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