High Resolution Melt (HRM) Analysis – A Practical Guide

High Resolution Melt (HRM) Analysis – A Practical Guide

Michael Tavaria, PhD

Scientific Applications Specialist

Australasia

2

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

3

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

4

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

5

What’s Happening during the Melt?

6

SYBR® Green dyeSaturating dye

Flu

ore

scen

ce

Temperature

Tm = 68 °CTm = 67°C

Saturating dsDNA binding dyes

7

HRM – determination of exact Tms

Melt & Anneal

G C

A T

Reformation of

homoduplexes

A TWT/WT gDNA

Melt & AnnealG CMT/MT gDNA

8

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

9

3 samples shown• WT/WT (green)

• MT/MT (blue)

• Het WT/MT (red)

HRM – different Tms & melt profiles

10

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

11




— SNP Genotyping


— Methylation


— Primer design

— DNA preparation


— Troubleshooting

12

• 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

13




— SNP Genotyping


— Methylation


— Primer design

— DNA preparation


— Troubleshooting

14

Occurrence of SNPs in the genome

HRM can detect all SNP classes

15

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

16

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

17

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)

18


• 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.

19


• 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 ???

20

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%

21


• 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.

22

• 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


23




— SNP Genotyping


— Methylation


— Primer design

— DNA preparation


— Troubleshooting

24

25

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)

26

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.

27

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

28

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

29

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

30




— SNP Genotyping


— Methylation


— Primer design

— DNA preparation


— Troubleshooting

31

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

32




— SNP Genotyping


— Methylation


— Primer design

— DNA preparation


— Troubleshooting

33

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

34

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

35

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

36

Primer Design

• Primer Express® Software v3.0 can assist

— Automates the design of PCR primers for amplifying targets

37

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

38

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

39




— SNP Genotyping


— Methylation


— Primer design

— DNA preparation


— Troubleshooting

40

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

41




— SNP Genotyping


— Methylation


— Primer design

— DNA preparation


— Troubleshooting

42

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

43

• 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)


44

• 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


45

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

46




— SNP Genotyping


— Methylation


— Primer design

— DNA preparation


— Troubleshooting

47

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?

48

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

49

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

50


• 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

51


• 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

52

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

53

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

54

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.

55

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.

Documents

High Resolution Melt (HRM) Analysis – A Practical Guide