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视频|PCR的原理与操作

作者: JoVE 发布: 2018-03-27 10,387阅读 320评论

聚合酶链式反应，或称PCR，是一种通过热循环反应来扩增DNA的技术，热循环是指在固定的时间间隔内温度变化的循环。采用热稳定的DNA聚合酶，PCR可以用DNA的合成原料，脱氧核苷三磷酸合成大量的DNA拷贝。PCR反应包括三步，变性，退火，和延伸。变性是PCR循环的第一步，它将DNA碱基之间的氢键打开来熔解DNA双链而产生单链DNA。退火则是将温度降到足够低，使寡聚核苷酸引物能够结合到DNA模板上。在延伸过程中，DNA聚合酶将合成新的双链DNA。本短片将介绍PCR的过程。除了逐步讲述如何准备一个常用的PCR反应，还将描述PCR的基本原理。视频展示了一个PCR反应的所需组分，以及引物设计的规则，并给出了保证PCR反应成功的一些有用技巧。

PCR介绍
聚合酶链式反应或称PCR, 是一种被广泛用来扩增DNA片段的方法。PCR要用到热循环，就是在三个被称为变性、退火和延伸的特定温度之间反复地加热和制冷。

反应
一旦将PCR试剂混合物置于热循环仪中，热循环反应就开始了。热循环仪是一种被设定来精确地加热和制冷反应的仪器。

PCR循环从变性开始，在95度持续20到30秒，它远高于DNA的熔解温度。溶解温度是当半数DNA处于双链螺旋，半数处于单链时的温度。变性温度远远高于熔解温度，就可保证所有互补碱基之间的氢键都被打开，这样只有单链DNA存在。成对的DNA单链被称为有义链和反义链。有义链，或称编码链，与最终编码蛋白的mRNA的序列相同。因此称为有义。它从左侧的5’端磷酸基开始到右侧的3’端羟基结束。反义链又称为互补链，则从左侧的3’端羟基开始到右侧的5’端的磷酸基结束。

反应第二步是退火。被称为引物的DNA短序列，特定地与有义链或反义链互补并通过氢键和它们结合。与反义链结合，和有义链有同样序列的引物称为正向或有义引物；与有义链结合，和有义链序列反向互补的引物是反向或反义引物。根据所用引物的长度，退火温度通常要比两个引物溶解温度中较低的那个温度低3到5度。退火温度一般在50度到65度之间，退火时间持续20到40秒。

引物结合到DNA上后，将引导 复制DNA的聚合酶结合到它的3’端羟基上。

下一步就是延长或延伸，发生在72度，它是聚合酶活性的最佳温度。一旦聚合酶结合，就开始按5’到3’顺序将游离的脱氧核苷三磷酸一个个加到引物末端，形成双链DNA。

延伸结束后会开始下一个循环。下一个循环中，引物会与上一步延伸形成的单链DNA相结合。当聚合酶用通过反向引物得到链作为模板，来延伸正向引物，或正好相反，您试图扩增的DNA短片段，又称为扩增子，就将最终形成。一旦产生，扩增片段的总量将会在余下的循环中成倍增长。根据你的需要，可以设定20到40个循环。

对于长的扩增片段，最后的延伸步骤通常是72度下进行5到15分钟，来保证所有的DNA都是双链。热循环仪中的最后一步会被设成4度，让DNA在被从热循环仪中取出来之前保持稳定。

反应组分
PCR反应需要几个关键试剂。首先是DNA模板，就是你需要扩增的DNA样品；其次是引物，就是DNA短片段或者叫做寡聚脱氧核糖核酸，用以引导起始聚合酶反应。

选择引物时需要考虑几个重要的因素。第一，它们必须和DNA模板上您想要扩增序列两头的 5’端和3’端互补。第二，引物的碱基数应当在15个到30个之间，含约50%的鸟嘌呤G和胞嘧啶C。第三，两个引物的熔解温度都应当在50度以上，并且相差不超过1-2度，这样它们能在同一退火温度下有效地结合到模板上。第四，正反向引物之间不能互补形成引物二聚体。第五，每个引物内不能含有退火时可自身形成的二级结构。

