Download Restriction Enzymes

Restriction Enzymes
• Restriction Enzymes scan the DNA sequence
• Find a very specific set of nucleotides
• Make a specific cut
Palindromes in DNA sequences
5
’
3’
3’
5
’
Genetic palindromes
are similar to verbal
palindromes. A
palindromic sequence
in DNA is one in which
the 5’ to 3’ base pair
sequence is identical
on both strands.
Restriction enzymes recognize and
make a cut within specific
palindromic sequences, known as
restriction sites, in the DNA. This is
usually a 4- or 6 base pair sequence.
Restriction Endonuclease Types
Type I- multi-subunit, both endonuclease and
methylase activities, cleave at random up to
1000 bp from recognition sequence
Type II- most single subunit, cleave DNA within
recognition sequence
Type III- multi-subunit, endonuclease and
methylase about 25 bp from recognition
sequence
Hae III
HaeIII is a restriction enzyme that
searches the DNA molecule until it finds
this sequence of four nitrogen bases.
5’ TGACGGGTTCGAGGCCAG 3’
3’ ACTGCCCAAGGTCCGGTC 5’
Once the recognition site is found Hae III will
cleave the DNA at that site
5’ TGACGGGTTCGAGGCCAG 3’
3’ ACTGCCCAAGGTCCGGTC 5’
These cuts produce
“blunt ends”
5’ TGACGGGTTCGAGG
3’ ACTGCCCAAGGTCC
CCAG 3’
GGTC 5’
The names for restriction enzymes come from:
• the type of bacteria in which the enzyme is found
• the order in which the restriction enzyme was identified
and isolated.
EcoRI for example
R strain of E.coli bacteria
I as it is was the first E. coli restriction enzyme to
be discovered.
“blunt ends” and “sticky ends”
Hae III produced a “blunt end”?
EcoRI makes a staggered cut and produces a
“sticky end”
5’ GAATTC 3’
3’ CTTAAG 5’
5’ GAATTC 3’
3’ CTTAAG 5’
5’ G
AATTC 3’
3’ CTTAA
G 5’
blunt end
sticky end
More examples of restriction sites of restriction
enzymes with their cut sites
Hind III: 5’ AAGCTT 3’
3’ TTCGAA 5’
Bam HI: 5’ GGATCC 3’
3’ CCTAGG 5’
Alu I: 5’ AGCT 3’
3’ TCGA 5’
Separating Restriction Fragments, I
Separating Restriction Fragments, II
Gene Cloning
• What is gene cloning? How does it
differ from cloning an entire
organism?
• Why is gene cloning done?
• How is gene cloning accomplished ?
• What is a DNA ‘Library’?
What is DNA cloning?
• When DNA is
extracted from an
organism, all its
genes are obtained
• In gene (DNA)
cloning a particular
gene is copied
(cloned)
Why Clone DNA?
• A particular gene can be isolated and its
nucleotide sequence determined
• Control sequences of DNA can be identified &
analyzed
• Protein/enzyme/RNA function can be
investigated
• Mutations can be identified, e.g. gene defects
related to specific diseases
• Organisms can be ‘engineered’ for specific
purposes, e.g. insulin production, insect
resistance, etc.
How is DNA cloned?, I
Blood sample
• DNA is extractedhere from blood
• Restriction enzymes,
e.g. EcoR I, Hind III,
etc., cut the DNA into
small pieces
• Different DNA pieces
cut with the same
enzyme can join, or
recombine.
DNA
Restriction enzymes
The action of a restriction enzyme, EcoR I
Note: EcoR I gives a ‘sticky’ end
DNA Cloning, II
• Bacterial plasmids
(small circular DNA
additional to a
bacteria’s regular DNA)
are cut with the same
restriction enzyme
• A chunk of DNA can
thus be inserted into the
plasmid DNA to form a
“recombinant”
DNA cloning, III
• The recombinant
plasmids are then
mixed with bacteria
which have been
treated to make them
“competent”, or
capable of taking in
the plasmids
• This insertion is called
transformation
DNA Cloning, IV
• The plasmids have
naturally occurring
genes for antibiotic
resistance
• Bacteria containing
plasmids with these
genes will grow on a
medium containing the
antibiotic- the others
die, so only transformed
bacteria survive
DNA Cloning, V
• The transformed
bacterial cells form
colonies on the medium
• Each cell in a given
colony has the same
plasmid (& the same
DNA)
• Cells in different
colonies have different
plasmids (& different
DNA fragments)
Screening, I
Screening can involve:
1. Phenotypic screeningthe protein encoded
by the gene changes
the color of the colony
2. Using antibodies that
recognize the protein
produced by a
particular gene
Screening, II
3. Detecting the DNA sequence of a cloned
gene with a probe (DNA hybridization)
Polymerase Chain
Reaction
PCR
PCR
• invented by Karry B.
Mullis (1983, Nobel
Prize 1993)
• patent sold by Cetus
corp. to La Roche for
$300 million
• depends on thermoresistant DNA
polymerase (e.g. Taq
polymerase) and a
thermal cycler
Heat-stable DNA polymerase
• Taq DNA polymerase
was isolated from the
bacterium Thermus
aquaticus.
• Taq polymerase is stable
at the high temperatures
(~95oC) used for
denaturing DNA.
Hot springs at Yellowstone
National Park, Wyoming.
DNA polymerase requirements
•
•
•
•
template
primer
nucleotides
regulated pH, salt concentration,
cofactors
Steps in DNA replication
1) template denatured
2) primers anneal
3) new strand elongation
Steps in a PCR cycle
1) template denatured:
94 C, 30 sec
2) primers anneal
45-72 C, depending on primer sequence
30 sec – 1 min
3) new strand elongation
72 C depending on the type of polymerase
1 min for 1000 nucleotides of amplified sequence
Number of specific DNA molecule copies grows exponentially
with each PCR cycle. Usually run 20-40 cycles to get enough
DNA for most applications (If you start with 2 molecules, after
30 cycles you will have more than a billion)
PCR Process
• 25-30 cycles
• 2 minute cycles
• DNA thermal
cycler
Template denatured
Annealing primers
New strand elongation
Uses for PCR
• Research
– Gene cloning
– Real-time PCR
– DNA sequencing
• Clinical
– DNA fingerprinting
• Crime scene analysis
• Paternity testing
• Archeological finds
– Genetically inherited
diseases
DNA Sequencing
Chain termination method (Sanger Method), sequence of
single stranded DNA is determined by enzymatic synthesis
of complementary strands which terminate at specific
nucleotide positions
Chemical degradation method (Maxam-Gilbert Method),
sequence of a double stranded DNA molecule is determined
by chemical treatment that cuts at specific nucleotide
positions
Dideoxynucleotide (ddNTP)
http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=hmg.figgrp.60
http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=hmg.figgrp.605
http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=hmg.figgrp.605
http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=genomes.figgrp.647
http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=hmg.figgrp.60
Costs and time for sequencing a
human genome (3.2 billion bp)