Download Polymerase chain reaction

POLYMERASE CHAIN REACTION
(PCR)
PCR is an exponentially progressing synthesis of the defined target DNA sequences in
vitro.
It is called “polymerase” because the only enzyme used in this reaction is DNA
polymerase.
It is called “chain” because the products of the first reaction become substrates of the
following one, and so on.
 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
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 thehyperthermophilic organisms
Thermusaquaticus, 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
Requirement for pcr
 Sample (+/- target DNA)
 Primers for the specific detection
 Nucleotides
 Enzyme
PERFORMING PCR
1. Put your tube in the apparatus
2. Let the program run (35 cycles)
3. If primers fit, there is amplification of target DNA
4. If primers do not fit, no amplification product
=>the DNA (micro-organism) was not in the sample
1. Detect if there is PCR product
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 (30-35 cycles)
 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
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)
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
Templates for PRC
Dried blood, semen stains, vaginal swabs, single hair, fingernail scrapings, insects in
amber, egyptian mummies, buccal swab, tooth brushes.
APPLICATIONS OF PCR
=Basic Research
• Mutation screening
• Drug discovery
• Classification of organisms
• Genotyping
• Molecular Archaeology
• Molecular Epidemiology
• Molecular Ecology
• Bioinformatics
• Genomic cloning
• Site-directed mutagenesis
• Gene expression studies
=Applied Research
• Genetic matching
• Detection of pathogens
• Pre-natal diagnosis
• DNA fingerprinting
• Gene therapy
APPLICATIONS OF PCR
=Molecular Identification
• Molecular Archaeology
• Molecular Epidemiology
• Molecular Ecology
• DNA fingerprinting
• Classification of organisms
• Genotyping
• Pre-natal diagnosis
• Mutation screening
• Drug discovery
• Genetic matching
• Detection of pathogens
=Sequencing
• Bioinformatics
• Genomic cloning
• Human Genome Project
=Genetic Engineering
• Site-directed mutagenesis
•
Gene expression studies