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常见限制性内切酶识别序列(酶切位点)

2023-03-21 10:14 来源:上海远慕生物试剂
常见限制性内切酶识别序列(酶切位点)(BamHI、EcoRI、HindIII、NdeI、XhoI等)

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。远慕先介绍一下什么是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ApaIType II restriction enzyme5'GGGCC^C 3'BamHIType II restriction enzyme5' G^GATCC 3'BglIIType II restriction enzyme5' A^GATCT 3'EcoRIType II restriction enzyme5' G^AATTC 3'HindIIIType II restriction enzyme5' A^AGCTT 3'KpnIType II restriction enzyme5' GGTAC^C 3'NcoIType II restriction enzyme5' C^CATGG 3'NdeIType II restriction enzyme5' CA^TATG 3'NheIType II restriction enzyme5' G^CTAGC 3'NotIType II restriction enzyme5' GC^GGCCGC 3'SacIType II restriction enzyme5' GAGCT^C 3'SalIType II restriction enzyme5' G^TCGAC 3'SphIType II restriction enzyme5' GCATG^C 3'XbaIType II restriction enzyme5' T^CTAGA 3'XhoIType II restriction enzyme5' C^TCGAG 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

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:



双酶切buffer的选择(MBI、罗氏、NEB、Promega、Takara)

远慕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:

①设计引物不必考虑选择什么酶切位点 ;

②不必考虑保护碱基的问题;

③不必每次都选择合适的酶来酶切质粒制备载体;

④而且不需要DNA连接酶;

⑤假阳性几率低(因为没有连接反应这一步,载体自连的问题没有了)。

另外,还有一个经济问题:如果一次性制备一批载体(小提一次质粒约80μL),并将之线性化(可双酶切亦可单酶切),然后每次做分子克隆实验时用一点,大约可以做约40次转化,用一年没问题!
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