Restriction Enzymes

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Restriction Enzymes
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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.

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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

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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

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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

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

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End Resulta DNA fingerprint
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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
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DNA sizes measured in Basepairs (bp) or
KilobasepairsKb
DNA samples
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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)

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Virtual Restriction Digestions of Plasmids

• http//www.vivo.colostate.edu/hbooks/genetics/bio
  tech/gels/virgel.html