Title: Restriction Enzymes
1Restriction Enzymes
2Bacteriophage Life Cycle
1.
2.
6.
3.
5.
4.
3Bacteriophage Lambda (l)
Capsid is icosahedral (20 faces) - Genome is
48,502 base pairs
4Bacteriophage Infection
T4 Phage
T4 phage infecting a bacterial cell
5Bacteria 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.
6REs Disable Viral DNA
7DNA Methylation Protects Bacterial DNA
Methyl groups (CH3) bind to the DNA and prevent
the restriction enzyme from attacking the cells
own DNA.
8Restriction Enzyme Binding to DNA
9Eco RI
- G AATTC
- CTTAA G
- E genus Escherichia
- co species coli
- R strain RY13
- I first enzyme isolated
10Bam HI
- A AGCTT
- TTCGA A
- B genus Bacillus
- am species amyloliquefaciens
- H strain H
- I first enzyme isolated
11HindIII
- G GATCC
- CCTAG G
- H genus Haemophilus
- In species influenzae
- d strain Rd
- III third enzyme isolated
12Mechanism of Action
ATP is required and is usually included in the
buffer system.
13Some REs Produce Sticky Ends
14Types of Cuts
Blunt Sticky ends
15Digestion 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.
16Lambda 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
17Gel Electrophoresis
DNA fragments can be sorted by size using a gel
matrix and electrical current
18Concentration0.5 mg/mlRecommended Load 1Â mg
19Restriction Enzyme Digestion Lab
- You will digest lambda DNA with 3 enzymes
- Eco RI, Hin dIII, and Bam HI
- What will your control be?
20(No Transcript)
21- http//web.onetel.net.uk/jbwhammond/REnz1.htm
22(No Transcript)
23 1 2 3
1. l/Hin dIII 2. l/Eco RI 3. l/Bam HI
24- 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.
25Plasmid A
2686 bp
26Plasmid B
5186 bp
27Plasmid C
6486 bp
28- The three different plasmids were all digested
with the restriction enzyme EcoR I and the
fragments were separated by electrophoresis using
an agarose gel.
29Gel
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).
30- 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. - Â
- Plasmid A of fragments_________________
- Size of fragments________________________
- Â
- Plasmid B of fragments_________________
- Size of fragments________________________
- Plasmid C of fragments_________________
- Size of fragments________________________
31- Label up the size of each of the fragments 1-6 on
your gel.
32- 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. - Â
- 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
33- 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. - Â
- 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.
34- 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.
35- Using the information you have determine which
plasmids are in the bacteria of each of the three
different tubes.
36- Orange Tube has plasmid C
- Pink tube has plasmid B
- Yellow tube has plasmid A
37Restriction Fragment Length Polymorphism
38Pedigree Analysis
39REs and Genetic Engineering
- http//www.dnai.org/text/mediashowcase/index2.html
?id549
40Recombinant DNA
Scissors and glue Enzymes are used to cut and
paste DNA Procedure takes advantage of a
natural defense mechanism
41Bacterial Plasmids
Bacteria have only one chromosome
Bacteria also contain circular DNA called
plasmids
Plasmids can act as vectors carrying additional
genes
42Polylinker
43A Plasmid as a Vector
44Typical e.coli cloning Vector
45pBR322
46Kpn 1 Pst 1 uncut 1 kb
ladder 100bp ladder
pBABE lab
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4132
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2615 2565
872
(176)
47Electrophoresis of Dyes