Post on 20-Dec-2015
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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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1. Denaturation of DNA to single strands
2. Annealing of primers to DNA
3. Extension by polymerase
4. Repeat 30-35 times
The basic protocol
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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
Target DNA
5’ 3’
3’ 5’
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PCR Primers
Identify gene sequence in DNA and mRNA sequence
select primers to use for PCR
CCAAGGTTGCACCATGGACAGGTGGCAGAAGTGGGATCTCATCCAAGAGTTACATCCCTGCCAAGGTTGCACCATGGACAGGTGGCAGAAGTGGGATCTCATCCAAGAGTTACATCCCTGCCTCTCACTTCCTCTCCTTACAGCCAAGGCTGATGACATTGTTGGCCCTGTGACGCATGACCTCTCACTTCCTCTCCTTACAGCCAAGGCTGATGACATTGTTGGCCCTGTGACGCATGAAATCTTTGAGAACAACGTCGTCCACTTGATGTGGCAGGAGCCGAAGGAGCCCAATGGTCTAATCTTTGAGAACAACGTCGTCCACTTGATGTGGCAGGAGCCGAAGGAGCCCAATGGTCTGATCGTGCTGTATGAAGTGAGTTATCGGCGATATGGTGATGAGGTAAGGCCCTTGACTCTGATCGTGCTGTATGAAGTGAGTTATCGGCGATATGGTGATGAGGTAAGGCCCTTGACTCTTGGGCATGCCCCTGCACACTTCAGCATGCCCCTTCAGAGTTGCACTTGGTACCTCCTTC
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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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1. Denaturation of DNA to single strands
2. Annealing of primers to DNA
3. Extension by polymerase
4. Repeat 30-35 times
The basic protocol
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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
Target DNA5’ 3’
3’ 5’
primers
AB Free
nucleotides
Taq DNApolymerase
Mg2+
Mg2+
Mg2+
Mg2+
Mg2+
Mg2+
Buffercontainingmagnesium
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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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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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Sequencing
A T A C T A T G A A A A A G G A G A A A A A
A T A C T A T G A A A A A N G A G A A A A A
A T A C T A T G A A A A A C G A G A A A A AHomozygous
Heterozygous
Wild type