Polymerase Chain Reaction (PCR2) fourth lecture Zoology department 2007 Dr.Maha H. Daghestani.

Polymerase Chain Reaction(PCR2)

fourth lecture Zoology department 2007

Dr.Maha H. Daghestani

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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• 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

CCAAGGTTGCACCATGGACAGGTGGCAGAAGTGGGATCTCATCCAAGAGTTACATCCCTGCCAAGGTTGCACCATGGACAGGTGGCAGAAGTGGGATCTCATCCAAGAGTTACATCCCTGCCTCTCACTTCCTCTCCTTACAGCCAAGGCTGATGACATTGTTGGCCCTGTGACGCATGACCTCTCACTTCCTCTCCTTACAGCCAAGGCTGATGACATTGTTGGCCCTGTGACGCATGAAATCTTTGAGAACAACGTCGTCCACTTGATGTGGCAGGAGCCGAAGGAGCCCAATGGTCTAATCTTTGAGAACAACGTCGTCCACTTGATGTGGCAGGAGCCGAAGGAGCCCAATGGTCTGATCGTGCTGTATGAAGTGAGTTATCGGCGATATGGTGATGAGGTAAGGCCCTTGACTCTGATCGTGCTGTATGAAGTGAGTTATCGGCGATATGGTGATGAGGTAAGGCCCTTGACTCTTGGGCATGCCCCTGCACACTTCAGCATGCCCCTTCAGAGTTGCACTTGGTACCTCCTTC

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Primers

Target DNA5’ 3’

3’ 5’

forward

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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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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The basic protocol--denaturation

Target DNA

95oC

5’ 3’

3’ 5’

5’ 3’

3’ 5’

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The basic protocol--annealing

~55oC

5’ 3’

3’ 5’

5’ 3’

3’ 5’

5’5’

Target DNA

A

B

primers

AB

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The basic protocol--extension

72oC

5’ 3’

3’ 5’

5’ 3’

3’ 5’

5’5’

Target DNA

Taq polymerase

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The basic protocol--extension

72oC

5’ 3’

3’ 5’

5’ 3’

3’ 5’

5’5’

Target DNA

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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 3

4 5 6

7 8 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

A A G G A A G G A C A T T T G T T T C A A

Heterozygous

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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

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