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Roche Applied Science1
SNP Genotyping IntroductionRoche Diagnostics Ltd. (Taiwan)
LightCycler 480 SystemRapid by Nature, Accurate by Design
www.roche-applied-science.com
Roche Applied Science2
Basic Introduction
SNP Identification by Melting Curve Technology
Two platforms for Melting Curve analysis
- LightCycler 2.0 & LightCycler 480 system
Summary
Content
Roche Applied Science3
SNP Analysis & TechnologySNP Analysis & Technology
Different SNP Analysis will need different tools
SNP Discovery- DNA sequencing, dHPLC, SSCP
SNP Validation- MALDI-TOFSNP Screen- DNA array, TaqMan, HybProbe,
Roche Applied Science4
Technology Platform Extension Technology Platform Extension for SNP Screeningfor SNP Screening
# of Individuals High
# o
f SN
Ps
Low
Low
High
Array
Mass Spec.
SBE
RFLPTaqMan
Roche Applied Science5
What is a Melting Curve
SNP Identification by Melting Curves
Two detection probe format
- HybProbe & SimpleProbe
Application
- Factor V, Apoiopoprotein B, Hemochromatosis,
SNP Identification- Melting Curve Technology
Roche Applied Science6
A melting curve is the slow increase of temperature (e.g.: 1C/0.1 sec) while constant measurement of fluorescence
It causes dissociation of double stranded DNA
The melting of double stranded DNA can be monitored by fluorescence dyes
What is a melting curve?
Roche Applied Science7
Melting Curve Analysis
Roche Applied Science8
sequence specific probes bind to target DNA
Fluorescence decreases as probes melt away from template DNA due to increased temperature
Single base changes lead to thermal destabilisation of Probe-target complex
Melting temperatures will be different for ampliconswith sequence differences (SNPs) = different Tm values
SNP Identification by Melting Curves
Roche Applied Science9
Anchor Probe Mutation Probe
Tm of Mutation Probe approx. 5 C lower than Tm of Anchor Probe
mismatch
perfect match
The Probe Design for SNP Detection
Roche Applied Science10
Mismatch
Perfect match
Temperature
low medium high
The principle of Melting Curve Analysis
Roche Applied Science11
Genotyping by Melting Peaks
Roche Applied Science12
SimpleProbe Format:Single fluorescent labeled probe. Fluorescence signal depending on hybridization status (FAM)
HybProbe Format:Dual Probe System utilizing FRET between hybridized labeled probes(Fluorescein & LightCycler Red 640/705)
Two detection probe formats
Roche Applied Science13
Hybridization Probes Format :
A FRET Model Approach
Roche Applied Science14
Fluorescence Resonance Energy Transfer (FRET)
Wavelength (Energy)
Quantity
Roche Applied Science15
Hybridization Probes Format
LC RedFluorescein
Roche Applied Science16
5 3-pQ
R
R = Reporter (Fluorescein)Q = Quencher
3 5
primer 5 3-pQ
R
A
If SimpleProbe is free in solution, emission of reporter dye is quenched
Single Label Probes Format
B
If the SimpleProbe is bound to target, Fluorescence from reporter dye increases
Roche Applied Science17
Application - Factor V
Background information about this gene:Factor V is involved in the blood clotting cascade
The G1691A point mutation leads to an arginine substitution by glutamine (R506Q)
This mutation reduces cleavage by activated protein C that normally inactivates this coagulation factor
Test features:DNA based test, Mutation detection
Single color detection of 1 point mutation (G1691A) in exon 10 with oneHybridization Probe pair
Current research - Factor V polymorphisms :Association of hetero- or homozygosity for a single point mutation in the factor V gene with APC-resistance phenotype
Background Information
Roche Applied Science18
Genotyping by Melting PeaksSingle Point Mutation: Factor V
Roche Applied Science19
Background InformationBackground information about this gene:
Family of molecules that comprise the protein moiety of lipoproteins
Genetically polymorphic
Genetic basis lies within codons 3500
Test features:
DNA based test, mutation detection
Single Color detection of 2 point mutations (C9774T, G9775A) withinone amplicon with one Hybridization Probe pair
Current research - Apo B polymorphisms:
Correlation between ApoB concentration of lipoproteins and risk of atherosclerosis
Relevance of ApoB in familial hypercholesterolemia
Application Apolipoprotein B
Roche Applied Science20
Mutation Analysis Single Color Genotyping of 3 Different Alleles : Apolipoprotein B
Template: Plasmid DNAs
Roche Applied Science21
Hemochromatosis Mutation Analysis Background Information
A LightCycler application was published by Bernard et al. (1998), AJP 153: 1055
C282Y: a G845A transition leading to the substitution of tyrosine for
cysteine associated with the majority of clinically confirmed cases of
hereditary hemochromatosis
H63D: a C187G transversion resulting in the substitution of aspartate
for histidine; not itself associated to iron loading syndrome but appears to
act synergistically
Roche Applied Science22
Mutation Analysis: HemochromatosisGenotyping of Two Different Loci (Single Color)
FluosLC
RED 640primer 1 3-P5
Amplicon 1
FluosLC
RED 640primer 2 3-P5
Amplicon 2
published by Bernard et al. (1998), AJP 153: 1055.
