Restriction enzymes, restriction endonucleases, or molecular scissors are bacteria-produced enzymes that can slice between two DNA strands at areas called recognition sites. Restriction enzymes were first discovered during Enterobacteria coli research. Type II restriction enzymes (REs) are of particular importance in the fields of molecular cloning, gene sequencing, and DNA mapping as this group can cut DNA very close to specific recognition sites and does not require energy in the form of ATP.
Restriction Enzyme Function
Restriction enzyme function in the natural world is to defend bacteria against specific viruses called bacteriophages. These viruses attack bacteria by injecting viral RNA or DNA into a bacterial plasmid (small, purple ring in the below image) and replicating there. If viral RNA or DNA is detected within a prokaryote cell, that cell can often stop the replication process by slicing through the foreign genetic information. This renders it useless. At the same time, bacterial DNA is protected from the cutting action of its restriction endonucleases within its restriction sites. This mechanism adds methyl (H3C) groups to the cytosine and adenine of bacterial DNA without affecting the coded DNA sequence.
To date, approximately 3500 restriction enzymes have been isolated from bacterial plasmids. Each enzyme recognizes a specific sequence of viral genetic code and will try to separate the new, mutated DNA strand close to or further away from the recognition site. This natural separation mechanism is also referred to as restriction enzyme digestion.
Not only the location and the method but also the type of cut can differ. Some REs leave uneven sticky ends (non-blunt ends) between slightly different areas of a double-strand that overhang; others leave blunt ends where base pairs are separated at the same point. For example, BamHI is a type II restriction enzyme obtained from Escherichia coli that recognizes the nucleotide sequence GGATCC and cleaves these sections of DNA leaving sticky ends. Cleaving, like cleaving a log with an ax, is the scientifically-accepted term for cutting a strand of DNA.
In the below image, a restriction enzyme called HindIII cleaves DNA at different points on the two strands to form a sticky end.
Once the double DNA strand has been separated, another enzyme called DNA ligase rejoins the DNA backbone as a sticky-end or blunt-end ligation. Single-stranded DNA that has been inserted into bacterial DNA by specific viruses can be removed by certain REs. DNA ligase then recombines the DNA by constructing a mirror copy of the bacterial sequence. In short, a restriction enzyme cleaves the foreign DNA and DNA ligase repairs the break to bring it back to its original form.
Since the discovery of genes, ways to manipulate them have been heavily researched. The action of removing a gene sequence and replacing it with another is known as gene recombination. The short restriction enzyme recognition sites usually number between four to eight nucleotides. This makes restriction enzymes ideal for use in the field of molecular biology.
Nucleotides in DNA consist of a nucleobase, a deoxyribose sugar, and a phosphate group. It is the phosphate and sugar groups that form the backbone of DNA, shown here in blue and turquoise.
Restriction Enzyme Groups
Natural restriction enzymes are arranged in five groups: type I, II, III, IV, and V. Type I REs, the first to be discovered, cut DNA sequences far from the recognition sites and require ATP to recognize, modify and/or digest asymmetrical sections. The distance from the recognition site makes type I restriction endonucleases less helpful in the field of genetic engineering.
Type II REs recognize and cut short sections of DNA close to restriction sites without ATP but using magnesium ions. Because of this, they are the most commonly used natural restriction endonucleases. Type II restriction enzymes are further categorized into subgroups and one of these subgroups is the high-precision IIS category.
Type III restriction endonucleases are rarely used in genetic engineering as they cut DNA sequences well outside of the recognition sequence and need to detect two separate sequences to achieve this. This means they are not always able to provide complete restriction enzyme digestion. Type IV restriction nucleases can only cleave methylated DNA (DNA that is not transcribed into a protein) and sequence specificity is weak.
Type V restriction enzymes require guide RNA (gRNA) to target specific sequences and it is these that are being modified or used in genome engineering methods such as TALENS and CRISPR-Cas9. CRISPR is the short form of clusters of regularly interspaced short palindromic repeats. CRISPR regions refer to repeated nucleotide and spacer patterns within a section of the DNA; it is within spacers that viruses incorporate their DNA. By inserting other genetic code into a spacer by artificial means it is possible to modify the genome of a living organism.
