PCR Polymerase Chain Reaction Revised

The Nobel Prize in Chemistry 1993 Kary B. Mullis "for contributions to the developments of methods within DNA-based chemistry" "for his invention of the polymerase chain reaction (PCR) method”

Taq polymerase

Thermus aquaticus

Thermal Cycler

Materials of PCR Materials of PCR target DNA target DNA Taq DNA polymerase Taq DNA polymerase 2 Primers 2 Primers ~20 nucleotides in length ~20 nucleotides in length Forward and reverse Forward and reverse the four DNTP’S the four DNTP’S Adenine Adenine Thymine Thymine Cytosine Cytosine Guanine Guanine cofactor MgCl 2. cofactor MgCl 2.

Locate the Target Sequence Scientists determine which GENE they are interested in studying Scientists determine which GENE they are interested in studying Locate Primers Upstream and Down stream of gene Locate Primers Upstream and Down stream of gene

Step 1 Denaturing 60 94°C

Step 2 Annealing 60 60°C Forward and Reverse Primers

Taq Polymerase Binds

Step 3 Extension 2 minute at 72°C DNTP’s

PCR Flash Animation

The exponential amplification of the gene in PCR.

Amplification

Cycle 3 Obtain two copies of target DNA Obtain two copies of target DNA

Cycle 30 1,073,741,764 target copies 1,073,741,764 target copies

Is there a gene copied during PCR and is it the right size ?

Applications of PCR quick, reliable method for detecting all manner of mutations associated with genetic disease - from insertions, to deletions, to point mutations. quick, reliable method for detecting all manner of mutations associated with genetic disease - from insertions, to deletions, to point mutations. Duchene muscular dystrophy Duchene muscular dystrophy Detect unwanted Genetic material Detect unwanted Genetic material Bacterial or viral infection Bacterial or viral infection HIV infection HIV infection Amplify degraded DNA samples Amplify degraded DNA samples Egyptian mummy Egyptian mummy Termite in amber Termite in amber

PCR animation

On-Line Sources Access Excellence. “Polymerase Chain Reaction (PCR)” Oct Access Excellence. “Polymerase Chain Reaction - From Simple Ideas” Oct Access Excellence. “Polymerase Chain Reaction - Xeroxing DNA ” Oct Cold Spring Harbor Laboratory.1994 Dolan DNA Learning Center. 18 Oct Oct Montpetit, Diane. “Electron microscopy” 17Nov Agriculture and Agri-Food Canada. 19 Oct Nobelprize.org. The Nobel Prize in Chemistry Nov Nobelprize.org. The PCR Method - a DNA Copying Machine. 19June Vierstraete, Andy. “Principle of the PCR” University of Ghent.19 Oct Ward, Laura. “Food for thought” 2004 LABORATORY NEWS magazine. 19 Oct

Book Sources Micklos, David, Greg Freyer and David Crotty. DNA Science a First Course Micklos, David, Greg Freyer and David Crotty. DNA Science a First Course. New York:Cold Spring Habor Laboratory Press, 2003.