November 15, 2014 (Vol. 34, No. 20)

Study Shows How Melting Point Analysis Infers the Presence of and Identifies SNPs

The melting temperature (Tm) of a PCR product is dependent on both the length and sequence of the DNA. Melting curve analysis has traditionally been used to identify nonspecific products or contamination following amplification in qPCR. 

During melting curve analysis, the temperature of the PCR product(s) is gradually raised until the double-stranded DNA (dsDNA) begins to dissociate. The temperature at which this takes place is dictated by the DNA sequence itself. While G-C base pairings have three hydrogen bonds between them, A-T base pairs have only two, causing them to break apart more easily. DNA with a higher G-C content will therefore have a higher melting temperature than DNA with a higher A-T content.

Information gathered through this technique can also be used to infer the presence of and identify single-nucleotide polymorphisms (SNPs).

Since the presence of a SNP will modify the sequence of the DNA it is likely that it will also change the Tm. Therefore, melting analysis in qPCR has become a widely used technique for characterizing PCR products in addition to checking for nonspecific products or contamination after amplification.

Completing Melting Point Analysis

During melting point analysis, a melting curve is generated by slowly denaturing the DNA in the presence of a dsDNA binding dye. Fluorescence data is collected at very small temperature increments, allowing the researcher to obtain the melting curves at extremely high resolution.

When variations in sequence are present, they result in differences in Tm—the point at which 50% of the DNA is double stranded and 50% is single stranded. These changes in Tm, together with the shape of the melting curves, can characterize genetic variations.

This technique requires the use of an intercalating dye, which will bind to the minor groove of dsDNA producing up to a thousand-fold increase in fluorescence.

Third-generation fluorescent dsDNA dyes are able to be used at higher concentrations than traditional dyes, thanks to their lower toxicity. This allows for greater saturation of the dsDNA and increased sensitivity, high fidelity, and better resolution in melting curve profiles, ensuring a more accurate analysis. This, combined with specific reagents and software, results in a powerful technique for high-throughput screening for genetic variation.

In the below investigation, a single base mismatch was identified using melting curve analysis comparing reagent kits containing both traditional and third-generation dsDNA intercalating dyes.


Two artificial 100-base single-stranded DNA (ssDNA) templates were synthesized based on the genomic sequence of Enterobacteria phage lambda. While the DNA sequence normally has a G base at position 16404, in this case one of the templates was synthesized with a mutant A base.

Primers were designed to amplify a 76-base-pair product within the templates, encompassing the base change. The final primer concentration was 0.2 μM and four different reagent kits were used for amplification and detection of the products (hereafter referred to as kit 1, kit 2 … kit 4). A passive reference dye (PRD) was added to each mix except for one, where the reagent kit already contained a PRD.

Approximately 5 × 105 copies of either template were used per reaction. Six replicates of either template were run for each kit plus two no-template controls (NTC).

The reactions were amplified for 35 cycles (initial denaturation, 95°C, 2 min; 35 cycles of 95°C, 5s; 60°C, 20s) using a PrimeQ real-time PCR system from Techne (part of Bibby Scientific). At the end of the amplification stage, a ramp stage was performed (72°C to 91°C in 0.2°C steps, hold time 10 seconds per step).


Single base changes in DNA, or SNPs, are classified into four groups (Table 1). G to A base changes, as used in this investigation, are designated as class 1 SNPs and are one of the easiest types to detect due to the relatively large shift in Tm; they are also one of the most frequently found classes of SNPs in the human genome.

The amplification curves obtained with kits containing third-generation dsDNA dyes continued to amplify in an extended linear phase when compared to kits containing traditional intercalating dyes and this resulted in a much higher final fluorescence at the plateau phase. This demonstrates the ability of third-generation dyes to reach much higher saturation levels with the DNA without causing inhibition of the PCR.

The ramp stage was analyzed using dissociation curve analysis. Figure 1 shows the melting curves and melting peaks for both the “wild-type” and “mutant” templates using each of the kits. In each case, the six replicates for each template type were clearly distinct.

Figure 1. Dissociation curves and melting peaks for each of the templates obtained with the reagents used. The wild type (G template) is shown in green and the mutant (A template) in red.

For further analysis, the raw fluorescence data for the samples amplified using kit 4 were exported to Excel. The melting curves were normalized between 78°C and 91°C such that the fluorescent signal at 78°C was set to 100% and the signal at 91°C, 0%. This “tidies” the data and aids in interpretation.

To further enhance the differences between curves, they can be plotted on a difference graph. A curve is selected as a reference and the difference in fluorescence between the samples and reference is plotted. These two graphs are shown in Figure 2.

Figure 2. Normalized melting curve data and difference curves for wild-type and mutant samples amplified using kit 4. The reference used for the difference plots was one of the wild-type melting curves.

The Tm of a product is strongly influenced by the reaction mixes, which may vary in additives and salt concentrations. This is apparent from the data presented in Figure 3, where the Tm for the wild-type sample was found to vary between 81.61°C and 83.76°C. The shift in Tm (ΔTm) also varied quite significantly depending on which kit was used. Of the reagents tested, those containing traditional intercalating dyes (kits 1 and 4) showed the greatest shift in Tm between the wild-type and mutant PCR products.

Figure 3. Tm values for the PCR products determined in each of the reaction mixes.


Melting analysis in qPCR has traditionally been used to identify nonspecific products or contamination following amplification. As an extension to this, the application of high-resolution melting (HRM) uses the small variations in Tm together with the shape of the melting curves to characterize genetic variations. Using reagents and dyes designed specifically for HRM, along with specialized software, results in a powerful technique for high-throughput screening for genetic variation.

The above investigation designed a class 1 SNP variation and demonstrated that it is possible to distinguish a single base pair mismatch (G/A) using the standard melting curve analysis software based on a change in Tm of the product. This analysis can be further enhanced by generating normalized and difference curves. In addition, the products were easily distinguishable using all of the intercalating dyes tested.

For more information, the application note detailing this study can be downloaded at

Jayne Bates ([email protected]) is technical support manager at Bibby Scientific.

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