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    Restriction Enzyme

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    Felix Simsung
    Restriction enzymes cut DNA molecules at specific recognition sites. They are used in biotechnology to manipulate DNA for applications like gene cloning and sequencing. In 1968, scientists discovered that bacteria produce endonucleases that degrade foreign DNA entering the bacterial cell. Later, the first restriction endonuclease was isolated from Haemophilus influenzae and shown to recognize a specific DNA sequence. Restriction enzymes are now widely used in techniques like recombinant DNA technology.

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    Restriction Enzyme

    Uploaded by

    Felix Simsung
    37 views6 pages
    Restriction enzymes cut DNA molecules at specific recognition sites. They are used in biotechnology to manipulate DNA for applications like gene cloning and sequencing. In 1968, scientists discovered that bacteria produce endonucleases that degrade foreign DNA entering the bacterial cell. Later, the first restriction endonuclease was isolated from Haemophilus influenzae and shown to recognize a specific DNA sequence. Restriction enzymes are now widely used in techniques like recombinant DNA technology.

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    Restriction enzymes cut DNA molecules at specific recognition sites. They are used in biotechnology to manipulate DNA for applications like gene cloning and sequencing. In 1968, scientists discovered that bacteria produce endonucleases that degrade foreign DNA entering the bacterial cell. Later, the first restriction endonuclease was isolated from Haemophilus influenzae and shown to recognize a specific DNA sequence. Restriction enzymes are now widely used in techniques like recombinant DNA technology.

    Copyright:

    © All Rights Reserved
    Download as DOCX, PDF, TXT or read online from Scribd
    Download as docx, pdf, or txt
    37 views6 pages

    Restriction Enzyme

    Uploaded by

    Felix Simsung
    Restriction enzymes cut DNA molecules at specific recognition sites. They are used in biotechnology to manipulate DNA for applications like gene cloning and sequencing. In 1968, scientists discovered that bacteria produce endonucleases that degrade foreign DNA entering the bacterial cell. Later, the first restriction endonuclease was isolated from Haemophilus influenzae and shown to recognize a specific DNA sequence. Restriction enzymes are now widely used in techniques like recombinant DNA technology.

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    © All Rights Reserved
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    Download as docx, pdf, or txt
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    Restriction enzymes

    The restriction enzymes also known as restriction endonucleases or restrictases are used to cut
    DNA at specific sites. DNA is usually cut into fragments by these enzymes at or near a specific
    site within the DNA molecule and this site is called the restriction site.

    Overall group of restriction enzymes is called endonucleases. These enzymes are the molecular
    scissors that is the reason they are given the name restriction enzymes. They cut the DNA by
    creating an incision at both strands of the DNA.

    Almost all of these restriction endonucleases are isolated from the bacteria. These endonucleases
    are the proteins produced by the bacteria to provide them protection against viruses like
    bacteriophages that attack bacteria and use their biosynthetic machinery for the synthesis of
    proteins and other substances that are required for their growth and survival. Bacteria cleave the
    viral DNA that enters in them using these restriction enzymes and in this way protects itself.

    Other than defense against the bacteriophages, restriction enzymes are also used in
    biotechnology in recombinant DNA technology and genetic engineering.

    Discovery of endonuclease:

    Before the discovery of restriction endonucleases, the phenomenon of restriction was well
    known. In 1968, two scientists named Matthew Meselson and Robert Yuan discovered that some
    enzymes are produced in E.coli that discard or digest the DNA that enters the bacteria from
    outside environment.

    They named these enzymes as restriction endonucleases. Later, they also discovered that these
    enzymes can degrade any double stranded DNA that is not protected by specific pattern of DNA
    bases known as methylation of DNA. Bacterial own DNA is usually methylated, which
    concluded that endonucleases can recognize the outside DNA by its non-methylation and in this
    way bacterial own DNA remains safe from endonucleases.

    Later in 1970, scientists named Hamilton O. Smith, Thomas Kelly and Kent Wilcox isolated first
    rectriction endonuclease from bacterium Haemophilus influenza known as HindII.