Finally, artificial restriction enzymes (AREs) are becoming ever more popular with geneticists as they can be modified to recognize and cut DNA sequences at predefined sites. CRISPR and TALENS use adapted restriction enzymes for increased accuracy; they can also edit many genes in a single process. Furthermore, commercially-available natural restriction enzymes are limited in number, and these fragment DNA into very short sections; it is rare that a smaller laboratory has access to the right enzymes. This is because different restriction enzymes are required to cleave the many separate areas of DNA that make up the code for a single gene.
The recent synthesis of artificial restriction enzymes using certain proteins such as Argonaute protein (PfAgo) provides an alternative technique that can cleave longer sticky-end sequence sequences with increased accuracy.
Restriction Enzymes in Molecular Cloning
In molecular cloning, molecular biologists insert a gene into a small, stable section of an organism’s DNA, allowing it to be replicated. When this gene is expressed, research on that gene’s effects on study organisms can be carried out.
Restriction enzyme cloning is one of the earliest techniques in the field of molecular cloning but remains popular due to a low cost-to-reliability ratio. After producing sticky or blunt ends, cleaved DNA is purified and inserted into the DNA of the host bacteria in a step called transformation. After transformation, the plasmid contains recombinant (recombined) DNA – a term used to describe the combination of extracted DNA fragments with DNA ligase enzymes. DNA ligase allows this section to be fixed into a plasmid. A host bacterium can then produce more DNA or express the inserted gene after protein synthesis.
Newer methods that do not require natural restriction enzymes but use synthetic versions are being increasingly implemented. The above-described technique is, therefore, commonly referred to as traditional cloning. DNA cloning should not be confused with the process used to create Dolly the sheep; only small strands of DNA are replicated in gene modification. Dolly was the result of complete genome cloning.
Restriction Enzymes in DNA Profiling
The discovery of restriction enzymes has made DNA profiling possible. Minisatellites are short, repetitive sequences of between ten and sixty base pairs that show greater variation between individuals than other sequences within the genome. This variation is determined by the number of repeated units (stutters) within a minisatellite sequence.
Multiple minisatellites provide a DNA fingerprint that identifies an individual. Earlier forms of DNA profiling used natural restriction enzymes to cut various-sized sections throughout the DNA. Pulsed-field gel electrophoresis separates these sections ready for identification. The separated sections representing minisatellites are blotted onto a membrane and pulled apart to produce single strands. This procedure requires opposing strands composed of radioactive phosphorous that link to their complementary (matching) strands on the membrane. An x-ray then produced an image of the DNA fingerprint – an image is possible due to the radioactive phosphorus copy.
Today, microsatellites of two to five base pairs are replicated many times over through a technique known as the polymerase chain reaction. This newer method provides results even with a tiny sample of DNA – something the earlier method was unable to do. Instead of radioactive phosphorous, primer RNA binds to both ends of those cut DNA sequences that show the most variation between individuals. RNA primers are labeled with fluorescent colors. Pulsed-field gel electrophoresis, multilocus sequence typing (MLST), and polymorphic amplified typing sequences (PATS) are technologies used to separate the resulting fragments. Lasers then provide different light wavelengths to produce a colorful DNA fingerprint.
While DNA profiling is most associated with the field of criminal forensic science, this identification method is also used to detect bacterial strains responsible for disease, provide a bacterial fingerprint that can be used to isolate and treat infection, or determine whether food or places where food is produced is free of pathogenic bacteria.
Traditional DNA Cloning Uses
Traditional DNA cloning using restrictive endonucleases has multiple uses. Expressed recombinant DNA (DNA sequences that code for protein synthesis), when inserted into the genetic information of bacteria, stimulate bacteria to produce the target protein.
This is the method whereby genetic engineers in pharmaceutical companies manufacture human insulin, human albumin, some vaccines, monoclonal antibodies, and human growth hormone at much lower cost that extracting these products from multicellular organisms. Recombinant DNA is also used to diagnose hereditary disease and produce antibiotics on a huge scale.
Gene analysis is a broad sector in which genetic engineers insert cleaved recombinant DNA sequences (rDNA) to help us understand what specific genes do. By understanding genes, we then have the data we need to make adjustments that can potentially eradicate disease.
Genetically-modified crops are the result of traditional molecular cloning techniques where resistance to insects and herbicides and more product per square hectare are the main goals. This method also improves nitrogen fixation in plants and crops.
The catering industry uses recombinant DNA in fermentation and cheese-making processes, and also to detect the presence of pathogenic bacteria and fungi on surfaces used for food preparation.
