Melting Point Analysis with the LightCycler® Carousel-Based System
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The temperature at which DNA strands melt (separate) when heated can vary greatly, depending on the sequence, length, and GC content. Even single-base differences in heterozygous DNA can change its melting profile. Thus, melting temperature profiles can be used to identify and genotype DNA products.
To determine melting profiles, the instrument steadily increases the sample temperature while monitoring its fluorescence. Sample fluorescence decreases as the temperature increases. In a SYBR Green I assay, this is because the double DNA strands separate and release SYBR Green I molecules. In a HybProbe assay, it happens because target-probe hybrids melt, leading to spatial separation of the dye molecules and a drop in fluorescence.
For SimpleProbe probes, fluorescence decreases during heating because the signal from the probe is quenched as the probe is displaced.
Figure 1: Melting chart of a HybProbe probes experiment.In the example shown,some sample curves have two peaks. These dual peaks indicate heterozygous samples. After DNA amplification, a heterozygous sample contains two DNA sequences, each of which melts at a different temperature, resulting in a two-peak curve.
You can use the Genotyping module in any experiment that contains a melt program. The Genotyping module determines the genotypes of unknown samples by analyzing the shapes of all sample melting curves and then grouping curves with similar shapes. The median curve from each group is designated the “genotype standard” for that group. Alternatively, the software allows you to include standards with known genotypes in the experiment or to import results from previously analyzed standards. LightCycler® Software 4.1/4.05 generates two numerical values, which are used to describe how well individual sample curves match similar curves (see Figure 2).
Figure 2: Genotyping module of the LightCycler® Software 4.1/4.05
The “score” value measures similarity between the sample and the standard that is the nearest match to the sample. A score of 1 indicates a sample has a melting curve identical to the melting curve of at least one standard, while a score of nearly 0 indicates a sample has a melting curve unlike that of any standard. The “resolution” value measures dissimilarity between the sample and the standard that is the second nearest match to the sample. If the resolution of a sample is nearly equal to the score of that sample, its melting curve is similar to the melting curve of only one standard. Alternatively, if the resolution of a sample is close to 0, the melting curve of the sample is similar in shape to the melting curves of two standards.
SNP Genotyping and Mutation Detection with SimpleProbe Probes
As the temperature increases during SNP analysis, the LightCycler® Instruments monitor the melting of the SimpleProbe probes by measuring the decrease in fluorescence. The more stable the hybridization between the SimpleProbe and the target, the higher the melting temperature. Mutations, such as SNP, weaken the binding of the SimpleProbe probe. When the results are analyzed with the LightCycler® Software 4.1/ 4.05 Genotyping or Tm Calling module, these probes can easily distinguish between wild type, mutant, and heterozygous samples.
Figure 3: Genotyping of a C/T polymorphism with the LightCycler® Software 4.1/4.05. The upper graph shows sample fluorescence versus temperature. A clear drop in the fluorescent signal can be seen for each sample as the temperature increases. The lower graph plots the first negative derivative of the sample fluorescence versus temperature, and shows the melting temperature of each sample as a peak.
Figure 4: You can use the Tm Calling module in any experiment that includes a melt program. The melting temperature, or Tm, is defined as the point at which half the probes (or dye) have melted off the DNA.
The results from a Tm Calling analysis include the melting temperature of each sample, the height and width of its melting peak, and the area under the melting peak.