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    Lecture - 1 - Restriction Enzymes

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    Restriction enzymes are molecular scissors that cut DNA at specific recognition sequences. They recognize short palindromic sequences and cut DNA to produce either blunt or sticky ends. The cuts allow manipulation and recombination of DNA from different sources. Restriction enzymes are categorized into four main types and have many applications in biotechnology and molecular biology.

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    Lecture - 1 - Restriction Enzymes

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    vikashlx0
    11 views27 pages
    Restriction enzymes are molecular scissors that cut DNA at specific recognition sequences. They recognize short palindromic sequences and cut DNA to produce either blunt or sticky ends. The cuts allow manipulation and recombination of DNA from different sources. Restriction enzymes are categorized into four main types and have many applications in biotechnology and molecular biology.

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    Lecture_1_restriction Enzymes

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    Restriction enzymes are molecular scissors that cut DNA at specific recognition sequences. They recognize short palindromic sequences and cut DNA to produce either blunt or sticky ends. The cuts allow manipulation and recombination of DNA from different sources. Restriction enzymes are categorized into four main types and have many applications in biotechnology and molecular biology.

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    © All Rights Reserved
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    Download as pdf or txt
    11 views27 pages

    Lecture - 1 - Restriction Enzymes

    Uploaded by

    vikashlx0
    Restriction enzymes are molecular scissors that cut DNA at specific recognition sequences. They recognize short palindromic sequences and cut DNA to produce either blunt or sticky ends. The cuts allow manipulation and recombination of DNA from different sources. Restriction enzymes are categorized into four main types and have many applications in biotechnology and molecular biology.

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    Molecular Scissors

    Restriction enzymes are


    molecular scissors

    DR. ABUL HASAN SARDAR


    Assistant Professor and Head
    Department of Microbiology
    Sarsuna College
    RESTRICTION ENZYMES
    • A restriction enzyme (or restriction
    endonuclease) is an enzyme that cuts double-
    stranded or single stranded DNA at specific
    recognition nucleotide sequences known as
    restriction sites.
    Property of restriction
    enzymes
    • They
    that link adjacent nucleotides in DNA
    molecules.
    HOW RESTRICTION ENZYMES
    WORKS?
    • Restriction enzymes recognize a specific
    sequence of nucleotides, and produce a
    double-stranded cut in the DNA, these cuts
    are of two types:

    • BLUNT ENDS.

    • STICKY ENDS.
    Blunt end

    Sticky end
    BLUNT ENDS
    • These blunt ended fragments can be joined to
    any other DNA fragment with blunt ends.

    • Enzymes useful for certain types of DNA


    cloning experiments
    “STICKY ENDS” ARE USEFUL
    DNA fragments with
    complimentary sticky ends
    can be combined to create
    new molecules which
    allows the creation and
    manipulation of DNA
    sequences from
    different sources.
    • While recognition sequences vary
    widely , with lengths between 4 and
    8 nucleotides, many of them are
    palindromic.
    PALINDROMES IN DNA SEQUENCES

    Genetic palindromes
    are similar to verbal
    palindromes. A
    palindromic sequence
    in DNA is one in
    which the 5’ to 3’
    base pair sequence is
    identical on both
    strands (the 5’ and 3’
    ends refers to the
    chemical structure of
    the DNA).
    PALINDROME SEQUENCES
    • The mirror like palindrome in which the same forward
    and backwards are on a single strand of DNA strand, as
    in GTAATG
    • The Inverted repeat palindromes is also a sequence
    that reads the same forward and backwards, but the
    forward and backward sequences are found in
    complementary DNA strands (GTATAC being
    complementary to CATATG)
    • Inverted repeat palindromes are more common
    and have greater biological importance than mirror-
    like palindromes.
    Star effect
    • Optimum conditions are necessary for the
    expected result.

    • Under extreme conditions such as elevated pH


    or low ionic strength, RE are capable of cleaving
    sequences which are similar but not identical
    to their recognition sequence.
    NOMENCLATURE OF RESTRICTION ENZYME
    • Each enzyme is named after the bacterium from
    which it was isolated using a naming system based
    on bacterial genus, species and strain.
    For e.g EcoRI
    Derivation of the EcoRI name

    Abbreviation Meaning Description

    E Escherichia genus

    co coli species

    R RY13 strain

    order of identification
    I First identified
    in the bacterium
    TYPES OF RESTRICTION ENZYMES
    • Restriction endonucleases are categorized into
    three general groups.

    • Type I
    • Type II
    • Type III
    TYPES OF RESTRICTION ENZYMES

    Type I Type II Type III

    Artificial
    Type IV restriction
    enzymes
    continue…..
    These types are categorization based on:
    • Their composition.
    • Enzyme co-factor requirement.
    • The nature of their target sequence.
    • Position of their DNA cleavage site relative to
    the target sequence.
    Type I
    • Capable of both restriction and modification
    activities

    • The co factors S-Adenosyl Methionine(AdoMet),


    ATP, and mg++are required for their full activity

    • Contain:
    two R(restriction) subunits
    two M(methylation) subunits
    one S(specifity) subunits
    • Cleave DNA at random length from recognition
    sites
    Type II
    • These are the most commonly available and used
    restriction enzymes

    • They are composed of only one subunit.

    • Their recognition sites are usually undivided and


    palindromic and 4-8 nucleotides in length,

    • They recognize and cleave DNA at the same site.

    • They do not use ATP for their activity

    • They usually require only mg2+ as a cofactor.


    Type III
    • Type III restriction enzymes cut DNA about 20-30
    base pairs after the recognition site.

    • These enzymes contain more than one subunit.

    • And require AdoMet and ATP cofactors for their


    roles in DNA methylation and restriction
    Type IV
    • Cleave only normal and modified DNA
    (methylated, hydroxymethylated and
    glucosyl-hydroxymethylated bases).

    • Recognition sequences have not been well


    defined

    • Cleavage takes place ~30 bp away from one


    of the sites
    ARTIFICIAL RESTRICTION ENZYMES
    • Generated by fusing a natural or engineered
    DNA binding domain to a nuclease domain

    • can target large DNA sites (up to 36 bp)

    • can be engineered to bind to desired DNA


    sequences
    Examples of Type II restriction
    enzymes
    EcoRI E = genus Escherichia
    co = species coli
    R = strain RY13
    I= first endonuclease
    isolated
    BamHI B = genus Bacillus
    am = species
    amyloliquefaciens
    H = strain H
    I = first endonuclease
    isolated
    HindIII H = genus Haemophilus
    in = species influenzae
    d = strain Rd
    III = third endonuclease
    isolated
    Isoschizomer
    • Restriction enzymes specific to the
    same recognition sequence. For
    example, SphI (CGTAC/G) and BbuI
    (CGTAC/G) are isoschizomers of each
    other.
    Neoschizomer
    • Enzyme that recognizes the same
    sequence but cuts it differently is
    a neoschizomer.

    • For example, SmaI(CCC/GGG) and XmaI


    (C/CCGGG) are neoschizomers of each
    other.
    APPLICATIONS
    They are used in
    gene cloning Restriction
    and protein enzymes are
    expression most widely used
    experiments in recombinant
    DNA technology.

    Detection
    of RFLPs

    Genotype a
    DNA DNA sample
    Mapping by SNP

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