Académique Documents
Professionnel Documents
Culture Documents
2006
Craig C. Mello and
Andrew Fire's
received a noble
Restriction Enzymes
2)
DNA Ligase
BIOLOGICAL ROLE OF RE
GGACGCTAGCTGATGAATTCGCATCGGATCCGAATCCGCTCTTTCAA
CCTGCGATCGACTACTTAAGCGTAGCCTAGGCTTAGGCGAGAAAGTT
Recognition Sequence
GGACGCTAGCTGATGAATTCGCATCGGATCCGAATCCGCTCTTTCAA
CCTGCGATCGACTACTTAAGCGTAGCCTAGGCTTAGGCGAGAAAGTT
Cleavage
GGACGCTAGCTGATG
CCTGCGATCGACTACTTAA
AATTCGCATCGGATCCGAATCCGCTCTTTCAA
GCGTAGCCTAGGCTTAGGCGAGAAAGTT
Sticky Ends
1)
2)
3)
Sticky Ends
DNA ligase bonds sticky ends cut with the same restriction
enzyme.
Sticky ends cut with different restriction enzymes will not bond
together.
Why?
Application Exercise
Make Recombinant
DNA Using Restriction
Enzymes
Circular DNA
Linear DNA
APPLICATION OF RESTRICTION
ENZYMES
Many possible
recombinant DNA
plasmids can be
produced, but this
was the desired
plasmid for the
experiment.
Transformation of
bacterial cells
through
electroporation.
TYPES OF RESTRICTION
ENDONUCLEASES
RESTRICTION
ENDONUCLEASES
Named for bacterial genus, species, strain, and
type
Example: EcoR1
Genus: Escherichia
Species: coli
Strain: R
Order discovered: 1
RESTRICTION ENDONUCLEASES
RECOGNIZE PALLINDROMIC
Palindrome-a word, verse, or sentence that reads
SEQUENCES
the same forwards or backwards.
RESTRICTION
ENDONUCLEASES
Recognition sites have symmetry
(palindromic)
Able was I, ere, I saw Elba
5-GGATCC-3
Bam H1 site:
3-CCTAGG-5
RESTRICTION
ENDONUCLEASES
Enzymes recognize specific 4-8 bp sequences
Some enzymes cut in a staggered fashion - sticky
ends
EcoRI
5GAATTC3
3CTTAAG5
5CAGCTG3
3GTCGAC5
Page 103
DIVERSITY OF ENZYMES
EcoRI
Esherichia coli R
G/AATTC
G/GATCC
HindIII
Haemophilus influenzae Rd
A/AGCCT
PstI
Providencia stuartii
PmeI
Psuedomonas mendocina
CTGCA/G
GTTT/AAAC
RECOGNITION SEQUENCES
EcoRI G/AATTC
BamHI G/GATCC
HindIII A/AGCCT
PstI
CTGCA/G
PmeI
GTTT/AAAC
HincII
GTY/RAC
FunII
G/AATTC
Features
Palindromic
Length
4 cutters, 6 cutters etc
Site of cleavage
Sticky ends
3 overhang
5 overhang
blunt end
Compatibility
Multiple Recognition sequence
Isoschisomers
Type II vs Type III RE
RESTRICTION MAP
http://www.phschool.com/science/bi
ology_place/biocoach/red/intro.html
http://wps.prenhall.com/wps/media/
objects/1144/1171557/17_5.html
RESTRICTION
ENDONUCLEASES
Restriction enzyme animation
http://www.dnai.org/b/index.html
Nuclease
s
Bal
31 and
exonuclease III (exonucleases), and
deoxyribonuclease I (DNase I) and
S1-nuclease (endonucleases).
(b)
(c)
(d)
Polymera
ses
POLYMERASES
Polymerase enzymes synthesise copies of
nucleic acid molecules and are used in many
genetic engineering procedures.
When describing a polymerase enzyme, the
terms DNA-dependent or RNA-dependent
may be used to indicate the type of nucleic
acid template that the enzyme uses.
Thus, a
DNA-dependent
into DNA,
an RNA-dependent DNA polymerase copies RNA
into DNA, and
a DNA-dependent RNA polymerase transcribes
DNA into RNA.
This
Major
Applications
DNA blunting by fill-in 5'-overhangs
Random-primed DNA labeling
Labeling by fill-in 5'-overhangs of dsDNA
DNA sequencing by the Sanger method
Site-specific mutagenesis of DNA with
synthetic oligonucleotides
Second strand synthesis of cDNA
DNA
Ligase
5AAGCTT3
5AAGCTT3
3TTCGAA5
3TTCGAA5
CCCGGG
AAATTT
CCCTTT
AAAGGG
BglIIA
TCTAG
ResultGGATCT
CCTAGA
GATCT
A
No longer
palindromic, so not
cut by BamHI or BglII
CONCLUSION
Group Assignment 1
organism used
The recombinant DNA product
The products application
Is the product an improvement compared to
similar non-recombinant products (explain)?
QUIZ 1
QUIZ 2
3
G