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Restriction Enzymes

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Restriction Enzymes. Tam Han. Nwamaka Okagbue. Intro into DNA Enzymology. History ... 1955: Arthur Kornberg and colleagues isolated DNA polymerase ... – PowerPoint PPT presentation

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Title: Restriction Enzymes


1
Restriction Enzymes
  • Tam Han
  • Nwamaka Okagbue

2
Intro into DNA Enzymology
  • History
  • 1955 Arthur Kornberg and colleagues isolated DNA
    polymerase
  • 1966 B. Weiss and C.C. Richardson isolated DNA
    ligase
  • 1968 H.O. Smith, K.W. Wilcox, and T.J. Kelley
    isolated and characterized the first sequence
    specific restriction enzyme
  • Phage growth restriction
  • Phages grow and infect one strain, but not the
    other strain
  • Methylation
  • Not modified get degraded by hosts enzymes
  • Methylase and Nuclease
  • Late 1960s, Stewart Linn and Werner Arber
    isolated two types of enzymes responsible for
    phage growth restriction in E. coli bacteria.
  • Methylase cleaved methylated DNA
  • Restriction nuclease cleaved unmethylated DNA
    at a wide variety of sites
  • Site-specific nuclease
  • 1968, Johns Hopkins University, H.O. Smith, K.W.
    Wilcox, and T.J. Kelley first restriction
    nuclease whose functioning depended on a specific
    DNA nucleotide sequence
  • HindII from Haemophilus influenzae bacteria
  • Always cut at a particular point within a
    specific sequence of six base pairs
  • 5 GTAC 3
  • 3 CATG 5

3
What are they?
  • Restriction enzymes are DNA-cutting enzymes found
    in bacteria (and harvested from them for use).
  • Restriction endonucleases
  • A restriction enzyme recognizes and cuts DNA only
    at a particular sequence of nucleotides.
  • Bacteria prevent their own DNA from being cut by
    modifying their nucleotides via DNA methylation.

4
How REs cut DNA?
  • Blunt ends
  • Cleave opposing phosphodiester bonds no unpaired
    bases on ends
  • Sticky ends
  • Staggerd cuts of two DNA strands leaving unpaired
    nucleotides at the ends
  • Base-pair with complementary sticky ends

5
  • Three Types of Restriction Enzymes
  • Type I cleave DNA at random sites that can be
    more than 1,000 bp from the recognition sequence.
  • Type II first isolated in 1970 (Hamilton Smith),
    require no ATP and cleave DNA within the
    recognition sequence.
  • Type III cleave about 25 bp from the recognition
    sequence.
  • Nomenclature

6
  • Restriction mapping
  • The process of obtaining structural information
    on a piece of DNA by the use of restriction
    enzymes.
  • A restriction map is a description of restriction
    endonuclease cleavage sites within a piece of
    DNA.
  • Restriction mapping involves digesting DNA with a
    series of restriction enzymes and then separating
    the DNA fragments by agarose gel electrophoresis.
  • The distance between enzyme sites can be
    determined by the size of the DNA fragments.

7
Restriction sites
  • Palindromes sequences can be read the same way
    in either direction

8
Sources
  • Nelson, David L., Michael M. Cox. Lehninger
    Principles of Biochemistry. 4th Ed. DNA-Based
    Information Technologies. pp.308-09.
  • Peters, Pamela, Ph.D., Access Excellence,
    Genentech, Inc. lthttp//www.accessexcellence.org/A
    E/AEC/CC/restriction.phpgt
  • Roberts, RJ, Belfort M, Bestor T, Bhagwat AS,
    Bickle TA, Bitinaite J, Blumenthal RM, Degtyarev
    SKh, Dryden DT, Dybvig K, Firman K, Gromova ES,
    Gumport RI, Halford SE, Hattman S, Heitman J,
    Hornby DP, Janulaitis A, Jeltsch A, Josephsen J,
    Kiss A, Klaenhammer TR, Kobayashi I, Kong H,
    Krüger DH, Lacks S, Marinus MG, Miyahara M,
    Morgan RD, Murray NE, Nagaraja V, Piekarowicz A,
    Pingoud A, Raleigh E, Rao DN, Reich N, Repin VE,
    Selker EU, Shaw PC, Stein DC, Stoddard BL,
    Szybalski W, Trautner TA, Van Etten JL, Vitor JM,
    Wilson GG, Xu SY (April 2003). "A nomenclature
    for restriction enzymes, DNA methyltransferases,
    homing endonucleases and their genes". Nucleic
    Acids Res. 31 (7) 180512. doi10.1093/nar/gkg274
    . PMID 12654995. lthttp//en.wikipedia.org/wiki/Res
    triction_enzymegt
  • Smith, H.O., D. Nathans (December 1973). "Letter
    A suggested nomenclature for bacterial host
    modification and restriction systems and their
    enzymes". J. Mol. Biol. 81 (3) 41923. PMID
    4588280. lthttp//en.wikipedia.org/wiki/Restriction
    _enzymegt
  • Dr. Jordan. Molecular Biology Tools. Lecture 1C.
    Slide 7.
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