I am working in the methylation status of some genes in cancer patients. We recruited clinical samples from patients with and without cancer. We designed methylation specific primer and unmethylated primers. When we amplified the target sequences some genes showed a smeared or multiple bands. Please see the picture below and comment to the best of your knowledge:
The cause of nonspecific bands in MSPCR,
Since, your amplifying your genomic DNA and also after bisulfite conversion its no more genomic DNA; the primer should be more specific to the region of interest and how much you used for your reaction; whether it forms dimers etc.
Polymerase which you used for your PCR, it should be Hot start polymerase.
Check with Positive and Negative control DNA with your Primer and hotstart pol and check the band.
All you need to standardize; complete bisulfide conversion without affecting the template much. Primer, polymerase etc.
And final thing you have to focus on your Annealing Temp. Set a gradient Protocol and check.
It will work.
All the very best Fawaz Naji Saleh Al-Shaheri
Dear Rajesh Parsnathan,
Thank you so much for your valuable advises.
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