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High Resolution Melt (HRM) Analysis – A Practical Guide
Michael Tavaria, PhD
Scientific Applications Specialist
Australasia
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A Practical Guide to HRM - Outline
• Basic Principles of HRM
• Overview of HRM Applications
— SNP Genotyping
— Mutation Screening
— Methylation
• Assay Development Guidelines
— Primer design
— DNA preparation
— Assay Optimisation
— Troubleshooting
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Why all the excitement?
• Lower cost alternative to TaqMan
• Can be performed with relatively low cost equipment
• Multiple applications possible
• Particularly suited to certain situations:
— SNPs close together
— Low frequency somatic mutations
— Inability to design TaqMan® Probes due to sequence limitations
— Large number of SNPs with low sample numbers
— Typing highly mutable samples where probe specificity may miss
some species
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Basic Principles of HRM
• High Resolution Melting (HRM) is a relatively new PCR based method to analyze DNA melt curves.
• HRM is different from a regular SYBR®Green I dye melt curve in three ways:
1. Chemistry: Uses brighter dsDNA binding dyes (LCGreen,
EvaGreen, SYTO9)
2. Instrument: More data points are collected than with a standard
melt curve
3. Software: New fluorescent normalization algorithms and plots
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What’s Happening during the Melt?
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SYBR® Green dyeSaturating dye
Flu
ore
scen
ce
Temperature
Tm = 68 °CTm = 67°C
Saturating dsDNA binding dyes
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HRM – determination of exact Tms
Melt & Anneal
G C
A T
Reformation of
homoduplexes
A TWT/WT gDNA
Melt & AnnealG CMT/MT gDNA
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Melt & Anneal
G C
A T
Reformation of
homoduplexes
A C
G T
Formation of
heteroduplexes
&
A T
G C
Het WT/MT gDNA
HRM – determination of exact Tms
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3 samples shown• WT/WT (green)
• MT/MT (blue)
• Het WT/MT (red)
HRM – different Tms & melt profiles
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Analysis of HRM melt curve data
1. Normalisation of signal intensity
2. Calculation of Tm
3. Determination of differences in melt curve shapes
Tm alone may not discriminate between some genotypes
eg. Wt (green) vs Het (blue)
virtually identical Tms but can be
easily distinguished based on the
shape of their melt curves
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A Practical Guide to HRM - Outline
• Basic Principles of HRM
• Overview of HRM Applications
— SNP Genotyping
— Mutation Screening
— Methylation
• Assay Development Guidelines
— Primer design
— DNA preparation
— Assay Optimisation
— Troubleshooting
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• SNP Genotyping— Use HRM to identify known variants — Confirm results with real-time TaqMan assays
• Mutation Screening— Use HRM to identify potentially new variants — Determine the genotype of the variant by DNA sequencing
• % Methylation
• Viral/Microbial Identification
• Many other applications……………
Overview of HRM Applications
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A Practical Guide to HRM - Outline
• Basic Principles of HRM
• Overview of HRM Applications
— SNP Genotyping
— Mutation Screening
— Methylation
• Assay Development Guidelines
— Primer design
— DNA preparation
— Assay Optimisation
— Troubleshooting
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Occurrence of SNPs in the genome
HRM can detect all SNP classes
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HRM Genotyping: Class 1 SNP data
• The red curves represent the
homozygous mutant population,
the green the wild-type and the
blue the heterozygote population
• The data is displayed in a
difference plot used to interpret
the HRM data more easily
* Class 1 SNPs include C/T and G/A mutations
and generally result in >0.5°C Tm shifts
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HRM Genotyping: Class 4 SNP (A/T) data
*Class 4 SNPs involve an A/T mutation and generally result in less than 0.1oC Tm shift
• The red curves represent the
heterozygous mutant population,
the green the wild-type and the
blue the homozygous mutant
population
• The data is displayed as
normalised melt curves to show
how little difference is required
for accurate genotyping
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HRM for Routine Genotyping
• HRM is most suitable for:
> SNP pre-screening (TaqMan assays can be costly)
> Routine genotyping in non-model organisms (TaqMan assays
not available)
> Genotyping polygenic organisms (populations <20% not easily
identified by TaqMan assays or sequencing)
> Genotyping somatic mutations (sensitivity nearly 100%)
> Detecting multiple SNPs close together (TaqMan assays cannot
be used)
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HRM for Routine Genotyping
• Cautions:
— New HRM assays should always be validated to the comparable
TaqMan assay before being used.
