Transcript Mechanism of Surface Stress due to DNA strands on Gold

Polymerase Chain Reaction
(PCR2)
fourth lecture Zoology department
2007
Dr.Maha H. Daghestani
PCR
I.
Definition of PCR
II. Requirements for PCR
III.PCR Process
A. Denaturing Stage
B. Annealing Stage
C. Extending Stage
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Polymerase Chain Reaction
(PCR
• One of the most powerful tools in molecular
biology
• Invented by Kary Mullis in 1983, resulting in
his Nobel Prize in Chemistry
• In essence, this process acts as a “copying
machine” for DNA
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PCR What is it?
The Polymerase Chain Reaction (PCR) is
an
in vitro method to amplify a specific
region of DNA.
PCR is extremely sensitive, with the
capability of amplifying minuscule
quantities of DNA.
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Advances due to PCR
-Study DNA sequencing
-Compare forensic samples
-Identify remains
• Disease diagnosis
• Paternity determination
-Unite living members of a separated family
-Determine tissue type for transplants
-Amplify cDNA fragments from the reverse
transcription products of mRNA (RT-PCR).
-Determine the SNPs and mutation in genes
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Tools for PCR
•
•
•
•
A small amount of DNA
DNA polymerace enzymes
Nucleotides
Primers
– Two different kind
– Usually about 20 nucleotides
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REQUIREMENTS
1. DNA sample
· very small amounts (ng or sometimes
less) if DNA is in good shape
· may be able to use DNA from only one
cell
· only a few molecules must be intact
samples with larger numbers of
molecules can be in poor shape or
degraded
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PCR REQUIREMENTS (cont’d.)
• Two primers
· flank region you are interested in
· you must know the sequence of the flanking regions so you can
order appropriate primers
• Heat stable polymerase
• Four dNTPs
• Reaction buffer (Tris, ammonium ions (and/or potassium ions),
magnesium ions, bovine serum albumin)
• Thermocycler (standard, but optional)
· changes temperature very rapidly for each cycle (denature,
anneal, extend)
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PCR METHOD
There are four basic steps in PCR
1. Denaturing Stage
2. Annealing Stage
3. Extending Stage
4. Replication
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The basic protocol
1. Denaturation of DNA to single
strands
2. Annealing of primers to DNA
3. Extension by polymerase
4. Repeat 30-35 times
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Melting Point Temperature
• Denaturation
– The more there is G or C, the higher
Tm
– The longer the primers, the higer Tm
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Primers
• Usually about 20 nucleotides in length
• Designed to flank the region to be amplified
• Melting point determined by G-C and A-T content
– Tm = 4 (G+C) + 2 (A+T)
– Ex: a primer with 10 G/C and 10 A/T would have a
Tm of 60oC
4(10) + 2(10)=60oC
5’
3’
3’
5’
Target DNA
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PCR Primers
• Go to Genbank, look up sequence of gene of interest
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PCR Primers
Identify gene sequence in DNA and mRNA sequence
select primers to use for PCR
CCAAGGTTGCACCATGGACAGGTGGCAGAAGTGGGATCTCATCCAAGAGT
TACATCCCTGCCAAGGTTGCACCATGGACAGGTGGCAGAAGTGGGATCTC
ATCCAAGAGTTACATCCCTGCCTCTCACTTCCTCTCCTTACAGCCAAGGCT
GATGACATTGTTGGCCCTGTGACGCATGACCTCTCACTTCCTCTCCTTACA
GCCAAGGCTGATGACATTGTTGGCCCTGTGACGCATGAAATCTTTGAGAA
CAACGTCGTCCACTTGATGTGGCAGGAGCCGAAGGAGCCCAATGGTCTAA
TCTTTGAGAACAACGTCGTCCACTTGATGTGGCAGGAGCCGAAGGAGCCC
AATGGTCTGATCGTGCTGTATGAAGTGAGTTATCGGCGATATGGTGATGAG
GTAAGGCCCTTGACTCTGATCGTGCTGTATGAAGTGAGTTATCGGCGATAT
GGTGATGAGGTAAGGCCCTTGACTCTTGGGCATGCCCCTGCACACTTCAG
CATGCCCCTTCAGAGTTGCACTTGGTACCTCCTTC
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Primers
forward
5’
3’
Target DNA
3’
5’
reverse
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Problems with primers
• ”hairpin” structure
– If 3’side is included in
structure, the primer
doesn’t work
• Primer dimers
– Only harm if the
binding is formed at
the 3’ends
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The basic protocol
1.
