M30 CytoDEATH Successfully applied to - NHRIcore.nhri.org.tw/userfiles/file/Roche SNP genotyping.pdf · Roche Applied Science 1 SNP Genotyping Introduction zRoche Diagnostics Ltd

Roche Applied Science1

SNP Genotyping IntroductionRoche Diagnostics Ltd. (Taiwan)

LightCycler 480 SystemRapid by Nature, Accurate by Design

www.roche-applied-science.com


Basic Introduction

SNP Identification by Melting Curve Technology

Two platforms for Melting Curve analysis

- LightCycler 2.0 & LightCycler 480 system

Summary

Content


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,


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


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


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?


Melting Curve Analysis


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


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


Mismatch

Perfect match

Temperature

low medium high

The principle of Melting Curve Analysis


Genotyping by Melting Peaks


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


Hybridization Probes Format :

A FRET Model Approach


Fluorescence Resonance Energy Transfer (FRET)

Wavelength (Energy)

Quantity


Hybridization Probes Format

LC RedFluorescein


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


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


Genotyping by Melting PeaksSingle Point Mutation: Factor V


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


Mutation Analysis Single Color Genotyping of 3 Different Alleles : Apolipoprotein B

Template: Plasmid DNAs


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


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.


LC - Apolipoprotein E Mutation Detection KitBackground Information

Background information about this gene:Family of molecules that comprise the protein moiety of lipoproteins


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


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


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


LightCycler NAT2 Mutation Detection KitBackground Information Background information about the NAT2 gene:

Encodes for a drug metabolizing enzyme


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


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


LightCycler NAT2 Mutation DetectionKitTypical Result: Control Template

wt wtwt wt

mutmut

mutmut

NAT5

NAT6 NAT14 NAT7

F3F2


LightCycler NAT2 Mutation DetectionKitTypical Result: Samples

F2 F3


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


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


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


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


Schematic of LightCycler480 Instrument


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


Summary


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


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


http://www.roche-applied-science.com

Innovation Profession Dedication Innovation Profession Dedication ServiceService

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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

Documents

M30 CytoDEATH Successfully applied to - NHRIcore.nhri.org.tw/userfiles/file/Roche SNP genotyping.pdf · Roche Applied Science 1 SNP Genotyping Introduction zRoche Diagnostics Ltd