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DNA Analysis Restriction Enzyme Digestion

Restriction Enzyme Digestion

Restriction digestion of recombinant plasmid constructs provides a fast, cost-efficient method of gaining indirect sequence information. Multiple plasmid constructs can be analyzed simultaneously for the presence or absence of an insert, orientation of the insert, plasmid size, and some site-specific sequence data. Purified plasmid DNA is digested with 1 or more restriction enzymes (REs) selected to give a distinct DNA band pattern that is easily resolved by electrophoresis. Restriction mapping tools, such as NEBcutter^®, allow the user to upload the expected sequence of a recombinant plasmid (vector + insert) and provide a predicted digestion pattern for all commercially available REs. Restriction enzymes that cut within the multiple cloning site (MCS) and result in a diagnostic pattern of 2-5 easy to resolve bands are typically selected. Methylation sensitivity of selected enzyme(s) should also be considered to assure that methylation from the host organism, such as dam, dcm, or CpG methylation, does not block cleavage. Comparison of analytical and predicted DNA digestion patterns indicates presence/absence of restriction sites at the expected position within the plasmid construct providing some information about the insert sequence. In addition, plasmid size can be confirmed by simply adding the size of each DNA band in the digestion. Finally, restriction digestion patterns can provide information regarding the diversity of a complex DNA library.

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FAQs for Restriction Enzyme Digestion

How do I create a list of restriction enzymes available from NEB in Geneious software?

Protocols for Restriction Enzyme Digestion

Tools & Resources

Feature Articles

Restriction Enzymes at NEB: Over 30 years of Innovation

Restriction Endonucleases: Molecular Cloning and Beyond

Type II Restriction Enzymes: What You Need to Know | NEB

Read about Type II restriction enzymes and the distinguishing properties of the four principle subtypes.

A Modern Day Gene Genie Sir Richard Roberts on Rebase

Whole genome assembly from next generation sequencing data using restriction and nicking enzymes in optical mapping and proximity-based ligation strategies

High throughput sequencing methods have revolutionized genomic analysis by producing millions of sequence reads from an organism’s DNA at an ever decreasing cost.

Publications related to Restriction Enzyme Digestion

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 (Bethesda); 6:4, 845-846. PubMedID: 26818077
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.
Loenen, W.A., Raleigh, E.A. (2014) The other face of restriction: modification-dependent enzymes. Nucleic Acids Res; 42, 56-69. PubMedID: 23990325

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Videos

What is a Type II Restriction Enzyme?

Type II restriction enzymes are most commonly used for molecular biology applications, as they recognize stereotypical sequences and produce a predictable cleavage pattern. Learn more about how Type II REs work.
What is a Type I Restriction Enzyme?

Type I restriction enzymes are a group of endonucleases that recognize a bipartite sequence, but do not produce a predictable cleavage pattern. Learn more about how Type I REs work.
What is a Type III Restriction Enzyme?

Type III restriction enzymes are a group of endonucleases that recognize a non-pallindromic sequence, comprising two inversely oriented sites. Learn more about these poorly understood enzymes.
Cloning With Restriction Enzymes

Restriction enzymes are an integral part of the cloning workflow, for generating compatible ends on fragments and vectors. This animation discusses three guidelines for determining which restriction enzymes to use in your cloning experiment.
Standard Protocol for Restriction Enzyme Digests

Let one of NEB's restriction enzyme experts help you improve your technique and avoid common mistakes in digest setup.
Why is My Restriction Enzyme Not Cutting DNA?

Not getting the cleavage you expected? Let an NEB scientist help you troubleshoot your reaction.
Restriction Enzyme Digest Problem: Too Many DNA Bands

Are you finding unexpected bands in your digestion reaction? Here are some tips to help you determine the cause.
What is Restriction Enzyme Star Activity?

Learn what Star Activity is, why it is detrimental to accurate restriction enzyme digestion, and how NEB's HF enzymes are engineered to avoid it.
Reduce Star Activity with High-Fidelity Restriction Enzymes

NEB has engineered HF® enzymes to eliminate star activity. Learn how, and what this means for your digests.
NEB^® Restriction Enzyme Double Digest Protocol

Double digestions can save you time, and this video can offer tips for how to achieve the best results, no matter which of NEB's restriction enzymes you're using.
Restriction Enzyme Digest Protocol: Cutting Close to DNA End

When cutting close to the end of a DNA molecule, make sure you know how many bases to add to the ends of your PCR primers.
Restriction Enzyme Digestion Problem: DNA Smear on Agarose Gel

Learn more about what causes this common problem, and how NEB's enzymes are QC'd to avoid DNA smearing.
Restriction Enzymes in Isothermal Amplification

Isothermal amplification generates many copies of a target sequence in a short period of time, at a constant temperature. Learn more about isothermal amplification.
Restriction Enzymes in Optical Mapping

Optical mapping is a method that allows production of restriction maps of whole chromosomes or genomes. Learn more about optical mapping.