— HRM has a much higher design and amplification failure rate and
requires more repeated samples to be run.
— HRM assays are more costly to develop and optimize compared
to off the shelf TaqMan assays.
— HRM genotyping assays should always use known controls for all
genotypes. The genotype of each control should be confirmed by
TaqMan assay or DNA sequencing .
— Any uncertain/equivocal results should be confirmed by TaqMan
assay or DNA sequencing.
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HRM for Routine Genotyping
• Summary:
— HRM can provide a lower cost method for routine genotyping.
— However, HRM is less specific and less robust than TaqMan assays.
> TaqMan assays give fewer incorrect/undetermined results
— HRM assays must be properly tested and validated.
> This takes time and money
— HRM results should always be confirmed with TaqMan assays or DNA sequencing to ensure accuracy.
> This takes time and money
Total Project Cost ???
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HRM for Mutation Scanning
• Identification of unknown mutations
• DNA sequencing every sample in a study may be too expensive
• Pre-screening required
• dHPLC, SSCP or CSCE traditionally used to pre-screen samples
• Can be effective at detecting most mutations
• Detects somatic/acquired mutations down to 1%
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HRM for Mutation Scanning
• One wild type control is used instead of a control for each genotype.
• Most samples are likely to be wild type
• Variants identified and grouped:
— eg. “variant 1”, “variant 2”, etc.
— Samples identified as new variants should be sequenced to characterize the mutation.
• HRM can also be used to identify more complex mutations, such as multiple base pair changes, insertions or deletions.
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• Wild-type is known
• Variants are assigned on a similarity basis within groups
Wildtype
Variant 1
Variant 2
For an example of mutation scanning: Krypuy M et al, 2006, BMC Cancer; 6; 295
HRM for Mutation Scanning
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A Practical Guide to HRM - Outline
• Basic Principles of HRM
• Overview of HRM Applications
— SNP Genotyping
— Mutation Screening
— Methylation
• Assay Development Guidelines
— Primer design
— DNA preparation
— Assay Optimisation
— Troubleshooting
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Methylation Sensitive (MS)-HRM
• Research goals
— To quantify amount of methylated DNA in samples
— Can also be used as a diagnostic application
• Methods:
— After bisulfite treatment, methylated and unmethylated DNA samples will have different melting profiles
(un-methylated Cs will have been converted to Ts)
— If compared to methylated and unmethylated reference samples, estimates of extent of methylation may be determined (Wojdacz et.al. NAR, 2007, Vol. 35, No. 6 e41)
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Bisulfite Conversion - Facts
• Bisulfite treatment of gDNA converts unmethylated Cytosine to Uracil.
— Methylated Cytosine are protected and remain C.
• After PCR amplification all Uracils are converted Thymine.
• Comparing the sequences of the native gDNA to the bisulfite treated
gDNA will show you the methylated bases.
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Principle of MS-HRM
0%Methylation
100%
Methylation
74°C 80.0°C
T T T T
C C C C
50%Methylation
C T C T
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MS-HRM Primer Design
C C C C C C C C C C C C
T T T T T T T T T T T T
methylated
non-methylated
1. CG-free Primers
• Even resolution from 0 - 100%
C C C C C C C C C CC C
T T T T T T T T T T T T
methylated
non-methylated
C
T
C
T
G
G
G
G
2. CG-containing Primers
• PCR bias toward methylated DNA
• Improved resolution of 0 - 10%
• Can adjust the resolution with annealing temp
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Effects of Primer Design & Cycling
• no CpG dinucleotides in primers• no bias towards methylated fragments
• even 0% - 100% resolution
• CpG dinucleotides in primers• bias towards methylated fragments• annealing temp raised to 63°C
- resolution adjustable via annealing temp• improved 0% - 10% resolution
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A Practical Guide to HRM - Outline
• Basic Principles of HRM
• Overview of HRM Applications
— SNP Genotyping
— Mutation Screening
— Methylation
• Assay Development Guidelines
— Primer design
— DNA preparation
— Assay Optimisation
— Troubleshooting
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HRM Assay Development – Important Factors
• Effective and efficient PCR
— PCR design and conditions critical to successful downstream HRM results
• Best laboratory practices
— Critical to have good laboratory technique
— Must have clean NTCs
• Inclusion of appropriate controls
— HRM clustering accuracy can improve with the use of controls
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A Practical Guide to HRM - Outline
• Basic Principles of HRM
• Overview of HRM Applications
— SNP Genotyping
— Mutation Screening
— Methylation
• Assay Development Guidelines
— Primer design
— DNA preparation
— Assay Optimisation
— Troubleshooting
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HRM Assay Development – PCR Design
— Primer specificity is critical
— Amplicon length should be kept short to maximise different melting behaviour of similar products:
> 70-150bp ideal for GT assays (50-250bp acceptable)
> With optimization it is possible to use longer amplicons (~400bp)
— Optimise Primer and Mg concentrations
— HPLC purified primers are preferred but may not be required
— Primer design has an affect on HRM assay
> Use SYBR Green primer design principles
> Test primers in different locations
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HRM Primer Design Strategy- SNP detection
Test combinations of primers eg.
F1/R1 F1/R2 F1/R3
F2/R1 F2/R2 F3/R3
F3/R1 F3/R2 F3/R3
R1 R3R2
F3F2F1
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HRM Primer Design Strategy- mutation scanning
• Entire gene length needs to be screened
• Amplicon length ~ 300bp (smaller = better resolution)
• Target sequences should overlap by at least 25bp to ensure complete coverage
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Primer Design
• Primer Express® Software v3.0 can assist
— Automates the design of PCR primers for amplifying targets
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Primer Design
• Methyl Primer Express® Software v1.0 can assist with the design of PCR primers for amplifying bisulfite converted DNA
www.appliedbiosystems.com/methylprimerexpress
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Primer Express Design Guidelines
GC Range: 30-80%
Tm Range: 58-60ºC
Avoid >3 consecutive Gs
Avoid GC clamp (<3 G or Cs in last 5 ntd)
Short Amplicons (70 – 150bp)
Assay Design and Testing Guidelines
Use high quality sequence data
Filter out primer self- or cross-dimers
Test multiple primer pairs
DNA sequence the product to verify specificity
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A Practical Guide to HRM - Outline
• Basic Principles of HRM
• Overview of HRM Applications
— SNP Genotyping
— Mutation Screening
— Methylation
• Assay Development Guidelines
— Primer design
— DNA preparation
— Assay Optimisation
— Troubleshooting
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HRM Assay Development – DNA template considerations
• Same DNA conc for all samples
> Good working concentration is 1-10ng of DNA per reaction
• Use same method and reagents
• High quality DNA
> Remove inhibitors
> Poor amplification leads to poor melt curves
• Match samples and controls
Consistency Critical
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A Practical Guide to HRM - Outline
• Basic Principles of HRM
• Overview of HRM Applications
— SNP Genotyping
— Mutation Screening
— Methylation
• Assay Development Guidelines
— Primer design
— DNA preparation
— Assay Optimisation
— Troubleshooting
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HRM Assay Optimisation
• AB MeltDoctorTM HRM Reagents:
— Specially formulated for high performance in HRM experiments
— Accuracy
— Reproducibility
— Precision
— Minimise risk of contamination
— Minimise assay set-up time and potential error
