Transcript Polymerase chain reaction (b)

POLYMERASE CHAIN
REACTION (PCR)
AND ITS APPLICATIONS
POLYMERASE CHAIN REACTION (PCR)
•
The Polymerase Chain
Reaction (PCR) was not a
discovery, but rather an
invention
• Kary Mullis, the inventor of
PCR, was awarded the 1993
Nobel Prize in Chemistry
WHAT IS PCR?
PCR is an exponentially progressing
synthesis of the defined target DNA
sequences in vitro.
WHY “POLYMERASE”?
It is called “polymerase” because the
only enzyme used in this reaction is DNA
polymerase.
WHY “CHAIN”?
It is called “chain” because the products
of the first reaction become substrates of
the following one, and so on.
POLYMERASE CHAIN REACTION (PCR)
• PCR is a technique which is used to amplify the number
of copies of a specific region of DNA, in order to
produce enough DNA to be adequately tested.
• The purpose of a PCR is to make a huge number of
copies of a gene. As a result, it now becomes possible
to analyze and characterize DNA fragments found in
minute quantities in places like a drop of blood at a
crime scene or a cell from an extinct dinosaur.
WHAT ALL PCR CAN DO ?
• Starting with one original copy an almost infinite number
of copies can be made using PCR
• “Amplified” fragments of DNA can be sequenced, cloned,
probed or sized using electrophoresis
• Defective genes can be amplified to diagnose any number
of illnesses
• Genes from pathogens can be amplified to identify them
(i.e., HIV, Vibrio sp., Salmonella sp. etc.)
• Amplified fragments can act as genetic fingerprints
THE “REACTION” COMPONENTS
1) Target DNA - contains the sequence to be amplified.
2) Pair of Primers - oligonucleotides that define the sequence to
be amplified.
3) dNTPs - deoxynucleotidetriphosphates: DNA building blocks.
4) Thermostable DNA Polymerase - enzyme that catalyzes the
reaction
5) Mg++ ions - cofactor of the enzyme
6) Buffer solution – maintains pH and ionic strength of the
reaction solution suitable for the activity of the enzyme
DNA SAMPLE
DNA consists of four elements: A, C, G and T
DNA molecule
• Double stranded DNA strands
• Bound together by chemical forces
–Exception: single stranded DNA/RNA viruses
DNA SAMPLE
Double stranded DNA:
…….A T G G C A T A T C G……..
…….T A C C G T A T A G C……..
PRIMERS
• Two short DNA fragment that stick specifically to each of the DNA strands
at some distance of each other
• Pair of primers:• One primer binds to the 5’ end of one of the DNA strands
• The other primer binds to the 3’ end of the anti-parallel DNA strand
• Delineate the region of DNA you want amplified
• Primers
• Can be specific for:
– A certain bacterium
– Bacterial species
– Genes (e.g., toxin gene)
DNA POLYMERASE
• DNA Polymerase is the enzyme responsible for copying
the sequence starting at the primer from the single DNA
strand
• Commonly use Taq, an enzyme from the
hyperthermophilic organisms Thermus aquaticus, isolated
first at a thermal spring in Yellowstone National Park
• This enzyme is heat-tolerant  useful both because it is
thermally tolerant (survives the melting T of DNA
denaturation) which also means the process is more
specific, higher temps result in less mismatch – more
specific replication
THE REACTION
PCR tube
THERMOCYCLER
INITIATION - FORMING THE REPLICATION EYE
Origin of Replication
5’
3’
3’
5’
3’
5’
5’
3’
3’
5’
5’
5’
3’
3’
5’
3’
3’
5’
5’
3’
EXTENSION - THE REPLICATION FORK
5’
3’
3’
5’
3’
5’
5’
3’
5’
Primase
Laging Strand
Okazaki
fragment
5’
RNA
Primers
3’
Single strand
binding
proteins
5’
DNA
Polymerase
5’
3’
Helicase
Leading Strand
5’
3’
WHAT YOU NEED FOR PCR
Put into one reaction tube:
• Sample (+/- target DNA)
• Primers for the specific detection
• Nucleotides
• Enzyme
PERFORMING PCR
1.
2.
3.
4.
Put your tube in the apparatus
Let the program run (35 cycles)
If primers fit, there is amplification of target DNA
If primers do not fit, no amplification product
=> the DNA (micro-organism) was not in the sample
5.
Detect if there is PCR product
PCR THERMOCYCLER
HOW PCR WORKS
• Protocol
– Put all reagents into a PCR tube
– Break the DNA ladder down the middle to create two strands, a 5’ to 3’ strand
and a 3’ to 5’ strand
• Melting or heat denaturation
– Bind each primer to its appropriate strand
• 5’ primer to the 5’ to 3’ strand
• 3’ primer to the 3’ to 5’ strand
– Annealing
– Copy each strand
• DNA polymerase
– Extending
WHAT HAPPENS?
• Sample DNA , nucleotides, DNA primers &
thermostable DNA polymerase placed in PCR
machine.
• Strands of sample DNA separated by heating to 95oC
• Mixture cooled to 50oC - 60oC to allow primers to
bind.
