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PCR의 기초 및 최신 분자진단 검사 기법 소개 한국애보트㈜ 분자진단사업부 박소양 April, 2015
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Topics PCR Overview PCR Applications PCR Formats
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PCR Overview What is PCR Principle PCR Reaction Components
How Does PCR Work Basics of measuring and analyzing real time PCR
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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 molecular diagnostic application
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Principle of Polymerase Chain Reaction
PCR Products are called AMPLICONS Nucleic Acid TARGET
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Utility of PCR Amplify large quantities of Nucleic Acid (e.g. DNA).
Analyze DNA fragments in complex mixtures Alter DNA sequence – directed mutagenesis.
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Principle Components of a PCR Reaction
Temperature Cycling A C T G Nucleic Acid Target Polymerase & Reagents Primer Pair
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Polymerase and Reagents
Template DNA Flanking Primers Thermo-Stable Polymerase Taq Pol dNTPs (the building blocks) dATP, dTTP, dGTP, dCTP PCR buffer (Magnesium salt, enhancers)
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Primer Pairs and Probes
Allele-specific priming (ASP) Primers provide target region specificity amplification start point. Additionally, they can embody the ability to discriminate certain molecular changes (e.g. Genotyping) Allele-specific probe (ASO) Probes are not used to specify start points of amplification. Probes are nested inside amplicons and provide independent means to Identify targets and discriminate changes of interest. 9 Company Confidential © 2009 Abbott
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The PCR Reaction is Temperature Cycling
(3) 94◦C (III) 72◦C 72◦C (II) 54◦C (II) n 25◦C 4◦C (III) (Hold) (min)
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Developing the Perfect PCR Reaction
Buffer Components 20mM Tris-HCL pH 8.4 50mM KCl 1.5 mM MgCl2 Salt (Magnesium) Optimal concentration of MgCl2 has to be selected . Too few Mg2+ ions result in a low yield of PCR product Too much increases non-specific products and promotes errors Potential Additives Helix Destabilizers - useful when target DNA has high G/C content. Examples: DMSO, DMF, urea Formamide Long Targets >1kb. Formamide and glycerol Low concentration of template: Polyethylene glycol (PEG) Temperature Cycling Primers Buffer Polymerase Salt Primer/Probe Design Length (17-28bp) 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 OPTIMIZE Enzyme Selection Taq, Vent, Pfu, others Native or Cloned Half-life & Attributes Taq 40 min vs. Vent 7 hour half-life 3’-5’ Exo nuclease – proofreading Fidelity (Error Rate). Taq 1/10,000nt, Pfu 1/1,000,000 Processivity and speed Bases per msec, Extra bases at end Temperature/Cycling Denaturation Trade off between denaturing DNA and Polymerase denaturing (e.g. 40min at 95 vs. 10min 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
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Phases in PCR 12
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Essential of Real-Time PCR 1
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Essential of Real-Time PCR 2
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Essential of Real-Time PCR 3
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Detection of Chemistry
< Detection System> < Filter Module- Dye Relationship> 16
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PCR Application 17
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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 18
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Realtime PCR Application 2- ( Absolute Standard )
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Realtime PCR Application- (Absolute Standard-Example)
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Realtime PCR Application- (Example)
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Realtime PCR Application- (Example)
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PCR Applications Clinically Relevant Molecular Targets Measured
Clinically Relevant Molecular Changes Measured Content vs. Analytical Needs for the Clinic AMs Qualitative and Quantitative Assays
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Clinically Relevant Molecular Targets Measured
Relevance Sample Template Application Preferred PCR Method
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Clinically Relevant Molecular Changes Measured
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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 PCR Next Gen Sequencing Realtime PCR Application 2- ( Absolute Standard ) Sensitivity Arrays FISH Sanger Sequencing Expanded Multiplex capability (Breadth) allows many interrogations at once. Multiplexing Capability
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PCR Assay Formats Other PCR methodologies
The Abbott Molecular Advantage
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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. Company
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FISH-Signal Enumeration
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Sanger Sequencing nbi HBV SMP 163, M204I Pol/RT mutant 30
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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. 31
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Companion Diagnostics
Collaborations with Market Leaders Abbott Offering CDx Collaborations Broad Technology Base Execution MAGE-A3 (NSCLC) MAGE-A3 (Melanoma) MAGE-A3 (HCC) FISH Real-time PCR Genotyping Multiplex analysis Sequencing Gene expression DNA methylation Nucleic acid composition Informatics Development Clinical trials Regulatory Reimbursement Commercialization Global reach ASL BAP (NSCLC) CML (Leukemia) Abbott Molecular is currently engaged with multiple pharmaceutical leaders including GlaxoSmithKline, Pfizer and Abbvie in the development of companion diagnostics for novel therapies. Historically the efficacy of oncology therapeutics has been less than 25% Biomarker development and CDx programs are improving efficacy and driving successful approval 35 CDx programs announced in the last five years. C-MET (Oncology) EGFR (Oncology) CMV (Vaccine) 32
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33 Abbott Molecular Breadth of Technology Solutions RealTime PCR FISH
Sequencing Bead Array New Technologies CMV, EBV CT/NG, CT HBV, HCV, HCV GT II HIV-1 HPV KRAS, BRAF Bladder Cancer Breast Cancer Chromosome Enumeration Genetics Hematology Oncology Solid Tumors Cystic Fibrosis Fragile X HARP Reagents HLA HBV Genotype & Drug Resistance HIV-1 Genotype cKit Factor II Factor V (Leiden) MTHFR Next Gen Sequencing Circulating Tumor Cells Microarrays Abbott Molecular continually advances and refines multiple molecular technologies, including FISH, real-time PCR, and sequencing as well as developing novel technologies. No single technology allows a laboratory to generate the most valuable results in every situation. Each lab should have the opportunity to choose the molecular platform that is best for their specific needs. All of our technology platforms make use of automation to optimize efficiency. We work to minimize hands-on time and risk of human error. Each of our technologies is accompanied by comprehensive training, a service plan and technical support to keep laboratory staff informed and running smoothly with maximum effectiveness. 33
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