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Restriction enzymes, also referred to as DNA manipulating enzymes, is an enzyme isolated from bacteria and that cuts DNA molecules at specific sequences.
- The restriction enzymes in the bacterial cell cleave foreign DNA which consequently eliminates the infecting organisms.
- It is used to perform DNA cloning, rDNA technolofy and gene modification in organisms.
- Example of restriction enzyme is EcoR1 and BamH1.
- Generally, in order to modify the genetic make up of an organism, DNA need to be cut.
- This can be acheived by using restriction enzymes.
- Two type of restriction enzymes are there namely, restriction endonuclease and exonuclease.
- Endonuclease will cut DNA sequence from the end while an exonucleases will cleave the sequence from middle of the base pairs.
| Table of Content |
Keyterms: Enzymes, DNA, bacterial cell, genetic engineering, microbe, hybridization, gene cloning, gene transfer, genes, endonuclease, exonuclease.
What are Restriction Enzymes?
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Restriction enzymes belong to a bigger group of enzymes and also called as molecular scissors due to the fact those enzymes cut DNA at unique sites.
- These enzymes will identtify a site from where cut needs to be made.
- This site is termed as recodnistion site.
- Each restriction enzyme type have different recognition sites.
- Some will recognize 2 base pair site while other will cleave it upto 4 base pairs.
- These cuts will generate two type of end- blunt end and sticky end.
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Types of Restriction Enzymes
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Type I Restriction Enzymes
These restriction enzymes have been the primary restriction enzymes to be identified. These enzymes are characterized with the aid of using their DNA cleavage sites.
- Type I enzymes reduce DNA some distance far from the diagnosed series inside the DNA molecule.
- They do now not have longer motive powerful fragmentation of the DNA and hence, are of now no longer a whole lot importance.
- Earlier, they have been notion to be uncommon in nature, however non-stop look at and studies proved that those kinds of enzymes are quite not unusual to place in nature.
- It is multifunctional as the restriction enzymes have 3 subunits that carry out limit digestion, reputation, and additionally change of the DNA with the assist of its cofactors like magnesium ions and ATP (adenosine triphosphate) that satisfy the catalyzing interest of the enzyme.
Type II Restriction Enzymes
These restriction enzymes are massively exceptional from Type I. For Type II restriction enzymes, the reputation of the series and the restriction digestion, i.e., the DNA cleavage, takes place in an equal place.
- These sites aren't exceptional from every other.
- For the cofactors, the Type II restriction enzymes typically use Magnesium ions for finishing the restriction system in DNA molecules. This kind is the maximum not unusual place restriction enzyme to be had and is used the maximum for wearing out restriction. Another foremost feature of the Type II enzymes is that those enzymes both reduce via the center of the DNA strand, inflicting blunt ends at each facet or developing cleaves at staggered positions leaving sticky ends. These restriction enzymes additionally have extra than simply one subunit, and those subunits carry out exceptional functions.
Type III Restriction Enzymes
These limit enzymes are multifunctional proteins. This form of restriction enzyme cuts the DNA far from the popular series. They have subunits that deliver the characteristic of DNA methylation or change and restriction digestion. These enzymes use the AdoMet cofactors usually for wearing out the restriction system.
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Applications of Restriction Enzymes
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The restriction enzymes are used in RFLP techniques in order to cut the DNA into smaller fragments, to study the fragment length differences among the individuals. They are also used in gene cloning. A gene is inserted into a plasmid during cloning. Restriction enzymes cut the plasmid producing single-stranded overhangs and the two DNA molecules are ligated with the DNA ligase to form a single DNA molecule.
Things to Remember
- The restricted enzymes reduce DNA at unique base collection, and that unique base collection is called the recognition collection.
- An endonuclease is a collection or sort of enzyme that enables cleaving nucleotide sequences in molecules.
- The restriction enzymes try and save you the replication of the DNA of the phage.
- Type I restriction enzymes have been the primary restriction enzymes to be identified.
- These enzymes are characterized with the aid of using their DNA cleavage websites.
