Restriction Endonuclease Digestion
Restriction Endonuclease Digestion - Related Videos
09:45
Restriction Enzyme Digests
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2023
Restriction enzymes or endonucleases recognize and cut DNA at a specific sequence. These enzymes occur naturally in bacteria as a defense against bacteriophages - viruses that infect bacteria. Bacterial restriction enzymes cut the invading bacteriophage DNA while leaving the bacterial genomic DNA unharmed due to addition of methyl groups. This video explains the basic principles of restriction enzymes including: how restriction enzymes are named and the types of recognition sites and...
09:16
In Vitro Directed Evolution of a Restriction Endonuclease with More Stringent Specificity
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2020
Restriction endonucleases with new sequence specificity can be developed from enzymes recognizing a partially degenerate sequence. Here we provide a detailed protocol that we successfully used to alter the sequence specificity of NlaIV enzyme. Key ingredients of the protocol are the in vitro compartmentalization of the transcription/translation reaction and selection of variants with new sequence specificities.
11:53
Substrate Generation for Endonucleases of CRISPR/Cas Systems
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Cited by 4 •
2012
CRISPR/Cas systems mediate adaptive immunity in Bacteria and Archaea. Many Cas proteins are proposed to act as endoribonucleases acting on crRNA precursors of varying length. Here we illustrate three different approaches to generate pre-crRNA substrates for the biochemical analysis of Cas endonuclease activity.
09:53
Single-Molecule Dwell-Time Analysis of Restriction Endonuclease-Mediated DNA Cleavage
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Cited by 2 •
2021
Using quantum-dot-labeled DNA and total internal reflection fluorescence microscopy, we can investigate the reaction mechanism of restriction endonucleases while using unlabeled protein. This single-molecule technique allows for massively multiplexed observation of individual protein-DNA interactions, and data can be pooled to generate well-populated dwell-time distributions.
06:58
Determining if DNA Stained with a Cyanine Dye Can Be Digested with Restriction Enzymes
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Cited by 5 •
2018
Staining DNA molecules for fluorescence microscopy allows a scientist to view them during an experiment. In the method presented here, DNA molecules are pre-stained with fluorescent dyes and digested with methylation and non-methylation sensitive restriction enzymes.
FAQs
Recognition-site placement determines the fragment pattern produced by Restriction Endonuclease Digestion. Because an enzyme cuts only where its particular nucleotide sequence occurs, the same DNA sample can yield a predictable set of fragments when the sequence locations are known. This sequence dependence allows researchers to infer structural differences by comparing fragment patterns rather than examining the entire molecule directly.
The distinction matters because the two end types offer different options for assembling DNA fragments. Sticky ends are useful when the exposed termini are compatible, allowing the fragments to be joined by DNA ligase during recombinant DNA construction. Blunt ends represent a different terminal product, so researchers must consider whether the selected cleavage sites produce ends suitable for the intended DNA assembly.
Many restriction endonucleases recognize sequences described as palindromic, so the sequence itself becomes a central criterion when choosing an enzyme. Researchers can select an endonuclease whose recognition site occurs at the desired positions in a DNA molecule. That choice controls where cleavage occurs and therefore which fragments become available for mapping, comparison, or downstream construction.
A practical workflow starts by selecting an enzyme whose recognition sequence is relevant to the DNA being studied. The DNA is then exposed to that endonuclease so cleavage generates fragments with defined ends. Researchers next use gel electrophoresis to separate and visualize the products, producing a fragment pattern that can support structural verification or comparison.
Restriction mapping uses cleavage positions to construct or examine an arrangement of sites along DNA, whereas DNA fingerprinting compares the resulting fragment patterns between samples. Both rely on predictable cleavage products, but they answer different questions: mapping focuses on DNA structure, while fingerprinting emphasizes pattern-based comparison. The same digestion can therefore support distinct analytical goals.
Researchers may digest plasmids to verify whether their structures match the expected fragment pattern before proceeding with molecular cloning. The same approach prepares DNA fragments for recombinant DNA construction, particularly when cleavage generates compatible ends that DNA ligase can join. These applications connect a sequence-specific cutting step with both quality control and assembly of engineered DNA.