High Resolution Melt

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High Resolution Melting (HRM) analysis• What is HRM ?

• Why use HRM in your research ?

• Applying HRM in genomics research

— HRM best suited to Mutation Scanning and SNP Discovery work

> Applicability to Genotyping

• The Applied Biosystems HRM solution

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What is HRM?High Resolution Melting (HRM) is a new method to analyzeDNA melt curves.

HRM is different from a regular SYBR®Green dye melt curve

in three ways:

1. Chemistry: Uses saturating and brighter dsDNA binding dyes

2. Instrument: More data points are collected than a standard melt curve

3. Software: New fluorescent normalization algorithms and plots

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SYBR® Green dyeHRM dye

Flu

ores

cenc

e

Temperature

Tm = 68 °CTm = 67°C

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HRM Melt Curve Dynamics

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Reading a high definition melt curve

3 samples shown• homozygous wt (green)• homozygous mutant (blue)• heterozygous (red)

The analysis is based on the difference in curve shape as well as Tm

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HRM Applications

• SNP Genotyping

• % Methylation

• Viral Strain Identification

• Screening of polygenic samples

• Gene screening for non-model organisms

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High Resolution Melting

HRM Applications and Assay Development

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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

• Inclusion of appropriate controls

— HRM clustering accuracy can improve with the use of controls

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SNP Genotyping

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SNP Genotyping: Class 1 SNP data • 7500 Fast Real-Time PCR

System

• Three replicates of each sample are displayed

• The red curves represent the homozygous variant 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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SNP Genotyping: Class 4 SNP (A/T) data

• 7500 Fast Real-Time PCR System

• Replicate samples = call accuracy of 100% using default HRM software conditions with wild-type and homozygote control

• The blue curves represent the homozygote population, the green the wild-type and the red the heterozygote population

*Class 4 SNPs include an A/T mutation and generally result in less than 0.1oC Tm shift

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Results of HRM

Genotyping: bad design or discovery?

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F-Primer R-Primer

Target site: G/T

Another SNP site

Genotyping – additional SNPs

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Lower mutation frequencies

• HRM appears to have a unique sensitivity at low mutant frequency – less than 1% reported*

• COLD PCR may be able to achieve down to 0.1%!

Useful in:

— Somatically acquired mutation detection

— Heterogeneous populations (e.g., virally infected cells, viral/bacterial cultures)

— Pooling of multiple samples

— Polygenic organisms

* Kristensen LS, et al, Nucleic Acids Research, 2008, 36(7): e42Snell C, et al, Breast Cancer Research; 2008, 10(1):R12

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Common Applications for HRM – Methylation Analysis

• Research goals:

— To detect presence or 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

— 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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HRM for Methylation

• Unknowns are estimated based on the nearest standard

• Advantages

— Simple

— High throughput

— Focusing sequence-specific analysis on informative samples

— Cost-effective

— Depending on primer design, low levels of methylation detectable

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Customer Example: HRM SNP GT followed by TaqMan®

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Why use HRM in your genotyping project ?

Applied Biosystems TaqManTM Assays remain the Gold Standard for accurate and reproducible PCR-based genotyping

• HRM can be another option and provides:— Closed system, on a real time PCR instrument – common to other

genomics projects

— A low cost investigation tool for analysis of nucleic acid variation for assays with possible un-known SNPs

— Inexpensive running costs

• HRM is well suited to some particular niches— Inability to design TaqManTM Probes due to inherent sequence limitations

— Large number of SNPs with low sample numbers in cost-conscious settings

— Typing highly mutable samples where probe specificity may miss some species

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HRM can be of interest for the following applications:

— Mutation scanning / new SNP discovery

> Identify predisposition genes

— Genotyping

> profile association study cohort & verify rare SNPs

— Small Insertions & deletions

> Loss of heterozygosity

— Mutation discovery in mixed samples

> Identify presence of somatically acquired mutations, or for polygenic species, pooling

— Percentage of Methylation

— Viral Strain Identification by comparative studies

Summary

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HRM on the 7500 Fast & 7900 HT Real-Time PCR Systems

• The HRM software is a standalone analysis-only software package

• The HRM software is compatible with melt curves run on 7500 Fast and 7900 HT Real-Time PCR systems

• The 7500 Fast and 7900 HT Real-Time PCR systems are capable of reading HRM specific dyes and can collect enough data points to provide “high” resolution curves

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HRM software Installation and Calibration

• The HRM Software is customer installable

• For the 7500 Fast

— All HRM calibrations and runs have to be run in either the SDS Software v1.4 or the 7500 Software v2.0

• For the 7900 HT

— All HRM calibrations and runs have to be run on the SDS Software v2.3

• All calibrations can be performed by the customer

— There is a Quick Sheet available for each compatible dye

On-site training course for purchase coming soon!

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User Guide and Quick Sheet

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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