High-Resolution Melt (HRM) PCR (CAT#: STEM-MB-0205-WXH)

High Resolution Melting Analysis (HRM) is a post PCR method. The region of interest within the DNA sequence is first amplified using the polymerase chain reaction. During this process, special saturation dyes are added to the reaction, that fluoresce only in the presence of double stranded DNA. Such dyes are known as Intercalating dyes.
During PCR, the region of interest amplified is known as Amplicon. As the amplicon concentration in the reaction tube increases the fluorescence exhibited by the double stranded amplified product also increases.
After the PCR process the HRM analysis begins. In this process the amplicon DNA is heated gradually from around 50°C up to around 95°C. As the temperature increases, at a point the melting temperature of the amplicon is reached and the sample DNA denatures and the double stranded DNA melts apart. Due to this the fluorescence fades away. This observation is plotted showing the level of fluorescence vs the temperature, generating a Melting Curve. Even a single base change in the sample DNA sequence causes differences in the HRM curve. Since different genetic sequences melt at slightly different rates, they can be viewed, compared, and detected using these curves.

• Detection of mutations, polymorphisms and epigenetic differences in double stranded DNA samples.
• gene scanning - the search for the presence of unknown variations in PCR amplicons prior to or as an alternative to sequencing.

In a Gene Scanning experiment, sample DNA is first amplified via real-time PCR in the presence of a proprietary saturating DNA dye. A melting curve is then performed using high data acquisition rates, and data are finally analyzed using a Gene Scanning Software, by three basic steps:
1.Normalization: the pre-melt (initial fluorescence) and post-melt (final fluorescence) signals of all samples are set to uniform, relative values from 100% to 0%
2.Temperature shifting: the temperature axis of the normalized melting curves is shifted to the point where the entire double-stranded DNA is completely denatured. Samples with heterozygous SNPs can then be easily be distinguished from the wild type by the different shapes of their melting curves.
3.Difference Plot: the differences in melting curve shape are further analyzed by subtracting the curves from a reference curve.

Intercalating dyes: the fluoresce only in the presence of double stranded DNA.