Roche Applied Science23
LC - Apolipoprotein E Mutation Detection KitBackground Information
Background information about this gene:Family of molecules that comprise the protein moiety of lipoproteins
Genetically polymorphic
Genetic basis lies within codons 112 and 158
Test features:DNA based test, mutation detection
Dual Color test: Detection of 2 point mutations (C3932T, C4070T) within oneamplicon with two Hybridization Probe pairs
Current research - Apo E polymorphisms:Association of Apo E4 allel with increased risk for coronary heart disease
Susceptibility to Alzheimers disease in association with Apo E polymorphisms
Influence of Apo E polymorphisms on outcome after traumatic brain injury and intracerebral hemorrhage
Roche Applied Science24
Mutation Analysis Dual Color Genotyping of Two Different Loci: Apolipoprotein E
ForwardPrimer
ReversePrimer
DNA
CGC CGC
LC Red 640 LC Red 705
Amplicon
Hyb Probe Pair 1 Hyb Probe Pair 2
Roche Applied Science25
Mutation Analysis Dual Color Genotyping of Two Different Loci: Apolipoprotein E
control: homozygous TGC at codon 112 and codon158control: homozygous CGC at codon 112 and codon158control: heterozygous at codon 112 and codon 158no template control samplesamplesamplesample
F2: codon 112 F3: codon 158
Roche Applied Science26
LightCycler NAT2 Mutation Detection KitBackground Information Background information about the NAT2 gene:
Encodes for a drug metabolizing enzyme
Genetically polymorphic
SNPs related to different (slow) acetylation of drugs
Test features:
DNA based test, mutation detection in human DNA blood samples
Amplification of two fragments (342bp and 479 bp) and Dual Color detection of 4 point mutations (191GA, 481CT, 590GA and 857GA) with fourHybridization Probe pairs by Melting Curve Analysis within one capillary
Current research - NAT2 polymorphisms:
Pharmacogenetics: Relevance of the NAT2 gene in hereditary differences in the acetylation of drugs and toxicants
Roche Applied Science27
LightCycler NAT2 Mutation Detection KitScheme: Amplification and Detection
DNA
Amplicon
ForwardPrimer
ReversePrimer
ForwardPrimer
ReversePrimer
191G-A
LC Red 705
NAT2* 14A
NAT2* 7A/B
LC Red 640 857G-ALC Red 640NAT2* 5A
LC Red 705
NAT2* 6A
590G-A481C-T
Amplicon
Fluorescein
Fluorescein Fluorescein
Fluorescein
Roche Applied Science28
LightCycler NAT2 Mutation DetectionKitTypical Result: Control Template
wt wtwt wt
mutmut
mutmut
NAT5
NAT6 NAT14 NAT7
F3F2
Roche Applied Science29
LightCycler NAT2 Mutation DetectionKitTypical Result: Samples
F2 F3
Roche Applied Science30
LightCycler 2.0 system LightCycler 480
real-time PCR and genotyping
moderate throughput specialised for SNP genotyping
increased throughput
Platforms for Melting Curve Technology
www.roche-applied-science.com
Roche Applied Science31
Real time resultsTime for Genotyping (include PCR reaction) approx. 30 minutes3 Detection Filters for different Probe FormatsDual color analysis for different loci mutation in one reactionno extra (manual) handling steps (clean-up of PCR products or secondary reactions)Simultaneous analysis of up to 32 samples ( 512 samples per day )
Key Features LightCycler 1.5
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Key Features LightCycler 2.0
6 Detection Channels for multicolor mutation detectionSoftware for automatic genotyping (allele calling)20 l and 100 l volume are available
Roche Applied Science33
Time for Genotyping (post-PCR ) approx. 10 minutes
Simultaneous analysis of up to 384 samples
Moderate to high throughput: up to 18,000 Genotypes
per day
6 Detection Filters for different Probe Formats
uses conventional PCR plates
Software for automatic genotyping (allele calling)
Key characteristics LightCycler 480
Roche Applied Science34
Schematic of LightCycler480 Instrument
Roche Applied Science35
Homogeneous PCR assays containing detection probes in conventional, white plates
no extra (manual) handling steps (clean-up of PCR products or secondary reactions)
Due to sequence-specific hybridisation and characteristic melting curve (and Tm-value) the technique provides high reliability
Roche Applied Science36
Summary
Roche Applied Science37
Ideal Genotyping Method
Genotype accuracy
Cost of assays and specialized instrument(s)
Assay development time and ease
Ability to automate
Time to perform assays
Ability to multiplex
Data accumulation and analysis
Should consider
Roche Applied Science38
Summary
Ease of use & convenient Ready-To-Genotype system (Instrument, Software,
Reagents) No special plastic consumables required Automated Grouping of genotypes
Flexible & Adaptable Use established, current 96- or 384-well thermal
cycler Easy assay development based on established
Melting curve technology
Fast Genotyping of one plate in approx. 10min (= 384
results) Up to 18.000 Genotypings in 8 hours
Roche Applied Science39Speed Flexibility Safety Reliability ComponentsSuccess
Roche Applied SciencePCR Workflow System
Instruments
Software
Reagents
Roche Applied Science40
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LightCycler 480 SystemRapid by Nature, Accurate by Design SNP Analysis & TechnologyWhat is a melting curve?SNP Identification by Melting CurvesThe principle of Melting Curve AnalysisGenotyping by Melting Peaks Application - Factor V Background Information Hemochromatosis Mutation Analysis Background InformationLC - Apolipoprotein E Mutation Detection Kit Background InformationMutation Analysis Dual Color Genotyping of Two Different Loci: Apolipoprotein EMutation Analysis Dual Color Genotyping of Two Different Loci: Apolipoprotein EPlatforms for Melting Curve Technology Summary