    They were later awarded Nobel Prize for the discovery of restriction endonuclease and their
    problem solving in molecular genetics.
    Properties of restriction enzymes:

    Restriction endonucleases have following properties:

     They are named according to the genus and specie of bacteria from which they are
    isolated and according to their class. For example, ECOR1 was isolated from E.coli and it
    belongs to class 1.
     They are proteins by nature.
     They only cut non-methylated DNA strands.
     They only cut at specific site, so, each site has a specific endonuclease.
     They create blunt ends, which can later be ligated.
     They can produce sticky ends
     They are non-palindromic.

    Properties of restriction Sites

    Restriction sites, or restriction recognition sites, are located on a DNA molecule containing


    specific (4-8 base pairs in length) sequences of nucleotides, which are recognized by restriction
    enzymes. These are generally palindromic sequences (because restriction enzymes usually bind
    as homodimers), and a particular restriction enzyme may cut the sequence between two
    nucleotides within its recognition site, or somewhere nearby.
    A palindromic sequence is a nucleic acid sequence in a double-
    stranded DNA or RNA molecule wherein reading in a certain direction (e.g. 5' to 3') on one
    strand matches the sequence reading in the same direction (e.g. 5' to 3') on the complementary
    strand. This definition of palindrome thus depends on complementary strands being palindromic
    of each other.
    Palindromic sequences may also have methylation sites.

    Enzyme Source Recognition Sequence Cut

    5'---G AATTC---3'
    EcoR1 Escherichia coli 5'GAATTC 3'CTTAAG
    3'---CTTAA G---5'
    Bacillus 5'---G GATCC---3'
    BamH1 5'GGATCC 3'CCTAGG
    amyloliquefaciens 3'---CCTAG G---5'

    5'---T CGA---3'
    Taq1 Thermus aquaticus 5'TCGA 3'AGCT
    3'---AGC T---5'

    5'---AG CT---3'
    Alu1* Arthrobacter luteus 5'AGCT 3'TCGA
    3'---TC GA---5'

    * = blunt ends

    Blunt Ends:
    Blunt end otherwise called as non cohesive restriction enzyme. In a blunt-ended molecule both
    strands terminate in a base pair. Blunt ends are not always desired in biotechnology since when
    using a DNA ligase to join two molecules into one, the yield is significantly lower with blunt
    ends

    Mode of action:
    The mode of action of restriction endonucleases varies in many aspects as the enzymes
    themselves. But the process of recognition of restriction site, binding of enzyme dimer to DNA
    strand, cutting and release of enzyme is similar.

    Restriction enzyme is used to cut double stranded DNA. During this it makes two incisions
    through both DNA strands creating blunt ends. Cuts are created at the sugar phosphate backbone
    of both of the strands of the double helix keeping that the nucleotides are not affected.

    Blunt end thus created are also known as sticky ends as they can again be ligated to a specific
    other or to the same DNA fragment because the nucleotides remain unaffected during the action
    of restriction enzyme.

    First, enzyme binds to the DNA strand with much less strength and specificity. Many proteins
    than interact with the enzyme that causes a conformational change to enzyme dimer increasing
    the strength and specificity. Enzyme than slides across the DNA strand searching for restriction
    site and restriction elements. On encounter to restriction elements the non-specific complex is
    converted into specific complex. Once the enzyme if properly bound to the DNA cleavage

    begins followed by the release of enzyme and sticky ends are created. Sticky ends are regions
    that are complementary overhangs and can be joined via complementation.
    Types of restriction enzymes:

    Restriction enzymes are usually divided into four types on the bases of differences in their
    structure, specific restriction sites, cleavage and cofactor. These types are as follows:

     Type 1 restriction enzymes


     Type 2 restriction enzymes
     Type 3 restriction enzymes
     Type 4 restriction enzymes

    Importance of restriction enzymes:

    They are used to manipulate DNA for various biological applications.

     They are used for isolation of genes


     Insertion of gene in vectors
     Gene cloning
     Protein production
     Gene sequencing
     DNA finger printing
     To detect mutations and SNPs
     In RFLP

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