Polymerase Chain Reaction 1998

PCR Evolution The future is amplifying! 1985First publication of PCR by Cetus Corporation in Science (R. Saiki, S. Scharf, F. Faloona, K. Mullis, G. Horn, H. Erlichand N. Arnheim). 1989The 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 * 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 Evolution The future is amplifying! 1991First 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 AMPLICOR ® Chlamydia trachomatis* and AMPLICOR HIV-1* are introduced as the first standardised DNA PCR kits for clinical diagnostic use 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 Evolution The future is amplifying! 1995COBAS 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. 1996AMPLICOR Mycobacterium tuberculosis** and AMPLICOR Enterovirus* tests are launched. AMPLICOR HIV-1 MONITOR™** test is launched. 1997COBAS AMPLICOR™** Analyser is launched. 1998COBAS AMPLICOR MONITOR™* System is launched. * European Launch ** US Launch

PCR Amplifies a Targeted Sequence Target Sequence DNA Strand Double Helix DNA Strand Supercoiled 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 Structure Hydrogen Bonds Cytosine Adenine Thymine Guanine Deoxyribose (Sugar molecule) Phosphoric Acid (Phosphate molecule)

DNA Double Helix

Deoxyribose and Phosphoric Acid DeoxyribosePhosphoric Acid

Adenine Cytosine Guanine Thymine

DNA Base Nomenclature BaseNucleosideNucleotideAbbreviationBase Ring Structure Adenine (A)AdenosineAdenosinedATPPurine Triphosphate Guanine (G)GuanosineGuanosinedGTPPurine Triphosphate Thymine (T)ThymidineThymidinedTTPPyrimidine Triphosphate Cytosine (C)CytidineCytidinedCTPPyrimidine Triphosphate DNA Base Nomenclature

The Nucleotide Sequence Hydrogen 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

Phosphate Molecule Bonding of Base, Sugar and Phosphate Groups Deoxyribose Sugar Molecule Bases Hydrogen Bonds Sugar-Phosphate Backbone 5’ 3’5’ 3’

PCR Cycle - Step 1 - Denaturation by Heat Target Sequence

PCR Cycle - Step 2 - Biotinylated Primer Pair Anneals to Ends of Target Sequence Target Sequence Primer 1 Primer 2 Biotin 5’ 3’ 5’ 3’ 5’ 3’

PCR Cycle - Step 3 - Taq DNA Polymerase Catalyses Primer Extension as Complementary Nucleotides are Incorporated Target Sequence Primer 1 Primer 2 Biotin 5’ 3’ 5’ 3’ 5’ 3’ Taq DNA Polymerase

End of the 1st PCR Cycle - Results in Two Copies of Target Sequence Target Sequence Biotin

Target Amplification No. ofNo. Amplicon CyclesCopies of Target ,048, ,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 RNADNA SugarRiboseDeoxyriboseAdenine (A) Bases Cytosine (C)Cytosine (C) Uracil (U)Thymine (T)Guanine (G) No. of strandsUsually singleDouble Heat stable?NoYes Difference between RNA and DNA

Reverse Transcription - Step 1 - Biotinylated Primer Anneals to Target RNA Sequence Target Sequence Primer Biotin 5’ 3’ 5’ 3’

Reverse Transcription - Step 2 - rTth DNA Polymerase Catalysing Primer Extension by Incorporating Complementary Nucleotides Target RNA Sequence Primer Biotin 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 cDNA Biotin

PCR Step 2 - Annealing of Primer to cDNA cDNA Biotin Primer Biotin

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 Amplicon 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