Critical Success Factors for HRM Performance

Factors influencing success in HRM experiments

 

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Outstanding performance in HRM analysis requires template purity, highly specific HRM PCR kits with a saturating dye, a real-time instrument with HRM capabilities that ensures superior temperature uniformity, and a powerful software package for data analysis. Click on the links below and read more about the critical factors that influence HRM performance.

Reaction chemistry
Template quality and amount
Successful primer and assay design
Instrument, software, and analysis settings

Reaction chemistry
Specific amplification is vital for reliable HRM analysis. It is critical to be certain that differences in the melting curves are due to variations in the sequence of the template DNA, and not due to the presence of any PCR artifacts. It is strongly recommended to check the PCR product by classical melting curve analysis before subjecting it to HRM analysis. Reliable PCR chemistry is essential for successful HRM analysis.

Reliable PCR enzymes
High-quality PCR, or amplification with high sensitivity and specificity, is a prerequisite for successful HRM results. Optimal reaction chemistry together with highly stringent HotStarTaq Plus DNA Polymerase is implemented in the Type-it HRM PCR Kit and the EpiTect HRM PCR Kit, ensuring highly reliable results.

PCR buffer for specificity
The innovative PCR buffer included in the Type-it HRM PCR Kit and the EpiTect HRM PCR Kit maintains specific amplification in every cycle of PCR by promoting a high ratio of specific-to-nonspecific primer binding during the annealing step in each PCR cycle. Owing to a uniquely balanced combination of KCl and(NH4)2SO4, the buffer provides stringent primer-annealing conditions over a wider range of annealing temperatures and Mg2+ concentrations than conventional PCR buffers. Optimization of PCR by varying the annealing temperature or the Mg2+ concentration is therefore often minimal or not required. The unique buffer ensures specific amplification of even the most challenging genomic loci (see figure Highly specific and successful amplification of difficult genomic loci). Watch this animated video and discover the benefits of QIAGEN's unique PCR buffer system.

Optimized HRM kits from QIAGEN
Unlike HRM kits from other suppliers, the unique features of the Type-it HRM PCR Kit ensure amplification and discrimination of even the most challenging, subtle sequence differences, including class IV SNPs (see figure Successful genotyping of an A/T class IV SNP). Highly sensitive detection of low levels of methylated DNA is enabled by the EpiTect HRM PCR Kit (see figure Highly sensitive results — detection of even low percentages of methylated DNA). 

Template quality and amount
Since HRM can reveal very subtle differences between samples, the effect of template purity on HRM analysis plays an important role. HRM can easily detect contaminants in samples, which can lead to misinterpretation of results. This is valid for a number of reagents that are typically used in home-brew DNA purification methods such as various salts or ethanols that are used for precipitation and washing of pellets.

Typical contaminants when using home-brew methods include: 
  • NaCl
  • KOAc
  • EDTA
  • ETOH
  • Isopropanol
  • Sodium citrate
  • Phenol

Effect of contaminants on HRM performance
As the concentration of contaminants increases, their effect in subsequent HRM analysis also increases. Although contaminant concentrations up to a certain level are not detectable, a clear shift in the melting temperature towards higher or lower values (depending on the contaminant) can occur (see figures Presence of salt increases the Tm  and Presence of alcohols decreases the T). Therefore, it is recommended to consistently use a standardized purification method to avoid data misinterpretation and false positives. QIAGEN offers a wide range of DNA purification products to meet these requirements. 

Successful primer and assay design
Meticulous assay and primer design is critical for setting up a successful HRM experiment. Follow these simple guidelines to ensure success in your HRM analysis.

Successful assay design
  • Design assays with PCR product lengths of 70–350 bp.
  • For SNP analysis, the use of PCR products of 70–150 bp is recommended. Larger products can be analyzed successfully, but usually provide lower resolution. This is because a single base variation has a greater effect on the melting behavior of, for example, a 100 bp PCR product than on a 350 bp PCR product.
  • Initially, determine the melting point for each new HRM PCR product. Run HRM analysis to span a temperature range from 65°C– 95°C, covering the full range of expected melting points. To save time in future experiments, after determination of the Tm, run HRM from 5°C below the lowest Tm of all expected PCR products to 5°C above the highest Tm of all PCR products in your experiment.
  • HRM assays can be designed using PyroMark Assay Design Software 2.0. The only requirement for HRM is PCR primers.
  • Design assays with a single melting domain. This can be supported by prediction tools such as MeltSim or Poland 8.
 