• Mixture heated to 72oC for replication (optimum temp
of DNA polymerase)
• Cycle repeats many times (~8mins /cycle)
How PCR Works
• Temperature Protocol
•
•
•
•
•
•
Initial Melt: 94ºC for 2 minutes
Melt: 94ºC for 30 seconds
Anneal: 55ºC for 30 seconds
Extend: 72ºC for 1 minute
Final Extension: 72ºC for 6 minutes
Hold: 4ºC
30-35
cycles
Denature (heat to 95oC)
Lower temperature to 56oC
Anneal with primers
Increase temperature to 72oC
DNA polymerase + dNTPs
POLYMERASE CHAIN REACTION
Temperature
PCR
100
Melting
94 oC
50
0
T i m e
3’
5’
5’
3’
Temperature
PCR
100
Melting
94 oC
50
0
T i m e
3’
5’
Heat
5’
3’
Temperature
PCR
100
Melting
94 oC
50
0
Melting
94 oC
Extension
Annealing
72 oC
Primers
50 oC
T i m e
3’
5’
5’
5’
5’
3’
Temperature
PCR
100
Melting
94 oC
50
0
Melting
94 oC
Extension
Annealing
72 oC
Primers
50 oC
T i m e
3’
5’
Heat
5’
5’
Heat
5’
5’
3’
30x
Temperature
PCR
100
Melting
94 oC
50
0
Melting
94 oC
Extension
Annealing
72 oC
Primers
50 oC
T i m e
3’
5’
5’
5’
5’
5’
5’
5’
5’
3’
30x
Temperature
PCR
100
50
0
3’
5’
5’
Melting
94 oC
Melting
94 oC
Extension
Annealing
72 oC
Primers
50 oC
T i m e
5’
5’
5’
3’
Heat
5’
5’
Heat
5’
30x
Temperature
PCR
100
50
0
3’
5’
5’
Melting
94 oC
Extension
Annealing
72 oC
Primers
50 oC
T i m e
5’
5’
5’
Melting
94 oC
5’
3’
5’
5’
5’
5’
5’
5’
30x
Temperature
PCR
100
Melting
94 oC
50
0
3’
5’
5’
T i m e
5’
5’
5’
Melting
94 oC
Extension
Annealing
72 oC
Primers
50 oC
5’
3’
Fragments of
defined length
5’
5’
5’
5’
5’
5’
30x
WHAT YOU NEED FOR PCR
• Apparatus to perform about 35 cycles of a three
temperature procedure
• 95 °C (denaturation of DNA)
• 50-60 °C (annealing of primers)
• 72 °C (extension of the primers)
Logaritmic multiplication
Logaritmic multiplication
Logaritmic multiplication
Logaritmic multiplication
ADVANTAGES OF PCR
• Quick
• Reliable
• Sensitive
• Relatively easy
• Specific
DISADVANTAGES OF PCR
• Need for equipment
• Taq polymerase is expensive
• Contamination
• False reactions
• Internal control
• Cross-reaction
• Enrichment steps in (contaminated) samples
• Capacity building needed
• Unspecific amplification
TYPES OF THE PCR
• Colony PCR
• Nested PCR
• Multiplex PCR
• AFLP PCR
• Hot Start PCR
• In Situ PCR
• Inverse PCR
• Asymmetric PCR
• Long PCR
• Long Accurate PCR
• Reverse Transcriptase PCR
• Allele specific PCR
• Real time PCR
APPLICATIONS OF PCR
• Classification of
organisms
• Genotyping
• Molecular archaeology
• Mutagenesis
• Mutation detection
• Sequencing
• Cancer research
• Detection of
pathogens
• DNA fingerprinting
• Drug discovery
• Genetic matching
• Genetic engineering
• Pre-natal diagnosis
APPLICATIONS OF PCR
Basic Research
Applied Research
• Mutation screening
• Drug discovery
• Classification of organisms
• Genotyping
• Molecular Archaeology
• Molecular Epidemiology
• Molecular Ecology
• Bioinformatics
• Genomic cloning
• Site-directed mutagenesis
• Gene expression studies
• Genetic matching
• Detection of pathogens
• Pre-natal diagnosis
• DNA fingerprinting
• Gene therapy
APPLICATIONS OF PCR
Molecular Identification Sequencing
Genetic Engineering
• Molecular Archaeology
• Bioinformatics
• Site-directed mutagenesis
• Molecular Epidemiology • Genomic cloning
• Gene expression studies
• Molecular Ecology
• Human Genome Project
• DNA fingerprinting
• Classification of organisms
• Genotyping
• Pre-natal diagnosis
• Mutation screening
• Drug discovery
• Genetic matching
• Detection of pathogens
MOLECULAR IDENTIFICATION:
SUMMARY
blood, fluid, semen,
hair root, saliva
68,719,476,736 copies
Gel Analysis,
Restriction
Digestion,
Sequencing
CONCLUSION
The speed and ease of use, sensitivity, specificity and
robustness of PCR has revolutionised molecular
biology and made PCR the most widely used and
powerful technique with great spectrum of research
and diagnostic applications.
THANK YOU
-PHARMA STREET