2. High resolution melting (HRM)
• HRM analysis is a new, simple and fast technique of identifying genetic variations in nucleic
acid sequences
• It is a post PCR technique
• Simple, fast and cost effective
• Based on PCR melting (dissociation) curve techniques
• Enables rapid, high-throughput identification of variants in the regions of interest without
sequencing
• Does not need fluorescent labels, and multiple alleles can be detected in a single amplicon.
Can reveal extremely minor differences in dsDNA such SNPs based on melting curve characteristics
1. Chemistry—HRM analysis uses brighter ds DNA binding dyes at higher concentrations
2. Instruments—requires instruments that collect fluorescence data at finer temperature resolution
3. Software—requires more sophisticated software which uses new fluorescent scaling algorithms
and plots
4. Concept of dissociation/ melt curve analysis
• dsDNA binding dyes are non specific- can bind to any dsDNA (specific product, ns
product, any artefact, primer dimers etc) and produce fluorescence- mislead
interpretations
• Gene expression levels are directly proportion to fluorescence intensity
• Was used as quality check in real time PCR
dF
(Change
in
fluorescence)
5. • In HRM analysis, the region of interest is amplified by PCR in the
presence of a fluorescent dsDNA-binding dye.
• Melt/ dissociation: PCR product is gradually melted (thermal melting 55-
95 ⁰C) leading to reduction in fluorescence. The gradually denaturing
amplicon releases the florescent dye
• HRM analysis: Rate of change of fluorescence is characteristic of
amplicon which is captured and analysed . A melting curve can be
charted from the diminishing fluorescence values plotted against
increasing temperature
In HRM analysis, the same fundamental principles of DNA melting are applied with further
refinements.
HRM- basics
Temperature
6. Normalization and alignment of plot
• The rate of fluorescence decrease is generally greatest near the Tm of the PCR product, a point in the
melt curve where 50% of the DNA is double-stranded and 50% is single-stranded (melted).
• Pre- and post-melt regions are defined by pairs of vertical bars placed before and after the active melt
region.
• pre-melt (initial) and post-melts (final) fluorescence signals of all samples are normalized to relative
values of 100 and 0%
• Eliminates difference in background fluorescence between the curves
100 % fluorescence 0 % fluorescence
All amplicons ds
All amplicons ss
Temperature
7. Difference plot
• In this plot, sample curves are subtracted from a single reference (typically a
wild type control or user defined sample), run in the same experiment
• Magnifies curve difference, sets a baseline, so small differences become
visible
8. Wild type, heterozygote or homozygote?
1. Neither allele contains a mutation (wild type)
2. One or other allele contains a mutation (heterozygote)
3. Both alleles contain a mutation (homozygote)
Allelic discrimination by HRM
9. • Two types of HRM profiles
• Melt curves that are similar in shape but that are distinguishable from each other by differences
in melting temperature (Tm) of the amplicon. – homozygous variant samples
• Another curve due to the presence of base-pairing mismatches (heteroduplexes) present in the
PCR product mix
10. Optimization of HRM assays
DNA Quality-
• All DNA samples in an analysis run should be prepared using the same method. DNA
input amount into the PCR should also be similar.
• salt carryover
• pH extremes
• HRM analysis should be performed immediately following PCR whenever possible. If
immediate analysis is not possible, the PCR products can be stored at –20˚C.
• Alternatively, PCR can be performed on a thermal cycler followed by transfer to a
real-time instrument for sample melt and data analysis
• Amplicon lengths of 100–300 bp are generally recommended for HRM
• For SNP analysis, It should be ideally 80–100 bp, including primers (target
sequence length of 30 bp in addition to two 25 bp primer sequences)
• The relative change in fluorescence intensity of smaller amplicons is greater
than for longer amplicons.
Amplicon length-
12. Primer Selection
• Should be specific (producing no primer dimers and nonspecific products.)
• Quick check specificity by BLAST analysis
• 2-3 primer sets can be designed to choose the most robust pair
• Primer Tm should be between 55- 60⁰C
Optimization of HRM assays
Dye Selection
• Selection can be made from vide variety of dyes available commercially
• Too little dye can result in low signal and inaccurate results
• Too much dye can stabilize the double-stranded form, artificially shifting the Tm
or inhibiting PCR
• Empirical testing with a titration experiment • EvaGreen® Dye
• SYBR® GreenER™ Dye
• SYBR® Green I Dye
• SYTO® 9 Green
13. PCR reagents
• Each PCR component should be optimized for best HRM results (primer sets, MgCl2
concentration, template concentration, and other components)
Optimization of HRM assays
Reaction volume can be scaled down to 10uL ..
5 ml (1000 rxn)
Taq
dNTPs
Dye
14. • Genotyping (SNP, SSR, Indels)
• SNP scanning/ discovery
• Methylation differences between genes
• DNA fingerprinting, barcoding, mapping genes, and marker-assisted selection
• Detection of alleles in pooled Samples
• It is used for DNA-based identification and differentiation of cultivars and closely
related species
• In addition to SNP discovery and validation, HRM has the ability to detect allelic
dosage in polyploid species.
Applications of HRM
SNP scanning
15. Analysis software
Precision Melt Analysis™ Software (for Biorad CFX Opus)
• Provide a view of the raw fluorescent melt data
• Provide a process to align that data
• View melt curve differences between samples
16. HRM analysis of six unique genotypes of summer
squash genotypes with EST–SSR marker CUTC002749.
PCR reaction mixture consisted of
20 ng genomic DNA,
1× PCR buffer,
2.5 mM MgCl2,
0.2 mM dNTP, 300 nM forward and reverse primers,
1.5 mM Syto® 9 green dye and
1 U Kapa Taq DNA polymerase
HRM in identification and classification of genotypes
17. • Genotyping and distinguishing Basmati from
Non- Basmati rice cultivars
• Presence of Basmati rice in commercial rice
products
• Quantify adulteration of Basmati rice
products with non-Basmati rice
HRM in identification in fragrance typing and detection of adulterants
18. Fragrance typing in rice varieties and products
• Seven commercial rice products and eight rice varieties were analyzed
• Genotyping method was based on 8 bp deletion in BAD2 gene. InDel
polymorphism between fragrant (95 bp) and non-fragrant (103 bp) was analysed
using HRM analysis
• DNA was isolated from 0.1g fine rice powder of all the products
19. Rice product traceability through HRM
HRM protocol was developed in
order to distinguish and quantify
the presence of non-fragrance
rice varieties in fragrance rice
products.
As reference
NB
B
20. Non- Basmati amplicon was mixed with a Basmati amplicon at varying
degrees
Rice product traceability through HRM: validation
23. HRM applications
• Universal plastid DNA sequence: trnH-psbA
• Present in all plant species
• Display Indels and variations unique to plant
genera and species present
24. • HRM is a doable technique in our laboratory (with some up gradations)
• Can be applied for genetic purity testing of lines and hybrids (SSR/ SNP based)
• Can be used in MAS (Markers reported for WM-GSB, Chilli- GMS, ty- Tomato etc)
• Can potentially help in identifying polymorphic markers in cases (crops/ varieties)
where SSRs mostly appear monmorphic
• GOT analysis ??
Conclusions
