PCR Polymerase Chain Reaction Revised 10-31-2006.
22
PCR PCR Polymerase Polymerase Chain Reaction Chain Reaction Revised 10-31-2006
-
Upload
marsha-brown -
Category
Documents
-
view
217 -
download
0
Transcript of PCR Polymerase Chain Reaction Revised 10-31-2006.
PCRPCR
Polymerase Polymerase Chain ReactionChain Reaction
Revised 10-31-2006
The Nobel Prize in Chemistry 1993The Nobel Prize in Chemistry 1993
Kary B. MullisKary B. Mullis
"for contributions to the developments "for contributions to the developments of methods within DNA-based of methods within DNA-based chemistry" "for his invention of the chemistry" "for his invention of the polymerase chain reaction (PCR) polymerase chain reaction (PCR) method”method”
Taq polymeraseTaq polymerase
Thermus aquaticusThermus aquaticus
Thermal CyclerThermal Cycler
Materials of PCR target DNAtarget DNA Taq DNA polymeraseTaq DNA polymerase 2 Primers 2 Primers
~20 nucleotides in length~20 nucleotides in length Forward and reverseForward and reverse
the four DNTP’S the four DNTP’S AdenineAdenineThymineThymineCytosineCytosineGuanineGuanine
cofactor MgClcofactor MgCl22..
Locate the Target SequenceLocate the Target Sequence
Scientists determine which GENE they Scientists determine which GENE they are interested in studyingare interested in studying
Locate Primers Upstream and Down Locate Primers Upstream and Down stream of genestream of gene
Step 1 DenaturingStep 1 Denaturing
60 seconds @ 94°C
Step 2 AnnealingStep 2 Annealing
60 seconds @ 60°CForward and Reverse Primers
Taq Polymerase BindsTaq Polymerase Binds
Step 3 ExtensionStep 3 Extension
2 minute at 72°CDNTP’s
PCR Flash AnimationPCR Flash Animation
The exponential amplification of the The exponential amplification of the gene in PCR. gene in PCR.
Amplification
Cycle 3Cycle 3
Obtain two copies Obtain two copies of target DNAof target DNA
Cycle 30Cycle 30 1,073,741,7641,073,741,764
target copiestarget copies
Is there a gene copied during PCR Is there a gene copied during PCR and is it the right size ? and is it the right size ?
Applications of PCRApplications of PCR quick, reliable method for detecting all quick, reliable method for detecting all
manner of mutations associated with manner of mutations associated with genetic disease - from insertions, to genetic disease - from insertions, to deletions, to point mutations. deletions, to point mutations. Duchene muscular dystrophyDuchene muscular dystrophy
Detect unwanted Genetic materialDetect unwanted Genetic material Bacterial or viral infectionBacterial or viral infection
HIV infectionHIV infection
Amplify degraded DNA samplesAmplify degraded DNA samples Egyptian mummyEgyptian mummy Termite in amberTermite in amber
PCR animation PCR animation
PCR animation
On-Line SourcesOn-Line SourcesAccess Excellence. “Polymerase Chain Reaction (PCR)” 1992.19 Oct 2004.
<http://www.accessexcellence.org/RC/VL/GG/polymerase.html>.
Access Excellence. “Polymerase Chain Reaction - From Simple Ideas” 1999.19 Oct 2004. <http://www.accessexcellence.org/RC/AB/BC/PCR_From_Simple_Ideas.htmlhttp://www.accessexcellence.org/RC/AB/BC/PCR_From_Simple_Ideas.html>.
Access Excellence. “Polymerase Chain Reaction - Xeroxing DNA ”1992.19 Oct 2004. <http://www.accessexcellence.org/RC/AB/IE/PCR_Xeroxing_DNA.html>.
Cold Spring Harbor Laboratory.1994 Dolan DNA Learning Center. 18 Oct 2004.<http://www.dnalc.org/resources/BiologyAnimationLibrary.htmhttp://www.dnalc.org/resources/BiologyAnimationLibrary.htm>.
Montpetit, Diane. “Electron microscopy” 17Nov 2003. Agriculture and Agri-Food Canada . 19 Oct 2004. . 19 Oct 2004. <http://www.agr.gc.ca/disc_e.phtml>.<http://www.agr.gc.ca/disc_e.phtml>.
Nobelprize.org. The Nobel Prize in Chemistry 1993. 1 Nov2000. <http://nobelprize.org/chemistry/laureates/1993/index.html>.
Nobelprize.org. The PCR Method - a DNA Copying Machine. 19June2004. <http://nobelprize.org/chemistry/educational/pcr/index.html>.
Vierstraete, Andy. “Principle of the PCR” 08-11-1999. University of Ghent.19 Oct 2004 <http://allserv.rug.ac.be/%7Eavierstr/principles/pcr.html>.
Ward, Laura. “Food for thought” 2004 LABORATORY NEWS magazine. 19 Oct 2004<Ward, Laura. “Food for thought” 2004 LABORATORY NEWS magazine. 19 Oct 2004<http://www.labnews.co.uk/features_5.htm>.>.
Book SourcesBook Sources
Micklos, David, Greg Freyer and David Crotty. Micklos, David, Greg Freyer and David Crotty. DNA Science a First CourseDNA Science a First Course. New York:Cold Spring Habor Laboratory Press, 2003.
