I have done methylation specific PCR for p21 and p27 with prostate cancer cell lines, LNCaP and DU145 but I didnot get any band for methylated as well as unmethylated allele for both the genes. I have taken primers from a paper:Methylation Analysis of Cyclin-Dependent Kinase Inhibitor Genes in Primary Gastrointestinal Lymphomas. Mod pathol 2003.I want to check whether these primers are appropriate or not? So I wanted to check them on meth primer but I dont know how to check ? Can somebody tell me how can we check whether these primers are working or not based on meth primer tool or if somebody has working primer sequence for methylation specific PCR of human P21 and P 27 then please provide me the sequence of primers or promoter seq of these two genes so that I can design primers for these using methprimer. I do not know how to get promoter seq . of genes? Please help me in this regard.
Thank you
First of all check if the sequence of primers is correct, compared to the human genome database. In many articles the primers are given with mistakes (mismatches, directions, etc). If all ok, then check primer's stability in gel, in some cases the primers are not properly synthesized (degradation or on different concentrations).
Here's a link to one of the sequences:
https://www.ncbi.nlm.nih.gov/nuccore/17981697
Check methprimer to see if your primers are compatible or create new. Make sure your DNA is completely bisulfite modified by sequencing your product.
Hi,
you can check the sequences of your primers with the bisearch tool (primer search, ePCR):
http://bisearch.enzim.hu/
Have you checked the quality of your bisulfite DNA with other primers?
Thank you Anrey sir and timonthy sir for replying but for designing primers for p21 , I need promoter seq as I want to check effect of my drug on promoter specific methylation of p21 and I donot know how to check primers on meth primer . I want to know how to check primers on meth primer?
I faced the same problem and it seems that it was in the bisulfite conversion ( I mean the efficiency of conversion). make sure to strictly follow kits instruction
The following links will help you to design primers for methylated DNA.
1- First link has all the human genome sequences, including the promotor regions
http://genome.ucsc.edu/
2- second link to detect CpG island in promotor region of the gene under study, you will copy the promotor sequence of the gene from the previous link and you will paste it in the empty box you will find in the following link.
http://www.uscnorris.com/cpgislands2/cpg.aspx
3- Third link to design primers for methylation either for MSP primers or for sequencing, We want primers for MSP.
You will copy and paste the region with high cpg Island in the box you will find in the following link
http://www.urogene.org/methprimer/index1.html
Good luck
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