前言
分子生物学中，连接是指通过形成磷酸二酯键将两个DNA片段连起来。连接反应是由一种叫连接酶的酶催化进行的。在细胞内，连接酶用于修复在DNA复制过程中的单链或双链的断裂。在实验室里，DNA连接酶用于分子克隆来连接插入片段和载体-它是宿主生物内复制目的片段的DNA载体分子。
本短片介绍了DNA连接。讲述了连接的基本原理以及准备一个常用的连接反应的逐步步骤，并讨论了连接反应中的关键因素，例如粘末端突出端的长度对反应温度的影响，如何调整DNA插入片段与载体的比例来防止自身连接。还将提及辅助连接的分子生物学工具如Klenow片段和虾的碱性磷酸酶SAP，并讲述连接的应用，例如邻位连接和为测序在片段上添加接头序列。
DNA连接的介绍
连接是指结合在一起，其生物术语是用来形容两个生物分子通过共价键结合起来的酶联反应。本短片讲述了DNA连接在分子生物学研究中的应用。
DNA连接：基本原理
在细胞内，连接酶识别并修复DNA链的断裂，催化DNA主链的 3’羟基和5’磷酸集团之间形成磷酸二酯键。连接反应属于正常细胞程序的一部分，如在复制过程中修复单链或双链DNA的断裂。
在实验室里，DNA连接酶经常用在分子克隆中，连接被内切酶消化的DNA片段，也称插入片段，与同样内切酶消化的载体-如质粒，这样目的片段就能进入宿主细胞内并得到复制。
核酸内切酶消化要用到限制性内切酶，也称限制性酶，它们会在DNA链的特定区域造成缺口。
这些缺口有些与单链断裂相似，有粘末端的3’和5’突出端，有些与双链断裂相似，没有任何突出端，被称为平端。粘端有利于连接，因为突出端的互补碱基会稳定片段间的连接反应。而平端由于没有任何互补的碱基配对，用酶将它们连接的效率要低且难度更大。通常情况下，粘端和平端之间不能进行连接。
但枯草杆菌蛋白酶裂解DNA聚合酶I后的产物Klenow片段能将粘端转变成平端。Klenow片段具有3’到5’的外切酶活性，可消化掉 3’突出端，它还具有聚合酶活性，可通过延长互补链的3’末端来补平5’突出端。
将一个基因插入到质粒的连接反应中，载体DNA的自我连接是一种常见的干扰结果。用碱性磷酸酶处理酶切后的载体DNA, 可以在片段两端去除掉5’磷酸集团， 从而防止这种干扰结果的出现。
准备连接反应
如我们在前面提到的，载体和插入片段DNA在连接前都要经过内切酶消化，然后跑胶纯化消化后的载体和插入片段，并用分光光度计测量纯化后DNA的浓度。
通过这个浓度，并根据每个DNA碱基的平均分子量以及每个片段中的碱基数目，我们可以算出插入片段或载体在1微升体积中的分子数目。根据得到的摩尔浓度，采用 3:1 的插入片段和载体的摩尔比来计算反应体系中要用的片段和载体的体积。这个片段与载体的比例最好为3:1，因为它增加了片段插入到载体的可能性。
现在我们已经知道如何确定反应中所用载体和插入片段的量，接下来让我们开始在冰上准备连接反应。向微量离心管中按以下顺序添加反应组分：使终体积达10微升的足够体积的无菌水，这里我们用了4微升；1微升10X浓度的连接缓冲液； 1微升10mM的ATP；通过计算得到的1微升载体和3微升插入片段DNA；最后是1微升DNA连接酶。将反应组分充分混合，离心后放置在合适的温度下孵育。
是否粘端还是平端连接对反应温度和孵育时间都有影响。例如，含6个碱基突出端的粘端连接可在室温下进行1小时，因为互补的末端会稳定片段之间的连接。而短的粘端或平端之间的连接则需在14到20度孵育过夜。
应用
现在我们已经学习了如何准备连接反应，让我们再来看看对这一过程的一些应用。
连接可以用来直接将PCR扩增的片段插入到一个线性的质粒中。这里您看到是研究人员从冻存的小鼠大脑中分离出基因组DNA，然后进行亚硝酸盐PCR，它是一种利用PCR来检测甲基化DNA的技术。接下来将PCR产物直接连接到质粒中，建立一个含该脑组织中所有被甲基化基因的文库。
连接还可用来在片段上加上含PCR引物结合位点的聚核苷酸接头，用于片段纯化。当操作肿瘤样品时，研究人员可使用这种方法来进行肿瘤全基因组测序，帮助识别导致肿瘤的突变。
本视频中，将从福尔马林固定的细胞中提取DNA，进行限制性酶切和Klenow片段处理后，与生物素进行连接，这样是为了接下来将连接后的DNA拉下来。然后如这里所显示的，用PCR技术扩增该DNA, 并测序产物来确定不同程度的染色质相互作用。
结语
您现在已经学习了DNA连接酶，在实验室中准备连接反应的各种原理，反应中可能遇到的问题及解决办法，和连接在分子生物学研究中的多种应用。感谢您的观看。
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