the polymerase chain reaction (pcr)microbiology.free.fr/presentations/pcr.pdfcomponents of...
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The Polymerase Chain Reaction(PCR)
Prepared by: H.M.Naimi
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Purpose
The PCR is a powerful technique that is capable of amplifying as little as single molecule of DNA in to a large amount by repeating a simple three step process:
Denaturation (95)
Annealing (Priming)(50-60)
Synthesis (72)
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PCR is frequently employed to detect pathogens in laboratory
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the PCR is done in an equipment namedThermo cycler
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Replication of DNA molecule
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Components of Polymerase chainreaction
Oligonucleotides: used for priming and should be at least 20-24 nucleotides in length.
Standard Buffer for PCR
Taq DNA polymerase: this enzyme purified from thermus aquaticus(a heat stable bacterium) and a genetically engineered form of the enzyme synsethized in E.coli (AmpliTaq) and have 5’----3’ polymerization activity.
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Deoxyribonucleoside triphosphate:dNTPs
Target sequence: DNA containing the target sequences can be added to the PCR mixture in single or double stranded form.
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Reaction:
Template 1ML Primer 1 2.5ML Primer 2 2.5ML dNTPs 4ML Taq enzyme 0.5-1ML Buffer 5ML H2O QSP 50MLMineral oil 1Drop
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Advantages
PCR has high sensitivity due to exponential amplification of template DNA
PCR is highly specific due to the specificity of primer annealing
The PCR techinque can be completed in one working day, providing rapid results
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Disadvantages
Minute mounts of contamination can lead to false positive results
The PCR techinque is expensive
Inhibitors of the PCR reaction can lead to false negative results
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Fluorogenic 5’ Nuclease PCR
Real Time PCR
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purpose
Sensitive and specific assay capable of quantitating PCR products.
Gaining popularity in diagnostic laboratories.
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method
DNA is isolated from the sample
Denaturation of DNA
Hybridization probes
Reporter fluorescent dye ant 5’ and quencher dye ant 3’
Annealing of the oligonucleotides primers
5’nuclease activity and synthesis of new DNA
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Advantages
Very sensitive and specific
The assay is quantitative
Rapid turn around time
No post PCR processing ( eg gel electrophoresis) is required
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Disadvantages
Cross contamination can easily lead to false positive results.
The technique is expensive and requires specialized instrumentation.