High resolution melting analysis is a post-PCR analysis method used to identify variations in nucleic acid sequences.

1. High Resolution
Melt Analysis (HRM)
Dr. Shatha S. Jumaah/ Lecturer
TISHIK International University
Faculty of Science
Medical Analysis Dept

2. HRM a novel post-PCR method to analyze
genetic variations in PCR amplicons.
It goes beyond the power of classical
melting curve analysis by allowing us to
study the thermal denaturation of a dsDNA .

3. 1. Post-PCR Analysis
2. Separate dsDNA based on sequence length,GC content or strand complementary
3. Detects a single base difference
4. Rapid, inexpensive sequence screening method
 Mutation Sequence can be unknown
 Sample are further processed to identify mutation sequence
5. Increase specificity and sensitivity
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Sample PCR HRM

4. 1. Nnl
2. N mm
3. nn
4. Species identification
5. DNA fingerprinting
6. Screening for loss of heterozygosity
7. Allelic prevalence in a population
8. Characterization of haplotype blocks
9. HLA compatibility testing
10. Identification of candidate predisposition genes
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Mutation discovery/gene scanning
SNP genotyping
DNA methylation analysis
95% of all applications

5. 1. HRM is to monitor the separation of strands in real-time achieved by using a
fluorescent dye known as intercalating dyes and have a unique property.
2. Use (PCR) prior to HRM analysis to amplify the DNA region in which their mutation
of interest lies.
3. The method is basically exact warming of the DNA amplicon from around 50 ˚C up
to around 95 ˚C.
4. If melting temperature of the amplicon is reached, the two strands of DNA separate
or "melt" apart.
5. Thermal melting of DNA is usually monitored by UV absorbance.
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6. The data plots as a graph showing the level of fluorescence vs the temperature:
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Thermal melting of DNA is usually
monitored by UV absorbance.

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8. HRM machine has the ability to monitor tiny difference, perhaps a fraction of a degree this process
in "high resolution“ & it is possible to accurately document these changes and therefore identify if a
mutation is present or not. 8

9. DNA methylation
• Is a process by which methyl group are added
to the DNA molecule & that can change the
activity of a DNA segment without changing the
sequence.
• 2-5%of our genome are methylated
• Considered as 5th base
• No effect on base pairing
• CpG islands 1000-2000 base long with 10-20
times higher CpG content.
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10. • Sodium bisulfite conversion of genomic DNA to differentiate and detect unmethylated
Vs methylated cytosines is the gold standard for DNA methylation analysis.
• Treatment of denatured DNA (i.e., single-stranded DNA) with sodium bisulfite leads to
deamination of unmethylated cytosine residues to uracil, leaving 5-mC or 5-hmC
intact. The uracils are amplified in subsequent PCR reaction as thymines, whereas 5-
mC or 5-hmC residues get amplified as cytosines.
• Converted DNA should be quantitated as RNA using a UV spectrophotometer
(NanoDrop) with Ab260 nm 1.0 = 40 µg/ml.
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15. 1. High sensitivity to detect even 1 nucleotide change
2. Cost Effective
3. Simple and Fast Work Flow
4. Fast and Powerful
5. Low Reagent Consumption
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16. Thanks
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