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Restriction Enzymes

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Restriction Enzymes What are restriction enzymes? Restriction enzymes are proteins that cut DNA. Because they cut within the molecule, they are often referred to as ... – PowerPoint PPT presentation

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Title: Restriction Enzymes


1
Restriction Enzymes
2
What are restriction enzymes?
  • Restriction enzymes are proteins that cut DNA.
    Because they cut within the molecule, they are
    often referred to as restriction endonucleases.

3
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4
Function
  • Found naturally in bacteria
  • Protects the bacteria from invading viruses
  • bacterium modifies its own restriction sites by
    methylation so enzyme cannot cut it
  • When virus injects its DNA the bacteria can
    cleave the viral DNA without affecting its own
  • Able to cut double stranded DNA molecules at a
    specific nucleotide pair sequence called the
    restriction site

5
Nomenclature
  • Over 600 restriction enzymes are commercially
    available
  • Named for the bacteria from which they were
    isolated
  • 3 letter system
  • Based on Genus and species that the enzyme was
    isolated from
  • Additional letters added to signify particular
    strains and order of discovery
  • Bgl II - Bacillus globigi
  • EcoR I - E. coli Strain RY13
  • Hind III - Haemophilus influenza

6
Probability and Non-Random Sequences
  • In a DNA sequence with a random distribution of
    nucleotides, there is a clear relationship
    between the number of nucleotide pairs in the
    recognition site and the frequency of cutting
  • Enzymes which recognize 4 bases (4 base cutter)
    will cut more frequently than enzymes which have
    a 6 base recognition sequence (6 base cutter) and
    so on.
  • In random DNA, the probability of finding one
    nucleotide pair is independent of any other pair
    in the sequence.
  • In general the probability of a recognition site
    is (1/4)n where n is the number of nucleotides in
    the recognition site
  • 4 bases (1/4)4 1/256
  • 5 bases (1/4)5 1/1024
  • 6 bases (1/4)6 1/4096
  • 8 bases (1/4)8 1/65,476
  • DNA in organisms is not randomly distributed
    however
  • There may be an organism bias in G/C content
  • There may be a difference in G/C content between
    coding and non-coding DNA

7
How were they discovered?
In 1970 Hamilton Smith accidentally found that a
DNase from the bacterium, Haemophilus influenzae,
cut DNA at specific PALINDROME DNA SEQUENCES
known as RESTRICTION SITES.
8
Restriction enzymes bind DNAat specific sites
9
In DNA, a PALINDROME SITE is a SEQUENCE OF BASE
PAIRS in double stranded DNA that reads the same
backwards and forwards across the double strand
Example The sequence of base pairs GAATTC is a
palindrome because both sequences of the double
strand READ THE SAME when read from either their
respective "G" or "C" ends.
5... G A A T T C 3 3... C T T A A G 5
10
Based on the TYPES OF CUTS they make, there are
two types of restriction enzymes.
  • BLUNT ENDS
  • STICKY ENDS

5... G A A T T C 3 3... C T T A A G
5
5... G A A T T C 3 3... C T T
A A G 5
11
Blunt Ends
12
Sticky Ends
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14
Sisters Sequence
15
Brothers Sequence
Perform a restriction digest on both siblings
how do they compare when you run the results out
on a gel?
16
Gel Electrophoresis(the separation of DNA based
on size)
agarose
wells
17
Loading a Gel
18
DNA is negatively charged (click once to run
animation)
Electrical charge

-
19
End Resulta DNA fingerprint
20
Gel Analysis
Ladder DNA of known sizes (used as a standard)
Large DNA pieces -these have a hard time
migrating through the gel pores
Small DNA pieces -these can migrate through the
gel pores easily and migrate farther
21
DNA sizes measured in Basepairs (bp) or
KilobasepairsKb
DNA samples
22
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23
Recombinant DNA (Chimeras)
  • making recombinant DNA from two different sources
    whether it be different species or different
    tissue sources is an important tool.
  • Using recombinant DNA technology can lead to
    important discoveries about the
    structure-function relationship of proteins, how
    mutations cause disease, how genes may be
    regulated, and to isolate a gene product for
    production (ex. Insulin)

24
Virtual Restriction Digestions of Plasmids
  • http//www.vivo.colostate.edu/hbooks/genetics/bio
    tech/gels/virgel.html
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