- For Type II limit enzymes, the reputation of the series and the restriction digestion, i.e., the DNA cleavage, takes place in an equal place.
- Type III restriction enzymes limit enzymes are multifunctional proteins.
Sample Questions
Ques: What is meant by restriction enzyme? (2 marks)
Ans: Restriction enzymes are the enzymes that are produced by certain bacteria having the property of cleaving DNA molecules at or near specific base sequences.
Ques: How is the restriction site recognized by the restriction enzymes? (2 marks)
Ans: Restriction enzymes are isolated from the prokaryotes which recognize the specific DNA sequences and bind to them. These are referred to as the restriction sites. On encountering its target sequence, the enzyme makes a double-stranded cut in the molecule.
Ques: How does a restriction enzyme protect its own DNA from cleavage? (2 marks)
Ans: The restriction enzyme methylates its DNA with the purpose that it is not recognized by the restriction endonuclease. Therefore, in this way, they prevent the restriction enzyme from cutting its own DNA.
Ques: What do you mean by exonucleases? (1 mark)
Ans: The exonucleases are the enzymes that remove nucleotides from the end of the DNA one at a time.
Ques: Does the modified DNA remain protected after replication? (3 marks)
Ans: One of the DNA strands remains methylated even after replication because of the semi-conservative mode of DNA replication. One methylated strand can protect the DNA from cleavage by restriction enzymes.
Ques: What are the types of restriction enzymes? (1 mark)
Ans: There are three types of restriction enzymes:
Type I restriction enzymes
Type II restriction enzymes
Type III restriction enzymes
Ques: Define type II and type III restriction enzyme. (4 marks)
Ans: Type II Restriction Enzymes
These restriction enzymes are massively exceptional from Type I. For Type II limit enzymes, the reputation of the series and the restriction digestion, i.e., the DNA cleavage, takes place in an equal place. These websites aren't exceptional from every other. Moreover, for the cofactors, the Type II restriction enzymes typically handiest use Magnesium ions for finishing the limit system in DNA molecules. This kind is the maximum not unusual place restriction enzyme to be had and is used the maximum for wearing out restriction.
Type III Restriction Enzymes
These limit enzymes are multifunctional proteins. This form of restriction enzyme cuts the DNA far from the popular series. They have subunits that deliver the characteristic of DNA methylation or change and restriction digestion. These enzymes use the AdoMet cofactors usually for wearing out the restriction system.
Ques: State the applications of restriction enzymes. (4 marks)
Ans: The restriction enzymes are used in RFLP techniques in order to cut the DNA into smaller fragments, to study the fragment length differences among the individuals. They are also used in gene cloning. A gene is inserted into a plasmid during cloning. Restriction enzymes cut the plasmid producing single-stranded overhangs and the two DNA molecules are ligated with the DNA ligase to form a single DNA molecule.
Ques: In 1963, two enzymes responsible for restricting the growth of bacteriophage in E. coli were isolated. How did enzymes act in order to restrict the growth of the bacteriophage? (All India 2011c)
Ans: Two enzymes that were responsible for restricting the growth of bacteriophage in coli are Exonucleases Add methyl group to DNA. Endonucleases Cut DNA at specific points.
Ques: Explain how does a restriction nuclease function? (All India 2014)
Ans: The restriction nucleases function by inspecting the length of DNA sequence, and binding to specific recognition sequences and then cutting the strands at sugar phosphate backbones.
Depending on their mode of action, these nucleases are of two types:
(i) Restriction exonucleases: These cut sequences at terminal ends of DNA.
(ii) Restriction endonucleases: These cut between the two bases of recognition sequence.
Ques: Why are molecular scissors so called? What are their use in biotechnology? (Foreign 2014)
Ans: Molecular scissors are so called as they cut DNA at specific sequences between base pairs.
As molecular scissors on restriction enzymes cut DNA at desired sequences and generate sticky ends facilitating them to join with host genome or vector DNA, they play an important role in genetic engineering or biotechnology. This is the reason why with the help of these enzymes we can cut the desired gene and introduce into vectors for expression.
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