除了引物和模板，DNA聚合酶也是PCR反应的重要组分。 PCR中最常用的是Taq DNA聚合酶。它是一种从生活在热温泉的嗜热细菌水生栖热菌中提取出来的热稳定酶。Taq DNA聚合酶可以耐受90度以上的温度。

脱氧核苷三磷酸，即dNTPs，也要被加到反应中，它在延伸链中形成碱基对。反应缓冲液，用来稳定反应并为聚合酶提供重要的反应辅助因子，也是反应中的必要组分，它可以稳定酸碱值并含有重要的离子，如锰离子、镁离子和钾离子。如所有反应一样，PCR也需要溶剂，因此会用到PCR级别的水，它不含任何抑制反应的离子。

准备反应
在开始PCR反应前，要保证工作环境清洁，避免污染。还需时刻戴上手套。

为了帮助记录您所需的不同反应组分，可以制作一个含每个样品，包括对照的试剂体积和浓度的表格。就体积而言，一个典型的反应应当包含5μL 10倍浓度的反应缓冲液，4μL 25mM的 MgCl2，1μL 10mM的dNTP混合物，以及50ng/μL的正向和反向引物各2μL，和0.3μL 5U/μL的Taq DNA聚合酶。您还需要加入足够量的模板使反应中模板量达100ng。最后，多数PCR反应的终体积是50微升，所以需要使用PCR级别的水来补足体积到50微升。

当在纸上计划好您的反应后，将所有试剂整理放到冰上。

接下来，将试剂加到PCR管中。首先是水，再是模板，引物，缓冲液，氯化镁，dNTP混合物，最后加入Taq DNA 聚合酶，彻底混匀。

反应准备好后，将样品置于热循环仪中，开始PCR程序。简单地说，热循环仪的组成包括一个加热板，它是放置PCR管或多孔板的地方，并能精确控制其温度变化；加热盖，它能够防止样品冷凝造成丢失；还包括一个操作界面，用来显示设计的PCR温度和循环时间。每次开始准备反应混合物前都要先将程序设定好。

热循环仪完成PCR程序后，取出您的反应物，用电泳来验证PCR产物。如果PCR反应成功，您应当会在正确碱基大小位置看到扩增片段。

有用的小技巧
接下来是一些操作PCR时有用的小技巧：当您试图从一些不同的模板来扩增相同的PCR产物时，会需要准备许多不同的反应。这时将反应体积放大来配制扩增预混液很有帮助。PCR扩增预混液是大体积的混合物，包含所有样品中都要用到的共用试剂，它随后被分装到多个反应管中。

多数时候，PCR反应都有一个起始的变性步骤，它在95度条件下进行1到9分钟。这一步骤保证所有的模板在第一个扩增循环时都是单链。

当您的样品被污染的可能性很高时，最好使用一个PCR工作间。为检查反应中是否有任何污染，准备一个负对照很有用，它不含DNA模板，所以在电泳胶中不应该出现PCR产物。

通常需要通过调整温度、氯化镁浓度、或尝试新的引物来优化PCR。一旦您的PCR条件成功，那么最好每次都使用一个肯定会有产物生成的正对照模板。

变化和应用
根据目的的不同，PCR可以有许多的变化和应用。

一种PCR的变化是热启动PCR，它让聚合酶在第一步变性步骤前没有活性发生反应，这样防止了循环前可能发生的非特异性扩增。

PCR还可经过改动，在一个PCR反应中使用多个PCR引物，来同时扩增多个DNA序列。这叫做多重PCR。

当结合使用荧光寡聚核苷酸探针时，PCR就能成为新技术，测量相对或绝对基因表达量，或者某个基因或某一组基因的mRNA的表达量，这种方法被称为qPCR。

PCR也可以用于检测一个生物中是否存在某特定DNA序列，这被称为基因型鉴定。例如，基因型鉴定可通过发现样品中是否存在某种群特异性序列来判断鱼样本的真实性。基因型鉴定还可用于法医分析来判断在现场发现的DNA样品是否和嫌疑人的吻合。

结语
您刚观看的是JoVE对PCR的介绍。本短片中，我们回顾了什么是PCR以及它是如何工作的，PCR的反应组分都有哪些，PCR扩增DNA的机理以及这个非常有用的技术的一些变化和应用。感谢您的观看。

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作者: JoVE

JoVE is the world's first and only peer-reviewed scientific video journal that increases productivity.



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