Restriction Enzymes for Modular Cloning

Restriction Enzymes for Cloning

Types of enzymes and their use in modular cloning

In the vector construction workshop, I discussed how restriction enzymes (REs) facilitate cloning by recognizing and cutting specific sites. This ability allows them to fragment inserts and linearize DNA for ligation into a vector or backbone.


Many common restriction enzymes are palindromic, meaning they cut their recognition sites in both forward and reverse orientations. As a result, when cloning with these enzymes, multiple enzymes are required to ensure the insert is positioned correctly.










In contrast, Type IIs enzymes have non-palindromic recognition sites with cut sites located outside the recognition sequence. 

This allows a single enzyme to digest multiple fragments while ensuring proper orientation and placement due to distinct overhangs. Using one enzyme is significantly more cost-effective and reliable than using three to five. Additionally, there are 256 possible overhangs available for use!


Available Type IIs Enzymes 

The most popular Type II RE is BsaI known for its efficiency, speed, and affordability. 


When a system uses multiple Type II enzymes, they will opt for using Esp3I, AarI, or BpiI. 



This process of using a type II RE to clone is called GoldenGate Cloning and is a very user-friendly modular cloning approach. 


See Misc. notes for domestication.



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