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Restriction Enzyme Digestion

Preparation of DNA for traditional cloning methods is dependent upon restriction enzyme digestion to generate compatible ends capable of being ligated together. The DNA to be cloned can vary widely, from genomic DNA extracted from a pure bacterial culture or a mixed population, to a previously cloned gene that needs to be moved from one vector to another (subcloning). Restriction enzymes can also be used to generate compatible ends on PCR products. In all cases, one or more restriction enzymes are used to digest the DNA resulting in either non-directional or directional insertion into the compatible plasmid.

Genomic DNA, regardless of the source, is typically digested with restriction enzymes that recognize 6-8 consecutive bases, as these recognition sites occur less frequently in the genome than 4-base sites, and result in larger DNA fragments. The desired insert size for the clone library determines which enzymes are selected, as well as the digestion conditions. Most often, a serial dilution of the selected restriction enzyme(s) is used to digest the starting material and the desired insert size range is isolated by electrophoresis followed by gel extraction of the DNA. This method of preparation provides DNA fragments of the desired size with ends compatible to the selected vector DNA. 

Subcloning requires the use of 1-2 restriction enzymes that cut immediately outside the insert fragment without cutting within the insert itself. Restriction enzymes that have a recognition site within the multiple cloning site (MCS) are commonly used since they do not cut elsewhere in the vector DNA and typically produce two easily resolved DNA fragments. The gene of interest is most commonly subcloned into an expression vector for improved protein expression and/or addition of a purification tag. In this case, it is essential that the gene be inserted in the correct orientation and in frame with the transcription promoter.

The Polymerase Chain Reaction (PCR) is commonly used to amplify a gene or DNA fragment of interest, from any source of DNA, to be cloned. In order to generate compatible ends, it is common to add restriction sites to the 5’ end of both PCR primers. When adding restriction sites to a PCR primer, it is recommended to include 6 bases between the recognition site and the 5’ end of the primer. These additional bases provide sufficient DNA for the restriction enzyme to bind the recognition site and cut efficiently. When selecting a restriction site(s) to add to the primers, it is important to determine which site(s) will be compatible with your selected vector, whether directional cloning is desired and, most importantly, confirm that the recognition site(s) does not occur within the gene or DNA fragment.


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FAQs for Restriction Enzyme Digestion

Protocols for Restriction Enzyme Digestion

    Publications related to Restriction Enzyme Digestion

  1. Fu YB, Peterson G. W., Dong Y 2016. Increasing Genome Sampling and Improving SNP Genotyping for Genotyping-by-Sequencing with New Combinations of Restriction Enzymes G3. 6:4, PubMedID: 26818077, DOI:
  2. Shah, S., Sanchez, J., Stewart, A., et al. 2015. Probing the Run-On Oligomer of Activated SgrAI Bound to DNA PLoS One. 10(4), PubMedID: 25880668, DOI: 10.1371/journal.pone.0124783.
  3. Loenen, W.A., Raleigh, E.A. 2014. The other face of restriction: modification-dependent enzymes. Nucleic Acids Res. 42, PubMedID: 23990325, DOI: