qiagen hrm for analysis of genetic variations

Sample & Assay Technologies

HRM – an innovative technology for analysis of genetic variations

Advanced applications and critical success factors

Dr. Andreas MisselAssociate Director, Research and Development


Overview

.Agenda

Introduction into HRM The principle Advantages Applications overview

HRM in detail Analysis Dye

Critical factors Reaction chemistry Instrument and software Cycling conditions Assay design Template

Application data and summary

The applications presented here are for research purposes. Not for diagnostic use.


The HRM principle

HRM characterizes double-stranded PCR products based on the melting behavior Different DNA sequences melt at different specific temperatures

This is measured in real time using special intercalating dyes The fluorescence decreases during DNA dissociation

The HRM software plots fluorescence decrease vs. temperature

Temperature

Flu

ore

scen

ce

78 79 80 81 82 83 84 85 86 87 88 89 90

100

80

60

40

20

Tm Melting Point


Advantages of HRM

Enables highly specific and accurate detection of genetic variations

Allows further downstream analysis of amplicon (e.g. by sequencing)

Less expensive than alternative methods

Easy to use

Fast

Flexible

Versatile


HRM applications

SNP analysis

Mutation detection and scanning

Pathogen detection

Methylation analysis

STR and VNTR analysis

Quantification of copy number variants and mosaicism

…


HRM – An emerging technology

HRM citations/year*

*source: google scholar

0

50

100

150

200

250

300

350

400

2004 2005 2006 2007 2008 2009 2010

Ririe, Rasmussen and Wittwer Product Differentiation by Analysis of DNA MeltingCurves during the Polymerase Chain ReactionAnal. Biochem., 1997, vol. 245

HRM is expected to become a standard technology for genotyping applicationsHRM is expected to become a standard technology for genotyping applications


Overview

.Agenda






HRM vs. classic melt curve analysis

Classic melt curve analysis

Monitors melting differences >> 1 °C

Can be done with any real-time cycler

Uses preferably SYBR Green

Main application: find non-specific amplicons

e.g. primer dimers

HRM analysis

Monitors melting differences < 0.5°C !

Requires special instrument & software

Needs saturating dyes (LCGreen, Eva Green)

Main application: identify DNA differences

Genotyping, methylation analysis,…

unspecificamplicons

HRM provides significantly more information down to single nucleotide differences HRM provides significantly more information down to single nucleotide differences


HRM Analysis - Five steps from PCR to result

Step 1: Amplification Was the amplification successful?

Step 2: Melt curve analysisCheck to verify amplification specificity

Step 3: NormalisationSelect suitable samples and analysis range

Step 4: Difference plotSelect the reference genotype

Step 5: Autocalling genotypesunknowns will be either related to known genotypes or will be marked as variation


HRM and SNP genotyping

SNP Class* Base Change Typical TM Shift Abundance (in humans)

I C/T and G/A Large>0.5°C

Very Small <0.2°C

64%

II C/A and G/T 20%

III C/G 9%

IV A/T 7%

.SNP (= Single Nucleotide Polymorphism)

Single nucleotide variation

Can have major impact on how individuals respond to

Disease

Environmental factors (e.g. bacteria, viruses, chemicals)

Drugs and other therapies

SNP maps help to identify multiple genes associated with complex diseases (e.g. cancer, diabetes, vascular disease)

Source: Wikipedia

Reliable SNP genotyping requires clear resolution of TM < 0.2°CReliable SNP genotyping requires clear resolution of TM < 0.2°C

* Classification according to Venter et. al., 2002


Allele 1

Allele 2

T

A

T

A

C

G

C

G

Different homozygous samples are differentiated by distinct melting pointsDifferent homozygous samples are differentiated by distinct melting points

weaker binding = lower meltingpoint

stronger binding = higher meltingpoint

SNP genotyping – homozygous samples

Wild Type

Mutant


C

G

Mixture of mismatches with very low melting point

T

GA

C

Mixture with intermediatemelting point

SNP genotyping – heterozygous samples

Heterozygote

Wild Type

Mutant

Heterozygotes form mixtures resulting in a different melt curve shape Heterozygotes form mixtures resulting in a different melt curve shape

Allele 1

Allele 2

T

A +


Traditional dye technology for melting analysis

SYBR® Green I

Some dye molecules relocate as melting begins and do not contribute to fluorescence decrease

. SYBR™ Green I is toxic to PCR,so low concentration is needed

. Unsaturated binding may allow dye relocation during melts, making it less suitable for HRM

Non saturating intercalating dyes,e.g. SYBR Green

Unoccupied positions


Novel dye technology for melting analysis

Saturating intercalating dyes,e.g. EvaGreen

Dye saturation leaves no room for relocation events during melting

. “Saturation” dyes are much less toxic, so concentration used can be higher

. This reduces dye relocation events and improve melting resolution

All positions occupied

Saturating dyes like EvaGreen are required for high resolution melt analysis Saturating dyes like EvaGreen are required for high resolution melt analysis


Overview

.Agenda






The amplification step in HRM analysis

Specific PCR products are vital for optimal results in HRM

Classical melt analysis is recommended in HRM method development

to monitor specificity of PCR amplification

High quality PCR is imperative for good HRM resultsHigh quality PCR is imperative for good HRM results

specific product unspecific products

Reactionchemistry


Components of the Type-Itand Epitect HRM kits

.The enzyme HotStarTaq Plus DNA Polymerase Activation within 5 minutes Unmatched specificity and sensitivity

.The buffer Unique combination of K+ and NH4

+ ions High specificity No optimization of PCR parameters necessary

The dye EvaGreen Saturating dye Distinct melting curves

.Q-Solution Improves amplification of difficult loci

.

Reactionchemistry


Successful genotyping of class IV SNPReactionchemistry

Only optimized chemistry allows clear resolution of minute TM differencesOnly optimized chemistry allows clear resolution of minute TM differences


Instrumentation prerequisites for HRM analysis

Temperature

Thermal variability from sample-to-sample must be minimal

Fluorescence

Low “noise” from detector system

High stability of excitation light

High intensity excitation tuned to the optimal dye wavelength

Combined prerequisites

High data density i.e. high number of data points per degree thermal transition

Instrument precision is imperative for accurate HRM results Instrument precision is imperative for accurate HRM results

Instrumentation


Instrument benchmarking by SNP differentiation

SNP class

Base change

Typical TM

Shift

Abundance (in humans)

I C/T and G/A

Large>0.5°C

Very Small <0.2°C

64%

II C/A and G/T

20%

III C/G 9%

IV A/T 7%

Herrmann et al

Clinical Chemistry 53, 2007

A: Rotor-Gene Q B: Instrument Supplier R

Temp uniformity: ± 0.01°CVery high precision all genotypes clearly resolved FULL HRM information

Instrumentation

Temp uniformity: ± 0.2°CIntermediate precisiongenotypes not clearly resolved Limited HRM information


Combined mode of cycler and chemistry

A: EpiTect HRM Kit and Rotor-Gene Q B: HRM Kit and Instrument from Supplier AII

Instrumentation &chemistry

Only the combination of optimal chemistry and instrumentation ensures reliable results Only the combination of optimal chemistry and instrumentation ensures reliable results

Confidence threshold:95%


Difference plot Auto calling Results

Data analysis - Automatically performed by software

ºC75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90

Nor

mal

ised

min

us w

t

0

-5

-10

Genotypes automatically called by comparing samples and controls in difference plot

Confidence value (%) is calculated as integrity check for auto called results

Samples below user-set confidence threshold will be marked as a variation (here: 95%)

Control (100%)

Variation (< 95%)

Rotor-Gene Q software conveniently identifies known genotypes or variations Rotor-Gene Q software conveniently identifies known genotypes or variations

Software


Rotor-Gene ScreenClust HRM Software

A new approach for highly reliable data analysis in HRM

Enhances main applications such as SNP and mutation detection Mutation scanning Species identification

Uses advanced statistics for data clustering in order to screen for genetic differences

Superior auto calling of genotypes

Automatic detection of new mutations

Minimized efforts and highly standardized processes

Software


ºC45 50 55 60

Nor

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us 7

/840

35

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0

-5

Real-life performance testHow many genotypes do you see?

Difference Plot

StandardHRM Software

unpublished customer field test data

Software


Real-life performance testHow many genotypes do you see?

ScreenClust HRM Cluster Plot

Rotor-GeneScreenClust HRM Software

unpublished customer field test data

Only ScreenClust HRM Software accurately separates all genotypes Only ScreenClust HRM Software accurately separates all genotypes

Software


HRM analysis – melting of amplicon at ≤ 72°C

72 °C

melting of amplicons at ≤ 72°C results in partial dye release already during the extension step

amplification plots display lower plateaus and therefore seem to indicate lower product yields

UB = 100% unmethylated, bisulfite converted human DNA

MB = 100% methylated, bisulfite converted human DNA

Target: CpG island of the MLH1 gene

Cycling

?


