Restriction Enzymes

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Restriction Enzymes
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Bacteriophage Life Cycle
1.
2.
6.
3.
5.
4.
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Bacteriophage Lambda (l)
Capsid is icosahedral (20 faces) - Genome is
48,502 base pairs
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Bacteriophage Infection
T4 Phage
T4 phage infecting a bacterial cell
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Bacteria Have a Primitive Immune System
Bacteria have evolved ways to protect themselves
from invasion by viruses. One method is to
produce an enzyme that chops up the foreign DNA
and disables it. In this way bacteria are able
to restrict the growth and replication of certain
viruses.
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REs Disable Viral DNA
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DNA Methylation Protects Bacterial DNA
Methyl groups (CH3) bind to the DNA and prevent
the restriction enzyme from attacking the cells
own DNA.
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Restriction Enzyme Binding to DNA
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Eco RI

• G AATTC
• CTTAA G
• E genus Escherichia
• co species coli
• R strain RY13
• I first enzyme isolated

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Bam HI

• A AGCTT
• TTCGA A
• B genus Bacillus
• am species amyloliquefaciens
• H strain H
• I first enzyme isolated

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HindIII

• G GATCC
• CCTAG G
• H genus Haemophilus
• In species influenzae
• d strain Rd
• III third enzyme isolated

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Mechanism of Action
ATP is required and is usually included in the
buffer system.
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Some REs Produce Sticky Ends
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Types of Cuts
Blunt Sticky ends
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Digestion of DNA with REs

• Reaction Temperature usually 37oC
• Storage Temperature -20oC, glycerol
• Buffer system pH, ions, Mg 2 NaCl
• Deactivation 65o C for 10-20 mins
• Unit of Enzyme Activity the amount of enzyme
  required to completely digest 1mg of DNA in 50ml
  at 37oC in 1 hour. (usually use 1 unit per mg
  DNA)
• Isoschizomer another enzyme that recognizes the
  same site.

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Lambda DNA Partial Sequence
gggcggcgac ctcgcgggtt ttcgctattt atgaaaattt
tccggtttaa ggcgtttccg ttcttcttcg tcataactta
atgtttttat ttaaaatacc ctctgaaaag aaaggaaacg
acaggtgctg aaagcgaggc tttttggcct ctgtcgtttc
ctttctctgt ttttgtccgt ggaatgaaca atggaagtca
acaaaaagca gctggctgac attttcggtg cgagtatccg
taccattcag aactggcagg aacagggaat gcccgttctg
cgaggcggtg gcaagggtaa tgaggtgctt tatgactctg
ccgccgtcat aaaatggtat gccgaaaggg atgctgaaat
tgagaacgaa aagctgcgcc gggaggttga agaactgcgg
caggccagcg aggcagatct ccagccagga actattgagt
acgaacgcca tcgacttacg cgtgcgcagg ccgacgcaca
ggaactgaag aatgccagag actccgctga agtggtggaa
accgcattct gtactttcgt gctgtcgcgg atcgcaggtg
aaattgccag tattctcgac gggctccccc tgtcggtgca
gcggcgtttt ccggaactgg aaaaccgaca tgttgatttc
ctgaaacggg atatcatcaa agccatgaac aaagcagccg
cgctggatga actgataccg gggttgctga gtgaatatat
cgaacagtca ggttaacagg ctgcggcatt ttgtccgcgc
cgggcttcgc tcactgttca ggccggagcc acagaccgcc
gttgaatggg cggatgctaa ttactatctc ccgaaagaat
ccgcatacca ggaagggcgc tgggaaacac tgccctttca
gcgggccatc atgaatgcga tgggcagcga ctacatccgt
gaggtgaatg tggtgaagtc tgcccgtgtc ggttattcca
aaatgctgct gggtgtttat gcctacttta tagagcataa
gcagcgcaac acccttatct ggttgccgac ggatggtgat
gccgagaact ttatgaaaac ccacgttgag ccgactattc
gtgatattcc gtcgctgctg
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Gel Electrophoresis
DNA fragments can be sorted by size using a gel
matrix and electrical current
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• lambda / BstEII

Concentration0.5 mg/mlRecommended Load 1  mg

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Restriction Enzyme Digestion Lab

• You will digest lambda DNA with 3 enzymes
• Eco RI, Hin dIII, and Bam HI
• What will your control be?

