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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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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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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: 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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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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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!