Post on 05-Jan-2016
Polymerase Chain Reaction
1998
PCR EvolutionThe future is amplifying!
1985 First publication of PCR by Cetus Corporation in Science (R. Saiki, S. Scharf, F. Faloona, K. Mullis, G. Horn, H. Erlichand N. Arnheim).
1989 The thermostable DNA polymerase, Taq, (enabling automation of PCR) declared molecule of the year by Science.Hoffmann-La Roche Inc. and Cetus agree to begin development of diagnostic applications for PCR.
1990
* European Launch** US Launch
Cetus scientists, D. Gelfand and S. Stoffel, named Distinguished Inventors for purifying Taq DNA polymerase.First forensic PCR kit is introduced for HLA DQA.Cetus scientists, H. Erlich and K. Mullis, recieve the Biochemical Analysis Award
from the German Society of Clinical Chemistry.
PCR EvolutionThe future is amplifying!
1991 First publication of TaqMan® method by Cetus.Hoffmann-La Roche Inc. acquires from Cetus worldwide rights and patents to PCR. Roche Molecular Systems, exclusively devoted to development of PCR, is founded. RT-PCR is developed using a single thermostable polymerase, rTth, facilitating diagnostic tests for RNA viruses.First publication on thermostable Reverse Transcriptase by Cetus Scientists.Cetus scientist, H. Erlich, recieves the Advanced Technology in Biotechnology (ATB’91) Milano Award from the International Federation of Clinical Chemistry.
1992 AMPLICOR® Chlamydia trachomatis* and AMPLICOR HIV-1* are introduced as the first standardised DNA PCR kits for clinical diagnostic use.
1993 AMPLICOR HCV* is introduced as the first standardised RNA PCR kit. Kary Mullis receives Japan Prize and shares Nobel Prize in Chemistry for conceiving the concept of PCR. AMPLICOR® C. trachomatis** is launched.
* European Launch** US Launch
PCR EvolutionThe future is amplifying!
1995 COBAS AMPLICOR™*, the first automated system for routine diagnostic PCR, is launched.First standardised quantitative PCR kits, AMPLICOR HIV-1 MONITOR™* and AMPLICOR HCV MONITOR™*, are launched.
1996 AMPLICOR Mycobacterium tuberculosis** and AMPLICOR Enterovirus* tests are launched. AMPLICOR HIV-1 MONITOR™** test is launched.
1997 COBAS AMPLICOR™** Analyser is launched.
1998 COBAS AMPLICOR MONITOR™* System is launched.
* European Launch** US Launch
PCR Amplifies a Targeted Sequence
Target Sequence
DNA StrandDouble HelixDNA StrandSupercoiled
DNA Strand
Chromosome
Cell Division Parent Cell
Prophase
Chromosomes align at the equatorial (metaphase) plate
Metaphase (Centromeres divide)
Sister chromatids separate during anaphase, becoming chromosomes
Two Daughter Cells
DNA StructureHydrogen Bonds
Cytosine
Adenine
Thymine
Guanine
Deoxyribose(Sugar molecule)
Phosphoric Acid(Phosphate molecule)
DNA Double Helix
Deoxyribose and Phosphoric Acid
Deoxyribose Phosphoric Acid
AdenineAdenine
CytosineCytosine
GuanineGuanine
ThymineThymine
DNA Base Nomenclature
Base Nucleoside Nucleotide Abbreviation Base Ring Structure
Adenine (A) Adenosine Adenosine dATP Purine Triphosphate
Guanine (G) Guanosine Guanosine dGTP Purine Triphosphate
Thymine (T) Thymidine Thymidine dTTP Pyrimidine Triphosphate
Cytosine (C) Cytidine Cytidine dCTP PyrimidineTriphosphate
DNA Base Nomenclature
The Nucleotide SequenceHydrogen Bonds
Cytosine (C)
Adenine (A)
Thymine (T)
Guanine (G)
Deoxyribose(Sugar molecule)
Phosphoric Acid(Phosphate molecule)
Cytosine (C)
Adenine (A)
Thymine (T)
Guanine (G)
5’ to 3’ Orientation of the Sugar - Phosphate Backbone 5’ end
3’ end
PhosphateMolecule
Bonding of Base, Sugar and Phosphate Groups
DeoxyriboseSugar Molecule
Bases
Hydrogen Bonds
Sugar-Phosphate Backbone
5’
3’ 5’
3’
PCR Cycle - Step 1 - Denaturation by Heat
Target Sequence
Target Sequence
PCR Cycle - Step 2 - Biotinylated Primer Pair Anneals to Ends of Target Sequence
Target Sequence
Target Sequence
Primer 1Primer 2
Biotin
Biotin
5’
3’
5’
5’
3’
5’
3’
3’
PCR Cycle - Step 3 - Taq DNA Polymerase Catalyses Primer Extension as Complementary Nucleotides are Incorporated
Target Sequence
Target Sequence
Primer 1
Primer 2
Biotin
Biotin
5’
3’
5’
5’
3’
5’
3’
3’
Taq DNA
Polymerase
End of the 1st PCR Cycle - Results in Two Copies of Target Sequence
Target Sequence
Target Sequence
Biotin
Biotin
Target Amplification
No. of No. Amplicon Cycles Copies of Target
1 2
2 4
3 8
4 16
5 32
6 64
20 1,048,576
30 1,073,741,824
1 cycle = 2 Amplicon
2 cycle = 4 Amplicon
3 cycle = 8 Amplicon
4 cycle = 16 Amplicon
5 cycle = 32 Amplicon
6 cycle = 64 Amplicon
7 cycle = 128 Amplicon
Ribose and Deoxyribose Structures
Ribose Deoxyribose
Difference between RNA and DNA
RNA DNA
Sugar Ribose Deoxyribose
Adenine (A) Adenine (A)
Bases Cytosine (C) Cytosine (C)
Uracil (U) Thymine (T)
Guanine (G) Guanine (G)
No. of strands Usually single Double
Heat stable? No Yes
Difference between RNA and DNA
Reverse Transcription - Step 1 - Biotinylated Primer Anneals to Target RNA Sequence
Target Sequence
PrimerBiotin
5’
3’
5’
3’
Reverse Transcription - Step 2 - rTth DNA Polymerase Catalysing Primer Extension by Incorporating Complementary Nucleotides
Target RNA Sequence
PrimerBiotin
5’
3’
5’
3’
rTth DNA Polymerase
End of Reverse Transcription - Step 3 - Results in Synthesis of Complementary DNA (cDNA) to the RNA Target Sequence
Target RNA Sequence
cDNA
Biotin
PCR Step 1 - Denaturation by Heat
Target RNA Sequence
cDNABiotin
PCR Step 2 - Annealing of Primer to cDNA
cDNA
Biotin
PrimerBiotin
PCR Step 3 - rTth DNA Polymerase Catalyses Primer Extension
cDNA
Biotin
Primer
Biotin
rTth DNA Polymerase
End of 1st PCR Cycle - Yields a Double-Stranded DNA Copy (Amplicon) of the Target Sequence
cDNA
Biotin
Amplicon
Biotin
PCR End of Second Cycle - rTth DNA Polymerase Catalyses Primer Extension
cDNA
Biotin
AmpliconBiotin
Biotin
Biotin
PCR - Exponential Amplification: Each New Cycle Doubles the Amount of Target, Resulting in an Exponential Increase in Amplicon
1 cycle = 1 (RNA/cDNA hybrid)
2 cycle = 2
3 cycle = 4
4 cycle = 8
5 cycle = 16
6 cycle = 32
7 cycle = 64
Single strand RNA