However, to produce results that may improve our health or food sources, our knowledge of the function of every gene is essential. Only once the function of a DNA sequence has been discovered can it be correctly used. Traditional DNA cloning was the first technique used in the field of genome mapping that has, over many years, taught us how genes are expressed. With new artificial restriction enzymes, genetic engineering can only be expected to move forward over the next few decades.
- Berg JM, Tymoczko JL, Stryer L. (2002). Biochemistry. 5th edition. New York: W H Freeman. Section 9.3, Restriction Enzymes: Performing Highly Specific DNA-Cleavage Reactions. Retrieved from:https://www.ncbi.nlm.nih.gov/books/NBK22528/
- Adrio, J. L., & Demain, A. L. (2010). Recombinant organisms for production of industrial products. Bioengineered Bugs, 1(2), 116–131. https://doi.org/10.4161/bbug.1.2.10484
- Pingoud A. Ed. (2004). Restriction Endonucleases. New York, Springer.
- Loenen WAM. (2019). Restriction Enzymes: A History. Huntingdon, Cold Spring Harbor Laboratory Series.
A restriction enzyme is a protein isolated from bacteria that cleaves DNA sequences at sequence-specific sites, producing DNA fragments with a known sequence at each end.What is a restriction enzyme quizlet? ›
Restriction Enzymes. Restriction enzymes or restriction endonucleases are enzymes used to cut within a DNA molecule. Restriction enzymes can be found within bacteria. They are also manufactured from bacteria. Restriction enzymes recognize and cut DNA at a specific sequence of nucleotides.Which sentence is correct for restriction enzymes? ›
So, the correct answer is 'Are synthesized by bacteria as part of defense mechanism'What are restriction enzymes ___ DNA? ›
Restriction enzymes are DNA-cutting enzymes. Each enzyme recognizes one or a few target sequences and cuts DNA at or near those sequences. Many restriction enzymes make staggered cuts, producing ends with single-stranded DNA overhangs.How does a restriction enzyme work? ›
How do restriction enzymes work? Like all enzymes, a restriction enzyme works by shape-to-shape matching. When it comes into contact with a DNA sequence with a shape that matches a part of the enzyme, called the recognition site, it wraps around the DNA and causes a break in both strands of the DNA molecule.What do restriction enzymes show? ›
Restriction enzymes, also called restriction endonucleases, recognize a specific sequence of nucleotides in double stranded DNA and cut the DNA at a specific location. They are indispensable to the isolation of genes and the construction of cloned DNA molecules.What are three restriction enzymes? ›
Type I restriction enzymes possess three subunits called HsdR, HsdM, and HsdS; HsdR is required for restriction digestion; HsdM is necessary for adding methyl groups to host DNA (methyltransferase activity), and HsdS is important for specificity of the recognition (DNA-binding) site in addition to both restriction ...What is a type of restriction enzyme? ›
Today, scientists recognize three categories of restriction enzymes: type I, which recognize specific DNA sequences but make their cut at seemingly random sites that can be as far as 1,000 base pairs away from the recognition site; type II, which recognize and cut directly within the recognition site; and type III, ...What are restriction enzymes also called? ›
Restriction enzymes are also called "molecular scissors" as they cleave DNA at or near specific recognition sequences known as restriction sites. These enzymes make one incision on each of the two strands of DNA and are also called restriction endonucleases.What are 2 examples of restriction enzymes? ›
Example of restriction enzymes includes EcoRI and smaI.
