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视频|分子克隆操作详解

作者: JoVE 发布: 2018-03-21 11,123阅读 382评论

分子克隆是将重组DNA插入到载体中的一套方法，载体作为DNA分子的携带者，将在宿主体内复制重组DNA片段。而DNA片段，可能是从原核或真核生物中分离得到一个基因。分离得到目的片段或称插入片段后，将其与载体用限制性酶切割并进行纯化。纯化后的片段通过一种叫做“连接”的技术连在一起。催化该连接反应的酶被称为连接酶。

本短片将按顺序介绍构成整个分子克隆操作过程的主要方法。并将讨论分子克隆技术的重要方面，例如分子克隆方案的必要性和如何追踪转化的菌落。也将提及其检测步骤，如用限制性酶切和测序来判断纯化的质粒中是否含有目的片段。

分子克隆是将来源于原核或真核的重组DNA插入一个可以复制的载体，如质粒或病毒载体中的一项技术。克隆是指制备大量拷贝的的目的DNA片段，比如一个基因。在这段视频中，您将了解分子克隆的不同步骤，如何进行操作，以及该技术的不同应用。

基本原理
克隆开始前，至少需要准备两个重要的DNA分子。首先，也是最重要的，是您想要克隆的DNA片段，也被成为插入片段。它可以是来自原核细胞，真核细胞，或者一种绝迹的生物，或者也可以在实验室中人工合成。通过分子克隆技术，我们可以对该基因的功能有更多的了解。

其次，你需要一个载体。载体是一种质粒DNA，在分子生物学中的被用作工具，来制备某个基因的大量拷贝。或者用来合成该基因编码的蛋白质。质粒就是载体的一种，它是独立于染色体的环状DNA，可以被细菌复制。

质粒通常具有多克隆位点MCS，此区域包含不同的限制性核酸内切酶也称为限制酶的识别位点。不同的插入片段可以通过一种叫链接的技术被整合到质粒中。质粒载体还含有复制起点，使得质粒能够在细菌中复制。此外，质粒还具有抗生素抗性基因。当细菌含有该质粒时，它就可以在包含抗生素的培养基上存活。这样就帮助筛选了成功转化的细菌。

插入片段和载体被导入宿主细胞种。分子克隆中最常见的宿主细胞是大肠杆菌，它生长迅速，可普遍获得，并且有有许多不同的克隆载体被商品化。真核生物如酵母也可以被用来作为一种宿主细胞。

普通分子克隆的第一步是获得想要的目的片段。它可以是来自于任何细胞类型的DNA或mRNA。根据插入片段类型和最终目的选择最佳载体和宿主细胞。通常使用基于PCR的方法来复制插入片段。然后通过一系列的酶促反应将插入片段与载体连和到一起，再导入到宿主细胞，来得到大量复制。复制后的载体从细菌中纯化出来，经过限制性酶消化，在电泳胶上进行分析。将纯化得到片段送去测序，来验证插入片段是所需DNA片段。

操作过程
让我们更详细一点看看如何进行分子克隆。在开始任何克隆实验前，需要计划好您的克隆方案。例如，任何一个质粒载体都只有有限的几个限制性酶切位点，用于将插入片段整合到多克隆位点中。你要选择一个在插入片段种没有的酶切位点，以防止片段被切开。您还有可能面临到连接一个带平端的片段和带有粘性末端的片段的情况。这时唯一的选择是，使用Klenow片段将突出端补平。然后用平末端连接，将插入片段放入到你想要的载体中。

分子克隆中的DNA可以是来自于任何细胞或是组织样品，通过简单的提取技术即可得到。提取之后，可以使用PCR来扩增插入片段。扩增之后，使用限制性内切酶对片段和载体进行酶切。酶切后，可对片段和载体进行电泳，并用凝胶纯化进行纯化回收。对于载体，这一步有助于分离酶切后的线性质粒和未被酶切的质粒。未被酶切的质粒，在凝胶上显示的一个高分子量的模糊带型。

酶切后，使用DNA连接酶将插入片段连接到质粒中。通常来讲，连接反应中的插入片段和载体的比例最好是3:1，这样可以保证只有少量的载体会发生自连。连接反应在冰上准备好后，可以在14-25˚C下孵育1小时或直至过夜。

接下来进行转化，将质粒载体倒入宿主中进行复制。将转化后的细菌涂板到含抗生素的琼脂板上，37℃培养过夜。 由于质粒包含了一个抗生素抗性基因，被成功转化的细菌在抗生素存在时，会生长形成菌落。可从转化板中挑取多个单克隆，接种到含有培养基的编号的管中，然后在摇床培养箱中扩增。少量的培养物用来在编号的琼脂板上涂板，剩余的用于质粒纯化。

经过纯化的质粒先用限制性酶进行切割，酶切产物进行凝胶电泳，以检查插入片段的存在。这样我们可以验证细菌菌落是被转化了含插入片段的质粒，还是转化了自连的质粒。被证明转化了插入片段的质粒可以用来扩大培养，以便进一步进行质粒纯化。测序用于最后验证目的基因得到了克隆。

应用
克隆可以有无数的应用。例如，当一个mRNA模版在反转录酶的作用下，经过反转录得到cDNA或者互补DNA，然后可使用PCR扩增该cDNA。这样通过分子克隆技术我们创建了一个cDNA文库，这一文库中包含某特定细胞类型中所有表达的基因。

分子克隆还能用来克隆一系列基因，或者来源于一个细菌菌株的基因簇。将它们重新组合到质粒上，在转化到另一个菌株中，这样可以重建整个生物合成途径，来生产复合物分子。

分子克隆可通过在一种特殊细菌菌株中，表达目的质粒来生成突变体文库，因为该菌株在特定温度下会使用易错聚合酶。用测序来鉴定这些突变体，然后用不同的药物或化学试剂来测试含突变基因的细菌转化体，看哪些细菌克隆产生了药物抗性。

分子克隆能将报告基因嵌入到DNA载体中。常见的报告基因是绿色荧光蛋白GFP，它在紫外灯照射下可发出绿色荧光。报告基因还可被插入到腺病毒中，来显示蚊子的转染和它在细胞中的可遗传性。

结语

以上为JoVEs视频的文字部分，您现在应该了解了如何操作分子克隆，以及该技术是如何在分子生物学中得到应用。感谢收看！

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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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