What is Restriction Enzyme ?
- Originally found in defense mechanism of bacteria which use to chop up foreign DNA into pieces to prevent infection by virus
- It recognize specific sequence in DNA fragment and bind to it.
- It cleaves DNA within or near to the recognized sequence.
- It cuts the DNA fragment end with 3’-OH and 5’-P group.
- It also known as restriction endonuclease.
- Generally. there are 4 types of restriction enzyme as follow :
- Type I enzymes - cleave the DNA approximately half-way between the two bi-partite recognition sites.
- Type II - cleave DNA within the sequence at both strand
- - recognize palindromic sequence (4-8 bases long)
- - can be divided into different types according to size of bases it recognize such as 4 base cutter, 6 base cutter and 8 base cutter.
- - most commonly used restriction enzyme
- - creating either sticky end (with overhang) or blunt end (without overhang) at restriction site
- Type III - consist of subunits Mod (modifies) and Res (cleave).
- Type IV - recognize and cleave methylated DNA.
Since Type II restriction enzyme is the most commonly used in gene cloning technique, we will mainly focus on it only.
Below are some of the example of Type II restriction enzyme :
Below are some of the example of Type II restriction enzyme :
How Restriction Enzyme Works?
- Extracted DNA will be cut by restriction enzyme to generate a smaller DNA fragment with desired gene.
- The same restriction enzyme will be applied on vector.
- Both DNA fragment and vector will have similar restriction site sequence and ready to be ligated.