The function of restriction endonucleases is mainly protection against foreign genetic material especially against bacteriophage DNA. The other functions attributed to these enzymes are recombination and transposition.What are two restriction enzymes? ›
- EcoRI and smaI are the two examples of restriction enzymes.What are the 4 types of restriction enzymes? ›
Traditionally, four types of restriction enzymes are recognized, designated I, II, III, and IV, which differ primarily in structure, cleavage site, specificity, and cofactors.Why are restriction enzymes important? ›
Restriction enzymes have proved to be invaluable for the physical mapping of DNA. They offer unparalleled opportunities for diagnosing DNA sequence content and are used in fields as disparate as criminal forensics and basic research.Do restriction enzymes cut DNA or RNA? ›
Restriction endonucleases naturally target DNA duplexes. Systematic screening has identified a small minority of these enzymes that can also cleave RNA/DNA heteroduplexes and that may therefore be useful as tools for RNA biochemistry.Why is it called restriction enzyme? ›
Restriction endonucleases are named from the fact that they stop bacteriophages from multiplying by recognizing and cutting DNA at specified locations.What is the source of restriction enzymes? ›
Definition. Restriction enzymes are a group of proteins that bacteria produce to cut up the DNA of invading viruses. Electron micrograph of Escherichia coli, close-up. Such bacteria are an important source for restriction enzymes.Where do restriction enzymes cut DNA? ›
Restriction enzymes are DNA-cutting enzymes found in bacteria (and harvested from them for use). Because they cut within the molecule, they are often called restriction endonucleases. To be able to sequence DNA, it is first necessary to cut it into smaller fragments.What are the best restriction enzymes? ›
The best characterized and most frequently used restriction enzymes are the classical Type II class. These enzymes recognize specific 4 to 8 nucleotide sequences that are typically palindromic and cleave within the recognition site leaving sticky (5′ or 3′ overhangs) or blunt ends.Do humans have restriction enzymes? ›
The HsaI restriction enzyme from the embryos of human, Homo sapiens, has been isolated with both the tissue extract and nuclear extract. It proves to be an unusual enzyme, clearly related functionally to Type II endonuclease.
Restriction enzymes are endonucleases, that is, enzymes that digest nucleic acids. Restriction enzymes recognize specific sequences of nucleotides in a DNA strand. Their use allows the detection of point mutations in DNA and eliminates the need for subcloning and sequencing.What is the difference between enzyme and restriction enzyme? ›
Restriction enzyme refers to an enzyme produced chiefly by certain bacteria, which has the property of cleaving DNA molecules at or near a specific sequence of bases, while restriction endonuclease refers to an enzyme that cleaves DNA into fragments at or near specific recognition sites within molecules known as ...What are restriction sites in DNA? ›
A restriction site is a sequence of approximately 6–8 base pairs of DNA that binds to a given restriction enzyme. These restriction enzymes, of which there are many, have been isolated from bacteria. Their natural function is to inactivate invading viruses by cleaving the viral DNA.What is a Type 1 restriction enzyme? ›
Type I restriction enzymes (REases) are large pentameric proteins with separate restriction (R), methylation (M) and DNA sequence-recognition (S) subunits.Which restriction enzyme is mostly used in cloning and why? ›
Type IIP enzymes  that generate identical, palindrome overhangs are commonly used in cloning experiments since the protruding ends of the two DNA fragments generated by these enzymes are generally compatible for joining by a DNA ligase.How many restriction enzymes are there? ›
Approximately 3,000 restriction enzymes, recognizing over 230 different DNA sequences, have been discovered. They have been found mostly in bacteria, but have also been isolated from viruses, archaea and eukaryotes.What are Type 1 and 2 restriction enzymes? ›
Type I restriction enzyme is a DNA restriction enzyme which cleaves DNA at random sites far from its recognition site. Type II restriction enzyme is a DNA restriction enzyme which cleaves DNA at defined positions close to or within the recognition site.What are Type 1 and 3 restriction enzymes? ›
The Type I and III restriction endonucleases are large, multimeric protein complexes with four enzyme activities; DNA methyltransferase, DNA endonuclease, ATPase and DNA translocase. It has been demonstrated that ATP-dependent protein motion along DNA is necessary for endonuclease activity.Do you need two restriction enzymes? ›
Using two different restriction enzyme sites can help ensure the correct orientation of the gene of interest when it is inserted and prevent the plasmid vector from ligating with itself.What is the purpose of restriction enzyme digestion? ›
Restriction digestion is usually used to prepare a DNA fragment for subsequence molecular cloning, as the procedure allows fragments of DNA to be pieced together like building blocks via ligation.
Restriction enzymes have proved to be invaluable for the physical mapping of DNA. They offer unparalleled opportunities for diagnosing DNA sequence content and are used in fields as disparate as criminal forensics and basic research.What happens during restriction enzyme digestion? ›
Introduction. Restriction enzyme digestion takes advantage of naturally occurring enzymes that cleave DNA at specific sequences. There are hundreds of different restriction enzymes, allowing scientists to target a wide variety of recognition sequences. For a list of many commonly used restriction enzymes, visit NEB.What role do the restriction enzymes play in DNA cloning? ›
Restriction enzyme cloning, or “restriction cloning,” uses DNA restriction enzymes to cut a vector and an insert at specific locations so they can be easily joined together by the enzyme DNA ligase to create recombinant DNA.