使用碱基A。另外，两条引物3端若互补，或者单条引物自身形成发夹结构也可能导致PCR反应失败。 5端序列对PCR影响不太大，因此常用来引进修饰位点或标记物。可在引物设计时加上限制酶位点、起始密码子、缺失或插入突变位点以及标记生物素、荧光素、地高辛等。通常应在5端限制酶位点外再加1-2个保护碱基。两引物间最好不存在4个连续碱基的同源性或互补性。Evaluation only.Evaluation only. Created with Aspose.Slides
7、 for .NET 3.5 Client Profile 5.2.0.0.Created with Aspose.Slides for .NET 3.5 Client Profile 5.2.0.0. Copyright 2004-2011 Aspose Pty Ltd.Copyright 2004-2011 Aspose Pty Ltd.引物设计注意事项引物序列的GC含量一般为40-60%，过高或过低都不利于引发反应，因为GC含量决定了DNA双链的解链温度（Tm）。另外，上下游引物的GC含量不能相差太大。 G值是指dna双链形成所需的自由能，该值反映了双链结构内部碱基对的相对稳定性。应当选用
8、3端G值较低（绝对值不超过9），而5端和中间G值相对较高的引物。引物的3端的G值过高，容易在错配位点形成双链结构并引发DNA聚合反应。 引物二聚体及发夹结构的能值过高（超过4.5kcal/mol）易导致产生引物二聚体带，并且降低引物有效浓度而使PCR反应不能正常进行。 Evaluation only.Evaluation only. Created with Aspose.Slides for .NET 3.5 Client Profile 5.2.0.0.Created with Aspose.Slides for .NET 3.5 Client Profile 5.2.0.0. Copyr
9、ight 2004-2011 Aspose Pty Ltd.Copyright 2004-2011 Aspose Pty Ltd.引物的设计的总原则：扩增的效率和特异性w 长度：1530bpw 碱基尽可能随机分布（G+C含量 40-60%）w 引物内部不能形成二级结构w 两引物间没有互补链存在w 3端一定要与模板严格配对w 5端可引入突变，加酶切位点等辅助软件：primer5，Oligo，DNAsis等PCR反应体系引物Evaluation only.Evaluation only. Created with Aspose.Slides for .NET 3.5 Client Profile
10、5.2.0.0.Created with Aspose.Slides for .NET 3.5 Client Profile 5.2.0.0. Copyright 2004-2011 Aspose Pty Ltd.Copyright 2004-2011 Aspose Pty Ltd.primer5.0操作界面Evaluation only.Evaluation only. Created with Aspose.Slides for .NET 3.5 Client Profile 5.2.0.0.Created with Aspose.Slides for .NET 3.5 Client Pr
11、ofile 5.2.0.0. Copyright 2004-2011 Aspose Pty Ltd.Copyright 2004-2011 Aspose Pty Ltd.Evaluation only.Evaluation only. Created with Aspose.Slides for .NET 3.5 Client Profile 5.2.0.0.Created with Aspose.Slides for .NET 3.5 Client Profile 5.2.0.0. Copyright 2004-2011 Aspose Pty Ltd.Copyright 2004-2011
12、Aspose Pty Ltd.Evaluation only.Evaluation only. Created with Aspose.Slides for .NET 3.5 Client Profile 5.2.0.0.Created with Aspose.Slides for .NET 3.5 Client Profile 5.2.0.0. Copyright 2004-2011 Aspose Pty Ltd.Copyright 2004-2011 Aspose Pty Ltd.Evaluation only.Evaluation only. Created with Aspose.Sl
13、ides for .NET 3.5 Client Profile 5.2.0.0.Created with Aspose.Slides for .NET 3.5 Client Profile 5.2.0.0. Copyright 2004-2011 Aspose Pty Ltd.Copyright 2004-2011 Aspose Pty Ltd.X mol/L OD260/ A(16000)+C(70000)+G(12000)+T(9600) X: 引物摩尔浓度,A、C、G、T: 引物中4种不同碱基个数。 PCR反应体系引物浓度计算一般PCR反应中的引物终浓度为0.2-1.0mol/L。引物
14、过多会产生错误引导或产生引物二聚体，过低则降低产量。利用紫外分光光度计, 可精确计算引物浓度，在1cm光程比色杯中，260nm下，引物浓度可按下式计算：Evaluation only.Evaluation only. Created with Aspose.Slides for .NET 3.5 Client Profile 5.2.0.0.Created with Aspose.Slides for .NET 3.5 Client Profile 5.2.0.0. Copyright 2004-2011 Aspose Pty Ltd.Copyright 2004-2011 Aspose Pt
15、y Ltd.dNTP应用NaOH 将pH调至7.0，并用分光光度计测定其准确浓度。dNTP原液可配成5-10mM/L并分装，-20贮存。一般反应中每种dNTP的终浓度为2.5mM/L。当dNTP终浓度大于50mM/L时可抑制TaqDNA聚合酶的活性。4种dNTP的浓度应该相等，以减少合成中由于某种dNTP的不足出现的错误掺入。 PCR反应体系 4种三磷酸脱氧核苷酸(dNTP)Evaluation only.Evaluation only. Created with Aspose.Slides for .NET 3.5 Client Profile 5.2.0.0.Created with As
16、pose.Slides for .NET 3.5 Client Profile 5.2.0.0. Copyright 2004-2011 Aspose Pty Ltd.Copyright 2004-2011 Aspose Pty Ltd.Mg2+浓度对TaqDNA聚合酶影响很大，它可影响酶的活性和真实性，影响引物退火和解链温度，影响产物的特异性以及引物二聚体的形成等。通常Mg2+浓度范围为0.5-2mmol/L。对于一种新的PCR反应，可以用0.1-5mmol/L的递增浓度的Mg2+进行预备实验，选出最适的Mg2+浓度。在PCR反应混合物中，应尽量减少有高浓度的带负电荷的基团，例如磷酸基团或EDTA等可能影响Mg2+离子浓度的物质，以保证最适Mg2+浓度。PCR反应体系 Mg2+Evaluation only.Evaluation only. Created with Aspose.Slides for .NET 3.5 Client Profile 5.2.0.0.Created with Aspose.Sli
