PCR Polymerase Chain Reaction Parviz Fallah Stem Cell Technology Research Centre.

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Presentation transcript:

PCR Polymerase Chain Reaction Parviz Fallah Stem Cell Technology Research Centre

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.

PCR Thermocycler

PROCEDURE …..

PCR Reagents 1X Buffer –10mM Tris-HCl, 50mM KCl MgCl 2 –1mM - 4mM (1.5mM) dNTPs –200μM Primers –100nM-1μM, 200nm (or less) for real time analysis DNA polymerase –Taq DNA polymerase is thermostable –1-4 Units (1 unit) DNA –10pg-1μg (20ng)

Polymerase Chain Reaction

PCR Melting 94 o C Temperature T i m e 5’3’ 5’

PCR Melting 94 o C Temperature T i m e 3’5’ 3’ Heat

PCR Melting 94 o C Annealing Primers 50 o C Extension 72 o C Temperature T i m e 3’5’ 3’ 5’ Melting 94 o C

PCR Melting 94 o C Melting 94 o C Annealing Primers 50 o C Extension 72 o C Temperature T i m e 30x 3’5’ 3’ Heat 5’

PCR Melting 94 o C Melting 94 o C Annealing Primers 50 o C Extension 72 o C Temperature T i m e 30x 3’5’ 3’ 5’

PCR Melting 94 o C Melting 94 o C Annealing Primers 50 o C Extension 72 o C Temperature T i m e 30x 3’5’ 3’ 5’ Heat

PCR Melting 94 o C Melting 94 o C Annealing Primers 50 o C Extension 72 o C Temperature T i m e 30x 3’5’ 3’ 5’

Fragments of defined length PCR Melting 94 o C Melting 94 o C Annealing Primers 50 o C Extension 72 o C Temperature T i m e 30x 3’5’ 3’ 5’

More Cycles = More DNA Number of cycles Size Marker

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