Fast and Accurate Genetic Analysis with HRM

Fast and accurate genetic analysis with HRM

Powerful and cost-effective solution for analyzing complex sequence variations

High-resolution melting (HRM) is a novel technology for fast and accurate genetic analysis. HRM is a powerful and cost-effective solution for analyzing previously unknown and even complex sequence variations in a multitude of applications.

HRM technology characterizes double-stranded PCR products based on their melting behavior, as they transition from double-stranded DNA to single-stranded DNA with increasing temperature. HRM offers unique advantages over other technologies that make it a definitive analysis and detection technology for various applications.

QIAGEN offers a complete solution for HRM-based genetic analysis. From dedicated HRM PCR kits to a real-time instrument with HRM capabilities and a powerful software package for data analysis — optimized solutions for HRM analysis are simply a click away!

Choose the right product for your needs with our helpful selection guide.

Selection guide

Application	Amplification kit with EvaGreen Dye	Instrument with HRM analysis software
SNP and mutation genotyping	Type-it HRM PCR Kit	• Rotor-Gene Q & Rotor-Gene ScreenClust HRM Software • LightCycler 480 • ABI 7500 • Other cyclers with HRM capability
Mutation screening	Type-it HRM PCR Kit	• Rotor-Gene Q & Rotor-Gene ScreenClust HRM Software • LightCycler 480 • ABI 7500 • Other cyclers with HRM capability
Pathogen detection	Type-it HRM PCR Kit	• Rotor-Gene Q & Rotor-Gene ScreenClust HRM Software • LightCycler 480 • ABI 7500 • Other cyclers with HRM capability
CpG analysis (with quantification of methylation status)	EpiTect HRM PCR Kit	• Rotor-Gene Q • LightCycler 480 • ABI 7500 • Other cyclers with HRM capability
CpG analysis (without quantification of methylation status)	EpiTect HRM PCR Kits	• Rotor-Gene Q & Rotor-Gene ScreenClust HRM Software • LightCycler 480 • ABI 7500 • Other cyclers with HRM capability

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.

Successful genotyping of an A/T Class IV SNP. HRM was performed using A a kit and instrument from Supplier R and using the B Type-it HRM PCR Kit and the Rotor-Gene Q. The Type-it HRM PCR Kit, in combination with the Rotor-Gene Q, results in clear discrimination and highly accurate results. In contrast, the Class IV SNP could not be detected using the kit and instrument from Supplier R. Red: wild-type (AA); blue: mutant (TT); green: heterozygous (AT).

Guidelines for successful HRM analysis. Successful HRM requires five steps – from PCR to result.

Presence of salt increases the Tm: The mere presence of the salt simulates differences between the samples, but these differences are not due to differences in the DNA sequence. The higher the NaCl concentration (100 mM), the higher the resulting Tm.

Presence of alcohols decreases the Tm: A clear decrease in the melting temperature is apparent with increasing amounts of alcohol.

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.

Analysis of homozygous and heterozygous samples by HRM. A Different homozygous samples are differentiated by distinct melting points. The base ‘T’ in wildtype samples (green) is substituted by a ‘C’ in mutant samples (red). All samples are homozygous for these SNPs, which means that both alleles are identical. As expected, a shift to a higher melting temperature can be clearly seen in the normalized melt curve upon exchange of an AT base pair by a GC base pair. This melting behavior also holds true for mutations such as deletions or insertions, as well as the melting profile of DNA from different microbial strains. B Heterozygotes form mixtures resulting in a different melt curve shape. When the same SNP is analyzed, but with samples that are heterozygous for this SNP (which means that one allele consists of a T whereas the other consists of a C), hybrids of DNA strands arising from both alleles will form towards the end of the PCR, after they melt and reanneal. Such hybrids will have a mismatch at the SNP position, destabilizing the double-stranded PCR product. When the amplification products from such heterozygous samples are subjected to HRM analysis, a characteristic melt curve is observed, clearly differing from the two different homozygous samples. The fluorescence change detected is a superposition of fluorescence changes for all PCR products present in the sample, resulting in this typical shape for a melt curve.

A typical HRM plot. The melt curve plots the transition from the high fluorescence of the initial pre-melt phase, through to the decrease in fluorescence of the melt phase, to the basal level of fluorescence at the post-melt phase. Fluorescence decreases as the DNA intercalating dye is released from dsDNA as it melts into single strands. The midpoint of the melt phase at which one half of the DNA duplex will dissociate and become single stranded, defines the melting temperature (Tm) of the DNA under analysis.

Differences between classical melt curve analysis and HRM analysis.

Highly specific and successful amplification of difficult genomic loci due to the unique HRM buffer system. A Type-it HRM PCR Buffer ensures highly specific amplification even of difficult genomic loci (Human ENG gene, Exon 11, 356 bp fragment, 64% GC). Only the expected specific PCR products are obtained. B In contrast, using the kit from Supplier R results in nonspecific products, even with prior optimization of Mg2+ and cycling parameters.

Principle of methylation analysis of the APC promoter by HRM. A Genomic DNA. All cytosines that are not part of CpG islands are symbolized by X. CpG islands containing potentially methylated cytosines are indicated as CG. B Bisulfite conversion of genomic DNA. All unmethylated cytosine residues are converted to uracil. Cytosines within CpG islands are indicated by Y and are either converted to U (if unmethylated) or remain unmodified (if methylated).

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.

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.