常見(jiàn)限制性內(nèi)切酶識(shí)別序列

1、常見(jiàn)限制性內(nèi)切酶識(shí)別序列(酶切位點(diǎn))（BamHI、EcoRI、HindIII、NdeI、XhoI等）Time：2009-10-22 PM 15:38Author:bioerHits: 7681 times 在分子克隆實(shí)驗(yàn)中，限制性內(nèi)切酶是必不可少的工具酶。 無(wú)論是構(gòu)建克隆載體還是表達(dá)載體，要根據(jù)載體選擇合適的內(nèi)切酶（當(dāng)然，使用T載就不必考慮了）。先將引物設(shè)計(jì)好，然后添加酶切識(shí)別序列到引物5' 端。常用的內(nèi)切酶比如BamHI、EcoRI、HindIII、NdeI、XhoI等可能你都已經(jīng)記住了它們的識(shí)別序列，不過(guò)為了保險(xiǎn)起見(jiàn)，還是得查證一下。 下面是一些常用的II型內(nèi)切酶的識(shí)別序列，僅供參

2、考。先介紹一下什么是II型內(nèi)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

3、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 system

4、s forming either C5-methylcytosine, N4-methylcytosine or N6-methyladenine. 酶 類型 識(shí)別序列 ApaI Type II restriction enzyme 5'GGGCCC 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' H

5、indIII 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

6、' 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' 要查找更多內(nèi)切酶的識(shí)別序列

7、，你還可以選擇下面幾種方法：1. 查你所使用的內(nèi)切酶的公司的目錄或者網(wǎng)站；2. 用軟件如：Primer Premier5.0或Bioedit等，這些軟件均提供了內(nèi)切酶識(shí)別序列的信息；3. 推薦到NEB的REBASE數(shù)據(jù)庫(kù)去查（網(wǎng)址：）當(dāng)你設(shè)計(jì)好引物，添加上了內(nèi)切酶識(shí)別序列，下一步或許是添加保護(hù)堿基了，可以參考：NEB公司網(wǎng)站提供的關(guān)于設(shè)計(jì)PCR引物保護(hù)堿基參考表下載（也可見(jiàn)圖片）雙酶切buffer的選擇（MBI、羅氏、NEB、Promega、Takara）再給大家推薦一種新的不需要連接反應(yīng)的分子克隆方法，優(yōu)點(diǎn)包括：設(shè)計(jì)引物不必考慮選擇什么酶切位點(diǎn)；不必考慮保護(hù)堿基的問(wèn)題；不必每次都選擇合適的酶來(lái)酶切質(zhì)粒制備載體；而且不需要DNA連接酶；假陽(yáng)性幾率低（因?yàn)?/p>