常见限制性内切酶识别序列.doc

常见限制性内切酶识别序列(酶切位点)（BamHI、EcoRI、HindIII、NdeI、XhoI等）Time：2009-10-22 PM 15:38Author:bioerHits: 7681 times 在分子克隆实验中，限制性内切酶是必不可少的工具酶。 无论是构建克隆载体还是表达载体，要根据载体选择合适的内切酶（当然，使用T载就不必考虑了）。先将引物设计好，然后添加酶切识别序列到引物5 端。常用的内切酶比如BamHI、EcoRI、HindIII、NdeI、XhoI等可能你都已经记住了它们的识别序列，不过为了保险起见，还是得查证一下。 下面是一些常用的II型内切酶的识别序列，仅供参考。先介绍一下什么是II型内切酶吧。 The Type II restriction systems typically contain individual restriction enzymes and modification enzymes encoded by separate genes. The Type II restriction enzymes typically recognize specific DNA sequences and cleave at constant positions at or close to that sequence to produce 5-phosphates and 3-hydroxyls. Usually they require Mg 2+ ions as a cofactor, although some have more exotic requirements. The methyltransferases usually recognize the same sequence although some are more promiscuous. Three types of DNA methyltransferases have been found as part of Type II R-M systems forming either C5-methylcytosine, N4-methylcytosine or N6-methyladenine. 酶 类型 识别序列 ApaI Type II restriction enzyme 5GGGCCC 3 BamHI Type II restriction enzyme 5 GGATCC 3 BglII Type II restriction enzyme 5 AGATCT 3 EcoRI Type II restriction enzyme 5 GAATTC 3 HindIII Type II restriction enzyme 5 AAGCTT 3 KpnI Type II restriction enzyme 5 GGTACC 3 NcoI Type II restriction enzyme 5 CCATGG 3 NdeI Type II restriction enzyme 5 CATATG 3 NheI Type II restriction enzyme 5 GCTAGC 3 NotI Type II restriction enzyme 5 GCGGCCGC 3 SacI Type II restriction enzyme 5 GAGCTC 3 SalI Type II restriction enzyme 5 GTCGAC 3 SphI Type II restriction enzyme 5 GCATGC 3 XbaI Type II restriction enzyme 5 TCTAGA 3 XhoI Type II restriction enzyme 5 CTCGAG 3 要查找更多内切酶的识别序列，你还可以选择下面几种方法：1. 查你所使用的内切酶的公司的目录或者网站；2. 用软件如：Primer Premier5.0或Bioedit等，这些软件均提供了内切酶识别序列的信息；3. 推荐到NEB的REBASE数据库去查（网址：/rebase/rebase.html）当你设计好引物，添加上了内切酶识别序列，下一步或许是添加保护碱基了，可以参考：NEB公司网站提供的关于设计PCR引物保护碱基参考表下载（也可见图片）双酶切buffer的选择（MBI、罗氏、NEB、Promega、Takara）再给大家推荐一种新的不需要连接反应的分子克隆方法，优点包括：设计引物不必考虑选择什么酶切位点；不必考虑保护碱基的问题；不必每次都选择合适的酶来酶切质粒制备载体；而且不需要DNA连接酶；假阳性几率低（因为没有连接反应