I would like to design primers for HRM methylation analysis. Did any of you use the Methyl Primer Express software to design HRM primers. The software has two options for designing such primers such as MSP or BSP primers. Is it necessary to use the MSP or BSP option for primer designing and whether the default settings of the software are sufficient for this action?
Dear Robert...
Here you will find some clues, it may help. For more details you can visit the following site:
https://www.thermofisher.com/iq/en/home/life-science/pcr/real-time-pcr/real-time-pcr-learning-center/genotyping-analysis-real-time-pcr-information/dna-methylation-analysis-using-hrm-dna-sequencing.html
"PCR primer design for MS-HRM is critical—the free online tool from Applied Biosystems, Methyl Primer Express® Software v1.0, was developed specifically for primer design in methylation studies (www.appliedbiosystems.com/methylprimerexpress). Authors recommended the designing of several sets of PCR primers for each genomic DNA region of interest, testing them for specificity and the ability to discriminate methylated from unmethylated DNA, and selecting the best-performing assay(s) for analysis of experimental samples. There are three main criteria to consider in designing PCR primers: amplicon length, number of potential methylation sites in the amplicon, and inclusion of CpG dinucleotides in the PCR primer sequence. All of these factors can have a big influence on the sensitivity of the assay, and in some cases they can be manipulated to discriminate large or small differences in methylation states".
Best luck
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