Modified HRM cycling protocol

3-step cycling withextension at 72°C

Cycling

3-step cycling withextension at 68°C

Ensure that cycling conditions allow amplification of all genotypesEnsure that cycling conditions allow amplification of all genotypes


Guidelines for assay design, cycling and analysis

.Assay design Design assays with PCR product lengths of 70–350 bp For SNP analysis, use of PCR products of 70–150 bp is recommended The melting temperature of primers used for PCR with subsequent HRM analysis

should be at least 56°C Design assays with a single melting domain

(http://www.bioinformatics.org/meltsim/wiki/Main/HomePage)

.Run Follow cycling guidelines as recommended in the Type-It and EpiTect HRM manual,

respectively. Initially determine the melting point for each new HRM PCR product. Run HRM

analysis to span a temperature range from 65°–95°C. For time savings, perform subsequent HRM analyses from 5°C below the lowest TM of all expected PCR products to 5°C above the highest TM of all PCR products.

.Analysis Check that the PCR result contains only specific product Exclude samples showing primer dimers or nonspecific products from analysis


Effect of template purity on HRM

.Typical Contaminants NaCl KOAc EDTA ETOH Isopropanol Na Citrate Phenol

Template


Template purity – effect of contaminantsNaCl

Template

Effect of NaCl on TM

71.6

72.0

72.4

72.8

73.2

73.6

None 10mM 50mM 100mM

Concentration [mM]

TM [°C]

Salts increase the TM of PCR ampliconsSalts increase the TM of PCR amplicons


DNA purity – effects of contaminants on HRM Isopropanol

Template

Effect of Isopropanol on TM

69.00

70.00

71.00

72.00

73.00

None 0,5% 1.0% 2.0% 4.0%

Concentration [%]

TM

Alcohols decrease the TM of PCR ampliconsAlcohols decrease the TM of PCR amplicons


Conclusions and recommendations

.Conclusions

Differences in final reaction compositions result in different melting behaviour

and lead to wrong genotype classification

.Recommendations

Use identical method for DNA isolation for all samples

Use identical batch of reagents for DNA isolation, including resuspension / elution

buffer for all samples

Be careful to avoid contamínant carryover in your sample

Template


Overview

.Agenda






Class III and IV SNP genotyping

.Highly suitable for all real-time cyclers with HRM capability.Class IV SNPs with Rotor-Gene Q only

.Highly suitable for all real-time cyclers with HRM capability.Class IV SNPs with Rotor-Gene Q only

Rotor-Gene Q Supplier R

wild type

heterozygote

mutant

Supplier AII

Class IV SNP

genotypes not to differ

Class III SNP

Class IV not detectable

Class IV SNP


Successful detection of point mutations

Assay source:Do et al.: High resolution melting analysis for rapid and sensitive EGFR and KRAS mutation detection in formalin fixed paraffin embedded BiopsiesBMC Cancer 2008, 8:142

Analysis of point mutations in the human KRAS geneAnalysis of point mutations in the human KRAS gene


Successful scanning of insertions/deletions

Assay source:Do et al.: High resolution melting analysis for rapid and sensitive EGFR and KRAS mutation detection in formalin fixed paraffin embedded BiopsiesBMC Cancer 2008, 8:142

Analysis of different insertions and deletions in exon 19 of the EGFR geneAnalysis of different insertions and deletions in exon 19 of the EGFR gene


Methylation analysis

Analysis of A CpG island from the promoter region of the APC gene(adenomatosis polyposis coli) Mixtures of methylated and unmethylated DNA as template

Clear resolution of similar methylation degreesClear resolution of similar methylation degrees


Pyrosequencing complements HRM analysis

.Pyrosequencing Sequence-based technology in real time Quantitative sequencing results from specific loci Detection and quantification of base variants in genotyping

and epigenetics

Application examples in genotyping SNP confirmation Cancer mutations Polyploidy

Perfectly suited For highly variable regions If quantitative info is needed

Homozygous A

Homozygous G

Heterozygous A/G


Summary

HRM is a powerful emerging technique to investigate genetic differences offering

Throughput & speed

Cost effectiveness & convenience

Broad application range

Outstanding performance in HRM analysis requires:

Reliable template purity

Highly specific HRM PCR kits with saturating dye

qPCR and HRM instrument with superior temperature uniformity

Powerful software package for any kind of data analysis


The complete solution for HRM analysis

gDNA preparation from any sample type Manual or automated for any throughput

Highly specific HRM PCR kits Mandatory for good HRM results

Most precise real-time cycler on the market Prerequisite for accurate HRM analysis

Powerful analysis software packages Easy and reliable data acquisition and

interpretation

Reliable results by dedicated solutions for an entire HRM workflow Reliable results by dedicated solutions for an entire HRM workflow

Cluster 1Cluster 2Cluster 3

2 –

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Principal Component 1



2 –

Pri

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–1 –

–2 –

–1

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–1

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–0.5

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–0.5 0

–

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Principal Component 1


Questions and Answers

For further information, please contactyour local technical service department at

www.qiagen.com

qiagen hrm for analysis of genetic variations

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