We would like to perform Methylation-Specific PCR using TaqMan primers and probes. With this method, we wanted to determine the DNA methylation of the promoters of the A (PGRA) and B (PGRB) isoforms of the progesterone receptor. The primers for methylated and unmethylated sequences were designed using Methyl Primer Express (Thermo Scientific), and the probes were further designed in the PrimerQuest Tool. The Tm temperatures of the primers and probes were chosen to attach at 600C in Life Technologies' Viia & and 7900 HT thermocyclers. Bisulfite-converted genomic DNA was used as the template. Real-time PCR reaction conditions: 950 C for 10 min initial denaturation and 50 cycles of 950 C for 15-sec denaturation and 600 C for 1 min primer attachment and elongation. Unfortunately, as a result, amplification occurred correctly only with primers and probes for unmethylated sequences while for methylated sequences primers and probes gave poor or no reads. However, agarose electrophoresis of the products after real-time PCR showed that the amplification products were also for primers and probes for methylated sequences. It seems that the problem is the probe that does not give the right signal. I don't know may also the problem that is too little DNA as a template or if that template DNA is after conversion. We used probes only because MS-PCR using primers showed amplification of many products in the melting curves of the PCR products. Perhaps you have encountered such a problem and could help. I could design new probes and primers but I am afraid of repeating the problem. In the attached file, I present amplification plots and a photo from agarose electrophoresis of the products after real-time PCR.
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology