Topics PCR Overview PCR Applications PCR Formats AMD00002875

Topics • PCR Overview • PCR Applications • PCR Formats AMD-00002875 2 Company Confidential © 2014 Abbott

PCR Overview • What is PCR • Principle PCR Reaction Components • How Does PCR Work • Basics of measuring and analyzing real time PCR AMD-00002875 3 Company Confidential © 2014 Abbott

PCR = Polymerase Chain Reaction • Genesis of modern biotechnology: - The game changer for putting Biological Principles to work Biology Dogma Kary Mullis invents PCR 1983 Nobel Prize in Chemistry, 1993 Today, PCR is essential or integral step in just about every AMD-00002875 molecular diagnostic application 4 Company Confidential © 2014 Abbott

Principle of Polymerase Chain Reaction PCR Products are called AMPLICONS Nucleic Acid TARGET AMD-00002875 5 Company Confidential © 2014 Abbott

Utility of PCR • Amplify large quantities of Nucleic Acid (e. g. DNA). • Analyze DNA fragments in complex mixtures • Alter DNA sequence – directed mutagenesis. AMD-00002875 6 Company Confidential © 2014 Abbott

Principle Components of a PCR Reaction Temperature Cycling ACTG Nucleic Acid Target Polymerase & Reagents Primer Pair AMD-00002875 7 Company Confidential © 2014 Abbott

Polymerase and Reagents • Template DNA • Flanking Primers • Thermo-Stable Polymerase v Taq Pol • d. NTPs (the building blocks) v d. ATP, d. TTP, d. GTP, d. CTP • PCR buffer (Magnesium salt, enhancers) AMD-00002875 8 Company Confidential © 2014 Abbott

Primer Pairs and Probes • Allele-specific priming (ASP) • Allele-specific probe (ASO) • Primers provide target region specificity amplification start point. • Probes are not used to specify start points of amplification. • Additionally, they can embody the ability to discriminate certain molecular changes (e. g. Genotyping) • Probes are nested inside amplicons and provide independent means to Identify targets and discriminate changes of interest. AMD-00002875 9 9 Company Confidential © 2009 Abbott Company Confidential © 2014 Abbott

The PCR Reaction is Temperature Cycling (I) 95◦C (3) 94◦C (III) 72◦C (II) 54◦C (II) 25◦C n 4◦C (Hold) (III) (min) AMD-00002875 10 Company Confidential © 2014 Abbott

Developing the Perfect PCR Reaction OPTIMIZE • Temperature • Cycling • Primers • Buffer • Polymerase • Salt Buffer Components Primer/Probe Design Length (17 -28 bp) GC content 50 -60% GC Clamp Tm’s between 55 -80 Avoid simple sequences – e. g. strings of G’s Avoid primer self complementary e. g. hairpins, homo-dimers, heterodimers Denaturation Trade off between denaturing DNA and Polymerase denaturing (e. g. 40 min at 95 vs. 10 min at 97. 5°). Annealing Trade off between efficient annealing and specificity 2 -5 ° below Tm Extension Temperature optimum for Taq Polymerase 72 ° Rate Number and speed of heating/cooling cycles Salt (Magnesium) Optimal concentration of Mg. Cl 2 has to be selected. Too few Mg 2+ ions result in a low yield of PCR product Too much increases non-specific products and promotes errors Enzyme Selection Temperature/Cycling 20 m. M Tris-HCL p. H 8. 4 50 m. M KCl 1. 5 m. M Mg. Cl 2 Potential Additives Taq, Vent, Pfu, others Helix Destabilizers - useful when target DNA has high G/C content. Native or Cloned Examples: DMSO, DMF, urea Formamide Half-life & Attributes Taq 40 min vs. Vent 7 hour half-life 3’-5’ Exo nuclease – proofreading Fidelity (Error Rate). Taq 1/10, 000 nt, Pfu 1/1, 000 Long Targets >1 kb. Formamide and glycerol Low concentration of template: Polyethylene glycol (PEG) Processivity and speed Bases per msec, Extra bases at end AMD-00002875 11 Company Confidential © 2014 Abbott

Phases in PCR AMD-00002875 12 Company Confidential © 2014 Abbott

Essential of Real-Time PCR 1 AMD-00002875 13 Company Confidential © 2014 Abbott

Essential of Real-Time PCR 2 AMD-00002875 14 Company Confidential © 2014 Abbott

Essential of Real-Time PCR 3 AMD-00002875 15 Company Confidential © 2014 Abbott

Detection of Chemistry < Detection System> < Filter Module- Dye Relationship> AMD-00002875 16 Company Confidential © 2014 Abbott

PCR Application AMD-00002875 17 Company Confidential © 2014 Abbott

Realtime PCR Application 1 1. Absolute quantification • Determine exact number of target nucleic acid molecules - Virus quantification - Transgene - Gene therapy 2. Relative quantification • Make quantification comparisons of a target nucleic acid • Describes as fold differences - Gene expression, Drug therapy - DNA damage AMD-00002875 18 Company Confidential © 2014 Abbott

Realtime PCR Application 2( Absolute Standard ) AMD-00002875 19 Company Confidential © 2014 Abbott

Realtime PCR Application(Absolute Standard-Example) AMD-00002875 20 Company Confidential © 2014 Abbott

Realtime PCR Application- (Example) AMD-00002875 21 Company Confidential © 2014 Abbott

Realtime PCR Application- (Example) AMD-00002875 22 Company Confidential © 2014 Abbott

PCR Applications • Clinically Relevant Molecular Targets Measured • Clinically Relevant Molecular Changes Measured • Content vs. Analytical Needs for the Clinic • AMs Qualitative and Quantitative Assays • AMD-00002875 23 Company Confidential © 2014 Abbott

Clinically Relevant Molecular Targets Measured Relevance Sample Template Application AMD-00002875 24 Company Confidential © 2014 Abbott Preferred PCR Method

Clinically Relevant Molecular Changes Measured AMD-00002875 25 Company Confidential © 2014 Abbott

Content vs. Analytical Needs for the Clinic Improved sensitivity (Depth) allows detection of rare alleles/events: needed for PGx, oncology, and viral resistance Digital PCR Next Gen Sequencing Sensitivity PCR Realtime PCR Application 2 - ( Absolute Standard ) Arrays FISH Expanded Multiplex capability (Breadth) allows many interrogations at once. Sanger Sequencing AMD-00002875 Multiplexing Capability 26 Company Confidential © 2014 Abbott

PCR Assay Formats • Other PCR methodologies • The Abbott Molecular Advantage AMD-00002875 27 Company Confidential © 2014 Abbott

FISH Technique- Molecular Cytogenetics • FISH is a cytogenetic technique that is used to detect and localize the presence or absence of specific DNA sequences on chromosomes in its native state. • The technique uses fluorescently-labeled DNA molecules (probes) to detect other DNA molecules (chromosomes or genes) of complementary sequence that can be seen using fluorescent microscopes. AMD-00002875 Company 28 Company Confidential © 2014 Abbott

FISH-Signal Enumeration AMD-00002875 29 Company Confidential © 2014 Abbott

Sanger Sequencing nbi HBV SMP 163, M 204 I Pol/RT mutant AMD-00002875 30 30 Company Confidential © 2014 Abbott

Next Gen Sequencing • The power of high–throughput DNA sequencing technologies is being harnessed by researchers to address an increasingly diverse range of biological problems. The scale and efficiency of sequencing that can now be achieved is providing unprecedented progress in areas from the analysis of genomes themselves to how proteins interact with nucleic acids. AMD-00002875 31 31 Company Confidential © 2014 Abbott

Companion Diagnostics Collaborations with Market Leaders Abbott Offering Broad Technology Base CDx Collaborations MAGE-A 3 (NSCLC) MAGE-A 3 (Melanoma) MAGE-A 3 (HCC) Execution FISH Development Real-time PCR Clinical trials Genotyping Regulatory Multiplex analysis Reimbursement Sequencing Commercialization Gene expression Global reach ASL BAP (NSCLC) CML (Leukemia) DNA methylation C-MET (Oncology) EGFR (Oncology) Nucleic acid composition Informatics CMV (Vaccine) AMD-00002875 32 32 Company Confidential © 2014 Abbott

Abbott Molecular Breadth of Technology Solutions Real. Time PCR FISH Sequencing Bead Array CMV, EBV Bladder Cancer Cystic Fibrosis Factor II CT/NG, CT Breast Cancer Fragile X HBV, HCV, HCV GT II Chromosome Enumeration HARP Reagents Factor V (Leiden) HIV-1 Genetics HPV Hematology HBV Genotype & Drug Resistance KRAS, BRAF Oncology HIV-1 Genotype Solid Tumors c. Kit HLA New Technologies Next Gen Sequencing Circulating Tumor Cells MTHFR Microarrays AMD-00002875 33 33 Company Confidential © 2014 Abbott

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