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如何让你的酶切反应“时半功倍”？

发布: 2016-01-15 1,574阅读 261评论

限制性核酸内切酶(以下简称限制性酶)是一类识别双链DNA中特定核苷酸序列的DNA水解酶，以内切方式水解DNA，产生5’-P和3’-OH末端。限制性酶主要分为三种类型：Ⅰ型限制酶为复合功能酶，具有限制-修饰两种功能，但在 DNA链上没有固定的切割位点，一般在离切割位点1kb到几kb的地方随机切割，不产生特异性片段。Ⅲ型酶与Ⅰ型酶基本相似，不同的是Ⅲ型酶有特异性的切割位点，但这两类酶对 DNA酶切分析的意义不大，通常所说的限制性内切酶是指Ⅱ型酶，它能够识别与切割DNA链上的特定的核苷酸顺序，产生特异性的DNA片段。

限制性酶切看似简单，但其实暗藏玄机。如果单单按照protocol上面操作，结果往往是不理想的。那么今天小编就会给你介绍一下酶切实验操作过程中需要注意的几个方面，哪怕是小细节，也至关重要。

1记得加牛血清白蛋白（Bovine Serum Albumin, BSA），达到稳定限制性酶的效果，并且减少内切酶粘附试管壁的情况。传统的方法是单独购买BSA溶液，现在越来越多的缓冲液例如Tango Buffer或是Mastermix都含有BSA，从而使酶切实验更为简便。

 

2如果限制性酶的反应效率在含盐缓冲液中(e.g., NEBuffer 3.1) 很低，说明很有可能这种酶对盐量敏感。在DNA 的制备过程中，要尽量清除掉溶液中的盐分。

 

3限制性酶的总体积要少于总反应液体积的10%。用于保存酶的甘油如果过多，会抑制酶切反应，大大降低酶切的效率。

 

4在保证第3条的前提下，加尽可能多的限制性酶来提升反应效率。这里要强调下一单位（1U）的定义，一单位酶可以在一小时内标准反应条件下酶切1 µg的DNA。如果担心甘油会抑制酶切，可以考虑购买单位浓度比较高的限制性酶。每一个生产商都会标明浓度单位，例如10U/µL。

 

5进行无酶切、单酶切测试。不管是什么实验，都要有控制实验来证明结果的有效性。不加入任何限制性酶，或是加入单一酶都可以很好的验证限制性酶的效率并且分辨出假阳性实验结果。

 

6查看限制性酶的反应效率表和相对应的保护碱基。限制性酶除了识别特定的DNA序列外，酶蛋白还要占据识别位点两边的若干个碱基，这些碱基对内切酶稳定的结合到DNA双链并发挥切割DNA作用是有很大影响的。每一个酶的工作效率和保护碱基的长短不尽相同，要根据个人使用的酶的特点来设计实验才能达到理想的效果。

 

以下链接是保护碱基数目和酶切活性对应值表，供小伙伴们参考。

http://wenku.baidu.com/view/312a400ff12d2af90242e6d3.html

https://www.neb.com/~/media/NebUs/Files/Chart%20image/cleavage_olignucleotides_old.pdf

以上技巧是小编根据个人经验和网上资料整理得来的，如果你有更好的建议和意见，也欢迎各位小伙伴来信告诉我们。

图片来自网络，内容由BioEngX原创，转载请注明！

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