What is PCR? : Why “Polymerase”?

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Research Techniques Made Simple: Polymerase Chain Reaction

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Presentation transcript:

What is PCR? : Why “Polymerase”? It is called “polymerase” because the only enzyme used in this reaction is DNA polymerase.

What is PCR? : Why “Chain”? It is called “chain” because the products of the first reaction become substrates of the following one, and so on.

What is PCR? : 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

The Reaction PCR tube THERMOCYCLER

Lower temperature to 56oC Anneal with primers Denature (heat to 95oC) Lower temperature to 56oC Anneal with primers Increase temperature to 72oC DNA polymerase + dNTPs

Applications of PCR Detection of pathogens Classification of organisms DNA fingerprinting Drug discovery Genetic matching Genetic engineering Pre-natal diagnosis Classification of organisms Genotyping Mutagenesis Mutation detection Sequencing Cancer research

MOLECULAR IDENTIFICATION:

Detection Of Pathogens Molecular Identification: Detection Of Pathogens

Detection Of Pathogens Molecular Identification: Detection Of Pathogens Sensitivity of detection of PCR-amplified M. tuberculosis DNA. (Kaul et al.1994)

Summary blood, chorionic villus, amniotic fluid, semen, hair root, saliva 68,719,476,736 copies Gel Analysis, Restriction Digestion, Sequencing

Conclusion The speed and ease of use, sensitivity, specificity of PCR essential tool of molecular biology and made PCR the most widely used and powerful technique with great spectrum of research and diagnostic applications.