Strengths of HRM

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One technology, multiple benefits
Advantages over traditional real-time PCR methods
Specific and accurate detection method 
Fast screening method for unknown mutations
Nondestructive analysis method 
Closed tube method — minimal contamination
Cost-effective method
Versatile technique
Advantages over traditional real-time PCR methods
HRM-based methods provide greater flexibility as there is no requirement for fluorescently-labeled detection probes (such as TaqMan probes) — simplifying assay design. Detection of previously unknown variants is straightforward and it is possible to simultaneously detect multiple SNPs or mutations in the same PCR reaction.

Specific and accurate detection method
PCR products can be discriminated according to sequence, length, GC content, or strand complementarity, down to single base pair differences. Previously unknown and even complex sequence variations — as seen in challenging genotyping applications — can be readily detected and characterized in an easy and straightforward way (see figure Successful genotyping of an A/T class IV SNP using the Type-it HRM PCR Kit).

Fast screening method for unknown mutations
Due to the high level of sensitivity afforded by HRM technology, it can be employed as an initial screen for samples suspected of harboring polymorphisms or mutations (see figure Successful detection of mutations by high-resolution melting [HRM] analysis). This would reduce the number of samples that need to be investigated further.

Nondestructive analysis method
The PCR product can be subjected to repeated melt analyses and it can be further used in downstream applications such as cloning or sequencing. 

Closed tube method — minimal contamination
Cross-contamination is minimized as HRM is a closed-tube technique. Unlike other genotyping methods such as denaturing gradient gel electrophoresis (DGGE), HRM does not require the use of hazardous reagents such as acrylamide, formamide, and ethidium bromide. 

Cost-effective method
HRM is cost effective as there is no requirement for fluorescently-labeled detection probes. All you need is a pair of standard oligonucleotides, PCR chemistry comprising of an HRM-compatible dye, and an instrument capable of monitoring the fluorescence decrease upon temperature increase with high thermal and optical precision. A cost-effective solution is the use of a real-time cycler that ensures high temperature uniformity (typically better than 0.1°C), together with dedicated HRM software.

Versatile technique
HRM is a versatile method that can be used in a large variety of applications. HRM technology addresses a wide range of genetic analyses such as mutation detection and mutation screening to analyze:

  • Insertions/deletions
  • Point mutations
  • Multiple mutations
These analyses can be performed for both known and unknown mutations.

Genetic variations that can be analyzed by HRM:

  • SNPs
  • CpG methylation status
  • Strain variations of microbes, plants, and animals
  • VNTRs (mini- and microsatellites)
  • Loss of heterozygosity
  • Genome polyploidy
  • Haplotypes
  • CNVs/gene copy number variations

Images
Successful genotyping of an A/T Class IV SNP using the Type-it HRM PCR Kit
Successful genotyping of an A/T class IV SNP using the Type-it HRM PCR Kit.
Typing the SNP (rs2270938) in the human GYS1 gene using the Type-it HRM PCR Kit results in highly reproducible and accurate results, but a strong variation is observed when using the master mix from Supplier QIII. A Normalized melting curve and difference plot showing successful and reliable discrimination of all 3 genotypes (wild-type, heterozygote, and mutant) of a class IV SNP using the Type-it HRM PCR Kit. B Wild-type and mutant of the same locus could not be resolved when performing the same experiment using the kit from Supplier QIII, resulting in unsuccessful genotyping. A and B Blue: wild-type; Green: heterozygous; Red: mutant.
Successful mutation detection.
Successful mutation screening.
Gene mutations (insertions and deletions in EGFR exon 19) were analyzed using the Type-it HRM PCR Kit on the Rotor-Gene Q. HRM analysis of the indicated genotypes is shown as a difference plot. Analysis was performed on the Rotor-Gene Q 5plex HRM cycler with Rotor-Gene HRM ScreenClust HRM Software. WT: EGFR Exon 19 wild type sequence, mut1: c.2235_2249del15, mut3: c.2237_2252del16insT, c.2237_2238ins18, mut4: c.2237_2238ins18, mut5: c.2239_2248del10insC, mut6: c.2240_2254del15, mut7: c.2240_2257del18.