2.
3.
4.
Denaturation of DNA to single strands
Annealing of primers to DNA
Extension by polymerase
Repeat 30-35 times
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What time does it take?
• Denaturation: 30 - 60 sec
• Annealing: 30 - 60 sec
• Doupling: 30 - 60 sec
• 25 - 35 cycles only (otherwise enzyme decay
causes artifacts)
• 72oC for 5 min at end to allow complete
elongation of all product DNA
Altogether: 7 min ( 8,5 min) * 25 (35) = 3h-5h
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The basic protocol—what’s in the
tube
5’
3’
Target DNA
3’
5’
A
B
Free
nucleotides
primers
Taq DNA
polymerase
2+
Mg2+ Mg2+
Mg
Mg2+
Mg2+
Mg2+
Buffer
containing
magnesium
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The basic protocol-denaturation
5’
3’
Target DNA
3’
5’
95oC
5’
3’
3’
5’
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The basic protocol--annealing
5’
3’
Target DNA
3’
5’
A
B
primers
~55oC
5’
3’
B
5’
3’
5’
A
5’
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The basic protocol--extension
5’
3’
Target DNA
3’
5’
72oC
5’
3’
Taq polymerase
5’
5’
3’
5’
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The basic protocol--extension
5’
3’
Target DNA
3’
5’
72oC
5’
3’
5’
5’
3’
5’
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One
One billion in about 2 hours!
• At the end of each cycle, the amount
of DNA has doubled
• By the end of 30 cycles, you will have
about 1 billion molecules from the
original one you started with!!
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What makes it work?
• Taq polymerase!
• Most enzymes would be killed at 95oC
• Taq was isolated from Thermus aquaticus, a
bacteria that grows in hot springs (~75oC)
• This organism’s enzymes have adapted to the
high temperature, so they can survive cycling
through the high temperatures
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The PCR machine
• Very rapidly changes the
temperature between the
various stages of the
PCR process
• Programmable for use
with many different
cycling parameters
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Has It Worked?
•
•
•
•
•
Check a sample by gel electrophoresis.
Is the product the size that you expected?
Is there more than one band?
Is any band the correct size?
May need to optimize the reaction conditions.
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Optimising the PCR
Reaction
•
•
•
•
•
•
Annealing temperature of the primers.
The concentration of Mg2+ in the reaction.
The extension time.
(The denaturing and annealing times.)
(The extension temperature.)
(The amount of template and polymerase
— “more is less”.)
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Theoretical Basis of Agarose
Gel Electrophoresis
 Agarose is a polysaccharide from marine alage that
is used in a matrix to separate DNA molecules
 Because DNA ia a (-) charged molecule when
subjected to an electric current it will migrate
towards a (+) pole
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Pouring an Agarose Gel
1
2
4
5
7
8
3
6
9
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Assessing the Integrity of
DNA
High Quality Genomic DNA
>95% DNA will be of high molecular
weight, migrating as intact band near
the top of the gel
Very little evidence of smaller
fragments indicated by a smear of
many different sized DNA fragments
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Gene sequencing
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Sequencing
Wild type
Heterozygous
A
A
G
G
A
A
G
G
A
C
A
T
T
T
G
T
T
T
C
A
A
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Sequencing
Wild type
A T A C T A T G A A A A A N G A G A A A A A
Heterozygous
Homozygous
A T A C T A T G A A A A A C G A G A A A A A
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Thank you
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