Transcript Realtime PCR Application

PCR의 기초 및
최신 분자진단
검사 기법 소개
한국애보트㈜
분자진단사업부 박소양
April, 2015
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Company Confidential ©2014 Abbott
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
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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
ACTG
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)
•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.
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© 2009 Abbott
Company Confidential ©2014 Abbott
The PCR Reaction is Temperature Cycling
(I)
(I)
95◦C (3)
94◦C
(III)
72◦C
72◦C
(II)
54◦C
(II)
25◦C
n
4◦C
(Hold)
(III)
(min)
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Developing the Perfect PCR
Reaction
OPTIMIZE
•
Temperature
•
Cycling
•
Primers
•
Buffer
•
Polymerase
•
Salt
Buffer Components
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
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
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
Enzyme Selection
Temperature/Cycling
20mM Tris-HCL pH 8.4
50mM KCl
1.5 mM MgCl2
Potential Additives
Taq, Vent, Pfu, others
Helix Destabilizers - useful when target DNA
has high G/C content.
Native or Cloned
Half-life & Attributes
Examples: DMSO, DMF, urea
Formamide
Taq 40 min vs. Vent 7 hour half-life
Long Targets >1kb.
3’-5’ Exo nuclease – proofreading
Formamide and glycerol
Fidelity (Error Rate).
Low concentration of template:
Taq 1/10,000nt, Pfu 1/1,000,000
Polyethylene glycol (PEG)
Processivity and speed
Bases per msec, Extra bases at end
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Phases in PCR
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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>
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PCR Application
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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
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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
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Preferred PCR Method
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
Next Gen
Sequencing
Sensitivity
PCR
Realtime PCR Application 2- ( Absolute
Standard )
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.
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FISH-Signal Enumeration
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Sanger Sequencing
nbi HBV SMP 163, M204I Pol/RT mutant
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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.
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Companion Diagnostics
Collaborations with Market Leaders
Abbott Offering
Broad
Technology Base
CDx Collaborations
MAGE-A3 (NSCLC)
MAGE-A3 (Melanoma)
MAGE-A3 (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)
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Abbott Molecular
Breadth of Technology Solutions
RealTime
PCR
FISH
FISH
Sequencing
New
Technologies
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
cKit
Next Gen
Sequencing
Circulating
Tumor Cells
MTHFR
HLA
Microarrays
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•Thank You
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