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• http//web.onetel.net.uk/jbwhammond/REnz1.htm

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1 2 3
1. l/Hin dIII 2. l/Eco RI 3. l/Bam HI
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• You are given a strain of E. coli bacteria which
  has 1 of 3 different plasmids. Each of the
  plasmids is a different size. The size of a piece
  of DNA (circular or linear) is measured in base
  pairs (bp). That's the number of nucleotide pairs
  which are formed between the two DNA strands.
  Take a look at the description of the three
  plasmids.

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Plasmid A
2686 bp
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Plasmid B
5186 bp
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Plasmid C
6486 bp
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• The three different plasmids were all digested
  with the restriction enzyme EcoR I and the
  fragments were separated by electrophoresis using
  an agarose gel.

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Gel
In lane M are the molecular weight markers which
are generated by digesting bacteriophage lambda
DNA with the restriction enzyme Hind III. The
numbers listed beside the fragments are the sizes
in base pairs (bp).
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• Look at each of the three plasmids and
  determine the size of the fragments which will be
  created if the plasmid is digested with EcoR I.
•  
• Plasmid A of fragments_________________
• Size of fragments________________________
•  
• Plasmid B of fragments_________________
• Size of fragments________________________
• Plasmid C of fragments_________________
• Size of fragments________________________

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• Label up the size of each of the fragments 1-6 on
  your gel.

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• In addition to the physical differences between
  the plasmids there are also differences in the
  genes they carry. These genotypic differences
  confer different phenotypic differences to the
  bacteria which carry the plasmid.
•  
• The same strain of E. coli will have different
  phenotypic characteristic depending on which of
  the three different plasmids it is carrying.
• The three different combinations will be
  designated by three label colors, Orange - Pink -
  Yellow

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• X-gal is a galactose sugar with a glycosidic
  linkage to a chromophor (colored) molecule. As
  long as the chromophor stays joined to the
  galactose, it remains colorless. In the presence
  of the enzyme beta-galactosidase (lac Z) the
  glycosidic link is hydrolyzed (broken by the
  addition of water). The free chromophor is now
  blue.
•  
• If the lac Z gene is intact colonies will be
  blue
• Colonies from the orange and pink tubes are
  white in the presence of X-gal.
• Colonies from the yellow tube are blue in the
  presence of X-gal.

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• The bacteria in the pink and yellow tubes grow
  colonies which appear dark under ultraviolet
  light.
• The bacteria in the orange tube grows colonies
  which fluoresce blue-green under UV light.

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• Using the information you have determine which
  plasmids are in the bacteria of each of the three
  different tubes.

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• Orange Tube has plasmid C
• Pink tube has plasmid B
• Yellow tube has plasmid A

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Restriction Fragment Length Polymorphism
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Pedigree Analysis
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REs and Genetic Engineering

• http//www.dnai.org/text/mediashowcase/index2.html
  ?id549

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Recombinant DNA
Scissors and glue Enzymes are used to cut and
paste DNA Procedure takes advantage of a
natural defense mechanism
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Bacterial Plasmids
Bacteria have only one chromosome
Bacteria also contain circular DNA called
plasmids
Plasmids can act as vectors carrying additional
genes
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Polylinker
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A Plasmid as a Vector
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Typical e.coli cloning Vector
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pBR322
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Kpn 1 Pst 1 uncut 1 kb
ladder 100bp ladder
pBABE lab
----
4132
----
2615 2565
872
(176)
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Electrophoresis of Dyes