前言
限制性酶或内切酶会识别并切开DNA上的某个特定序列。这些酶在细菌中天然存在，用来抵御感染细菌的病毒-噬菌体的攻击。细菌中的限制性内切酶会切开入侵噬菌体的DNA，而细菌本身的DNA由于有甲基化，将不会受到影响。
本短片讲述了限制性内切酶的基本原理：内切酶是如何命名的，已有的酶切位点和突出端类型。并将讲解常用的限制性内切酶消化的逐步操作过程，包括所需组分，添加各组分到混合物中的顺序，以及常用的消化温度和时间。还将提及灭活限制性内切酶来抑制非特异性活性的重要性。短片还给出了操作多酶消化及酶切中使用对照的小技巧。
概述
限制性酶或称限制性内切酶，在分子生物学中的应用多种多样。这些酶识别并切开DNA上被称为限制性酶切位点的某个特定序列。接下的视频将为您介绍这些神奇的分子，并演示如何准备限制性酶消化。
限制性酶的背景及命名
限制性酶究竟从何而来？这些酶被发现是细菌适应产生的一种防御机制，用来抵御被称为噬菌体的病毒。由于细菌本身的DNA上这些酶切位点被添加了甲基化集团，限制性酶只能识别并消化病毒的DNA, 从而有效防止了感染。
限制性酶的名字有些奇怪，象HindIII, NotI, EcoRI,还有BamHI。限制性酶名字的前三个字母是代表酶提取的生物来源。例如，EcoRI是从大肠杆菌E.coli中分离出来。有时有第四个字母，它代表分离细菌的菌株。罗马数字代表该酶是从这个生物中分离出来的第一个，第二个还是第三个酶。
基本原理
限制性酶通常识别一段长度为4-8个碱基的核苷酸序列，也称识别位点。在该序列中的某个特定核苷酸位置，酶将打开DNA链的磷酸二酯键。识别位点通常是回文结构，意思是正向和反向读的序列相同。DNA互补链中出现的回文序列被称之为反向重复回文结构。
限制性酶切开DNA后会留下不同类型的末端：粘性末端和平端。粘性末端有3’和5’突出端，而平端则没有突出端。末端的类型决定了如何将DNA酶切回收后的片段与其他DNA片段相连，这一过程叫连接。
准备限制性内切酶消化
做限制性酶切消化需要细心的准备。一个消化反应系统里通常含以下组分：去离子水，将被酶切的DNA, 所用酶特定的缓冲液，有时也要加上牛血清白蛋白BSA。BSA用来防止酶与反应溶液所在的管壁结合。每个限制性酶都可能需要不同的缓冲液条件，反应温度和是否需要BSA的要求。限制性酶的厂家会有相关资源帮助您查到所需的所有信息。
准备消化之前，先将限制性酶从冻箱或冰箱中取出，放置在冰上或恒冷的容器中保证酶不会失活。再向一个微量离心管中按以下顺序添加反应组分。首先是一定体积的不含核酸酶的无菌水，使最终体积达到20微 升。然后加入10X浓度的酶缓冲液，再根据需要添加BSA，然后是1微克的DNA, 和2-10个单位的酶。一个酶单位是指在50微升反应体系中，37度反应60分钟能完全消化1微克对照DNA所需的酶量。在微量离心管中加入所有组分后，震荡混匀，然后12000g快速离心，使所有溶液集中在管底部。然后在加热板上用酶的反应最佳温度，通常是37度孵育1 到4小时。
消化完成后，最好是将反应混合物放在65度加热来失活限制性酶。尽管多数时候限制性酶会进行特异性切割，但过长的孵育时间会引起酶的非特异性活性，而切割与其特定消化位点相似但不同的位点。
酶失活后，应将DNA在琼脂胶上跑电泳来确定消化是否成功。
小技巧
这里有几个关于如何操作酶消化并保证消化成功的有用的小技巧。
有时您会需要用到多个酶来消化获得特定的DNA片段。这时，您需要检查两种酶的缓冲液和温度条件是否匹配。如果是，则可在同一条件下使用两个酶进行双酶切。然而，有些时候这两种酶的反应条件并不匹配，这时就得进行分步酶切。例如，先用一种酶进行消化，然后改变缓冲液中组分浓度使其达到第二种酶的最佳反应条件。克服缓冲液条件不匹配的另一种方法是将第一个酶切后的DNA进行纯化后再进行第二个酶的酶切。
使用对照可以很好地帮助分析为何消化不成功。比如，不含酶的对照反应体系可用来检查DNA样品是否完整，从而判断是否存在核酸外切酶活性。使用已知含酶切位点的对照DNA可以帮助检测酶活性。
应用
现在我们已经知道了如何进行酶切反应，让我们来 看看使用限制性酶的多种用途。
限制性酶可用于临床诊断，用来鉴定特殊的样本。将消化产物上样到一种特制的芯片上，然后把芯片插入到称之为生物分析仪的机器上。研究人员可以检测酶切消化产生的片段大小，来判断鱼样本的真实性。这种不同亲缘样本的同一基因产生不同酶切带型图谱，称之为限制性片段长度多态性。
限制性酶还用于分子克隆，将从一个质粒中消化回收的片段连接到另一个质粒上，这样目的片段可在细菌中得到复制。
使用聚合酶链式反应PCR，可在基因的特定位置添加酶切位点，限制性酶还可用于判断同一基因的等位基因序列中是否存在单个核苷酸的区别。这种单核苷酸多态性SNP很难仅通过PCR和凝胶电泳来检测。在SNP中引入了酶切位点后，简单的酶切消化就可以区分不同的等位基因。
结语
您刚观看的是JoVE关于限制性内切酶的短片。您学习了这些酶的来源，它们的工作原理，并观看了如何准备酶切反应和在分子生物学中如何使用限制性酶。我们一如既往地感谢您的观看。
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