— Master Mix or Reagent kit formulations
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• Magnesium chloride concentration is critical
— MgCl2 concentration is one of the first troubleshooting steps
— Usually 1.5 – 3.0 mM
• Choice of polymerase
— MeltDoctorTM HRM Enzyme
— Ultra Pure, high activity
— Too little enzyme can effect results
> Use at least 1.25-1.5U/µl
• Use AB MeltDoctorTM HRM Dye (Syto9)
HRM Assay Optimisation
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• Amplification on standard thermal cycler is possible, however there is large value in having real-time PCR results for troubleshooting
— Aim for CT values of 20-25
— Look for all samples amplifying at a similar cycle
— Strong and steep exponential phase
— Reaction should reach a strong plateau
— Add cycles if necessary, but beware non-specific amplification
Ensure efficient PCR
HRM Assay Optimisation
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HRM Assay Development – Summary
• Primer design critical
— Design and test several primer combinations
• DNA Quality and quantity critical
• Assays may require optimization
— Primer concentration
— Mg concentration
— PCR conditions
• Focus on maintaining good laboratory practice
— Accurate pipetting
— Appropriate use of replicates and controls
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A Practical Guide to HRM - Outline
• Basic Principles of HRM
• Overview of HRM Applications
— SNP Genotyping
— Mutation Screening
— Methylation
• Assay Development Guidelines
— Primer design
— DNA preparation
— Assay Optimisation
— Troubleshooting
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Troubleshooting the PCR
• Less than optimal PCR performance will lead to more population spread in HRM Software
SOLUTION:
Troubleshoot the PCR
— Not enough enzyme?
— Not enough MgCl2?
— Not enough primer?
— Increase extension time?
— Degraded template?
— Set up one ice?
— New primer sequence?
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Troubleshooting the PCR
• Contamination in PCR
— Note the extra peak in the raw data plot on the bottom
— These tasked NTCs are automatically omitted from the analyzed data in the top plot
— The contamination may be present in other samples and might be affecting their melt behavior
— Primer Dimers
SOLUTION:
— Repeat experiment (optimize if necessary)
— Run a primer matrix
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Troubleshooting the HRM files
• NTCs not tasked & failed samples
— Note erratic curves in the Normalized Melt Curve plot
— Default Pre- and Post-melt settings may not be ideal
• SOLUTION:
— Task NTCs & omit failed samples
OR
— Omit wells in HRM Software and manually set pre- and –post melt regions
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Troubleshooting the HRM files
• Pre-melt region set too far away
— Noise in pre-melt region affects the cluster call
— Additional variants are called
SOLUTION:
— Bring pre-melt region closer to inflection point
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Troubleshooting the HRM files
• Pre-melt region set too close
— Miss the first part of the curve, flattening it out in the difference plot
— Will result in incorrect cluster calls
• SOLUTION:
— Move bars out to flat region before the melt begins
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HRM Assay Troubleshooting - Summary
• Is amplification profile and Ct value normal?
• Does melt curve suggest single product?
Remove inhibited PCRs, failed reactions, outliers
Check PCR Conditions and reagents optimal
• Poorly clustered samples, poor data quality
Check Amplification controls (NTC etc)
Check/modify analysis parameters
Use genotype controls
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HRM Handbook is Now Available!
• Technical guide with detailed
information on HRM and
associated applications
• Please download at:
www.appliedbiosystems.com/hrm
• Also Quick Reference Cards for:
— Genotyping
— Mutation Scanning
— Methylation
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Legal Statement
© 2009 Life Technologies Corporation. All rights reserved. Trademarks of Life Technologies Corporation and its affiliated companies: AB(Design)®, AB Logo™, Applied Biosystems®, Life Technologies™ (Stylized), MeltDoctor™, POP-4™, POP-6™, Primer Express®, SYBR®. TaqMan is a registered trademark of Roche Molecular Systems, Inc. All other trademarks are the sole property of their respective owners.
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LegalFor Research Use Only. Not for use in diagnostic procedures.
Notice to Purchaser: License Disclaimer
Purchase of this software product alone does not imply any license under any process, instrument or
other apparatus, system, composition, reagent or kit rights under patent claims owned or otherwise
controlled by Applera Corporation, either expressly, or by estoppel.
Applera, Applied Biosystems and AB (Design) are trademarks of Applera Corporation or its subsidiaries
in the US and/or certain other countries.
2006 Applied Biosystems. All rights reserved.