How to Choose the Right Restriction Enzyme?

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Restriction endonucleases are a class of enzymes that can recognize specific nucleotide sequences in double-stranded DNA molecules and cut phosphodiester bonds in DNA chains at specific positions. Different restriction endonucleases recognize different DNA sequences. They can cut DNA within the recognition sequence or at a position not far from the recognition sequence, and different types of products are formed after the restriction enzyme cleavage.

It is well known that restriction enzymes are most commonly used in molecular cloning experiments. But in addition to cloning, it can also be used in vaccine development and production, gene sequencing, SNP identification, ddPCR and other fields.

Classification of restriction endonucleases
According to the complexity of the structure, the mode of action and the difference between cofactors, restriction enzymes can be divided into four categories. The following summarizes the characteristics and typical enzymes of each category:

Type I
1. It has both cleavage recognition and modification activities.
2. It can recognize specific DNA sequences, and the cutting site can be thousands of bases away from the recognition site.
3. ATP is required for action.
Typical enzymes: EcoB, Ecok, etc.

Type II
1. It only has the function of identifying cuts.
2. The recognition sequence is mostly a short palindrome sequence (usually 4-8bp), and the enzyme cleavage site is usually the recognized sequence, which is specifically cut.
3. Mg2+ is required for action.
4. The most commonly used type of restriction enzymes in molecular cloning.
Typical enzymes: HindII, NotI, etc.

Type III
1. It has both cleavage recognition and modification activities.
2. The cleavage site is about 24-26bp away from the recognition site.
3. ATP is required for action.
Typical enzyme species: Hinflll, etc.

Type IV
1. Only cut DNA sequences modified by methylation.
2. The cleavage site is about 30 bp away from the recognition site.
Typical enzymes: MrcA, McrBC, etc.

How do I choose which restriction enzymes to use?
Restriction endonucleases originate in bacteria as a defense against bacteriophages (viruses that attack bacteria), i.e. the recognition sequences in any particular DNA sequence are universal. This allows restriction enzymes to be used to insert genes from any organism into plasmids. However, before choosing an endonuclease for this process, it is necessary to consider two factors.

1. The size of DNA fragments
In a given genome, shorter restriction sites containing 4 base pairs are more likely to occur, while longer sites containing 6 to 8 consecutive base pairs occur less frequently. Therefore, most genomic DNA should be digested with restriction enzymes that recognize and cut longer restriction sites, as this will result in larger DNA fragments that are easily examined by gel electrophoresis.

2. Location and frequency of cuts
Restriction enzymes should be located in two positions: 1) on either side of the gene of interest; 2) within the plasmid backbone. This will create compatible sticky ends on the two DNA fragments that can be religated to construct recombinant plasmids. Carefully screen your plasmid vector and gene of interest to ensure that the restriction enzyme you choose does not cut any other sites that might interfere with your gene or plasmid function. If the restriction endonuclease cuts within the sequences of the gene of interest or important plasmid elements (such as origins of replication, selectable markers or promoter regions, etc.), these interrupted sequences will no longer function or encode functional products.

For example, the endonuclease ApaII is not a good choice for cloning the RFP gene because there is an ApaII recognition site inside the RFP gene. Digestion with ApaII will cleave the RFP gene, rendering it incapable of expressing the RFP protein.

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