Recommended primer specifications
  • Design primers allowing specific amplification. Perform a BLAST search to ensure specific primer binding. Check similarity to other known sequences with BLAST or primer-BLAST.
  • Primers should be 18-30 nucleotides in length.
  • The melting temperature of primers used should be at least 56°C.
  • The melting temperature of primers can be calculated using the formula:

        Tm = 2°C x (number of [A+T]) + 4°C x (number of [G+C])

  • Whenever possible, design primer pairs with similar Tm values.
  • Primers should have a GC content of 40-60%.
  • Avoid complementarity of 2 or 3 bases at the 3' ends of primer pairs to reduce primer dimer formation.
  • Avoid mismatches between the 3' end of the primer and the target template.
  • Avoid runs of 3 or more G and/or C at the 3' end.
  • Avoid complementary sequences within primers and between primer pairs.
  • Ensure primer sequence is unique for your template sequence.
  • Check the concentration and integrity of primers before starting. Typically, standard-quality primers are sufficient.

 


Instrument, software, and analysis settings
 
Instrument precision is imperative for accurate HRM results. The thermal variability from sample-to-sample must be minimal and very high levels of temperature uniformity are required over the entire HRM run. In addition, there are enhanced requirements for the detection optics. A combination of minimal detection noise, high stability of excitation, and optimized detection ensure comparability between the samples. A high density of data points per degree thermal transition is also needed. Only when all these prerequisites are fulfilled, can subtle differences from sample-to-sample be revealed in a reliable manner. Click on the links below to learn more.

Instrumentation prerequisites include:

Thermal precision

Thermal variability from sample-to-sample must be minimal. For example, to reliably detect homozygous SNP mutants, which can easily differ in Tm by <0.2°C (class IV SNPs), a temperature uniformity of 0.1°C or better is required.

Optical precision

  • Low "noise" from detector system
  • High stability of excitation light
  • High intensity excitation tuned to the optimal dye wavelength
  • High data density (i.e., a high number of data points per degree thermal transition)

 

List of cyclers with HRM capabilities

  • Rotor-Gene Q
  • Applied Biosystems 7500, 7900, ViiA 7 cyclers
  • Bio-Rad (CFX96 and 384)
  • Roche Lightcycler 480
  • LightScanner System (IdahoTechnology Inc.)
  • Eco Real-Time PCR System (Illumina)

QIAGEN's Rotor-Gene Q real-time PCR cycler, with its unique and innovative rotary design, is particularly well-suited for all HRM applications, including detection of class IV SNPs (see figure Successful genotyping of an A/T class IV SNP). The outstanding HRM performance of the Rotor-Gene Q is powered by an unrivalled low-temperature uniformity of less than 0.02°C and a special optical design. The superiority of Rotor-Gene Q technology for all HRM applications has been independently confirmed by various researchers and is documented in many publications.

Software prerequisites
To exploit the extensive information content of HRM experiments and enable reliable HRM analysis and data interpretation, powerful HRM software packages are required. Typical HRM data analysis discriminates between genotypes by comparing the position and shape of melting curves of different samples. For example melting curves of different homozygotes differ in their melting points (Tm) and melting curves of different heterozygotes differ in their shape and melting points (see figure HRM analysis of homozygous and heterozygous samples). In standard HRM software packages, variations in melt curve shape and Tm position compared to a control are used to differentiate between samples and to assign the corresponding genotype. A typical workflow as performed with the Rotor-Gene Q standard software package is depicted in the figure Guidelines for successful HRM. While this standard method of HRM analysis works well for many genetic variations, it has some limitations. Often, difficult-to-interpret results require additional time-consuming manual data interpretation and controls are required for accurate genotype classification. In contrast, Rotor-Gene ScreenClust HRM Software uses innovative mathematical algorithms to characterize samples and group them into clusters. This statistical approach exploits the entire information content of the HRM experiment, which enables discrimination of even the most difficult class IV A/T SNPs with differences in melting temperatures as low as 0.1°C in a standardized way. Additionally, the statistical approach allows a classification of genotypes without control samples using the unsupervised mode of the Rotor-Gene ScreenClust HRM Software. This hypothesis-free method enables discovery of new mutations in the data, when there is limited or even no knowledge of the genotypes present in the samples.


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