Wide Variety of Applications using HRM Technology

Versatile technology for a multitude of applications

HRM is a versatile and cost-effective method that has been shown to be successfully used for a diverse range of applications, as illustrated by various publications. From analysis of SNPs, mutations, insertions, deletions, and genetic variations such as microsatellites and haplotypes, to detection of changes in CpG methylation status, exploit the full potential of HRM for your research. QIAGEN offers a complete solution for HRM analysis, no matter what your application!

Fast and accurate detection of class IV SNPs

The Type-it HRM PCR Kit provides the ultimate solution for fast and accurate genotyping via HRM analysis. The kit is optimized to even enable successful analysis of genomic loci that are difficult to amplify.

Procedure: HRM analysis with EvaGreen Dye
Template: Wild-type, heterozygous, and mutant DNAs (1 ng each) for SNP rs 2270938
PCR kit: Type-it HRM PCR Kit
Instrument: Rotor-Gene Q
Assay: HRM assay for SNP rs 2270938
Analysis: Rotor-Gene ScreenClust HRM Software

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 detection of mutation

Gene mutations (insertions and deletions) are readily detected and reliably distinguished using the Type-it HRM PCR Kit, resulting in distinct melting curves.

Procedure: HRM analysis with EvaGreen Dye
Template: Wild-type and mutant DNA sequences in EGFR exon 19
PCR kit: Type-it HRM PCR Kit
 Instrument: Rotor-Gene Q 5plex HRM
Assay: HRM assay for EGFR exon 19
Analysis: Rotor-Gene HRM Software and Rotor-Gene HRM ScreenClust HRM Software

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. 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.

Reliable pathogen detection

HRM can be successfully utilized in microbiology applications such as pathogen typing. Using HRM, 8 different bacterial strains were identified, discriminating the samples accurately by analyzing the 16S rRNA gene.

Procedure: HRM analysis with EvaGreen Dye
Template: Bacterial DNA from various strains
PCR kit: Type-it HRM PCR Kit
Instrument: Rotor-Gene Q 5plex HRM
Assay: HRM assay for the 16S rRNA gene
Analysis: Rotor-Gene HRM Software

Pathogen typing using robust HRM detection. HRM primers were designed to recognize and differentiate the 16S ribosomal DNA of 8 different bacterial strains. Microbial DNA (10 pg) from each strain was used as template and amplified using the Type-it HRM PCR Kit. PCR products were sequenced to confirm the results and the robustness of the HRM detection method for this application.

Highly sensitive DNA methylation analysis

Using the EpiTect HRM PCR Kit and the Rotor-Gene Q, even low percentages of methylated DNA are detected reliably.

Procedure: HRM analysis with EvaGreen Dye
Template: Mixtures of methylated and unmethylated DNA
PCR kit: EpiTect HRM PCR Kit
Instrument: Rotor-Gene Q 5plex HRM
Assay: HRM methylation assay for the APC (adenomatosis polyposis coli) gene promoter region
Analysis: Rotor-Gene HRM Software

Highly sensitive results with detection of even low percentages of methylated DNA. Mixtures of methylated and unmethylated DNA (EpiTect Control DNA) of varying ratios were used as template. A CpG island from the promoter region of the APC gene (adenomatosis polyposis coli) was amplified and the degree of methylation was determined by HRM methylation analysis on the Rotor-Gene Q using the EpiTect HRM PCR Kit. A A standard normalized melt curve and a B difference plot normalized to the 50% methylated sample are shown.