I have a primer for a Methylation-Specific PCR (MSP) taken from the previous study and I want to design a primer for sequencing bisulfite-converted DNA for the same region that MSP primer bind to the DNA. So, how can I find the region and design the primer and what is the difference between two primers (MSP primer and sequencing primer)?
The main difference between BS and MSP primers is that the later are designed to selectively amplify a DNA región based on its methylation status, they deliverately contain CpG dinucleotides within their sequence in order to give a signal if a certain condition is met (e.g. ATTACCACG will work only if the "C" in the CpG dinucleotide is methylated; primer sequence in Bold). Considering the previous example your PCR will be successful only if there is methylation on the CpG dinucleotide.
In the other hand, BS primers will be designed to amplify a given region, independently of its methylation status. In this case it is recommended to avoid CpG dinucleotides within your primer sequence in order to avoid amplification bias due to differences in annealing temperatures of the primers depending on the methylation status of the template, but even in this cases it is normally accepted to include, at most, one CpG dinucleotide within your BS primers and if this is the case you will need to design primers to amplify the template in both scenarios (methylated or unmethylated).
To find the region that you want to amplify you need to know which gene (or original sequence) you are trying to analyze. If you only have the MSP primers sequence (already desinged to amplify bisulfite converted DNA) it will be more difficult, since you will need to determine the primer sequence as if it was designed to amplify normal DNA. If the later is your case i will use blast and try to identify the region that the MSP primers will amplify, it will not be that simple though.
Once you have identified the region you wish to analyze it will be quite simple to obtain the BS primers. Most people use http://www.urogene.org/methprimer/ to desing primers for DNA methylation analysis, it works quite good. You will just need to input your sequence and the plataform will make the rest.
Hope this helps.
The main difference between BS and MSP primers is that the later are designed to selectively amplify a DNA región based on its methylation status, they deliverately contain CpG dinucleotides within their sequence in order to give a signal if a certain condition is met (e.g. ATTACCACG will work only if the "C" in the CpG dinucleotide is methylated; primer sequence in Bold). Considering the previous example your PCR will be successful only if there is methylation on the CpG dinucleotide.
In the other hand, BS primers will be designed to amplify a given region, independently of its methylation status. In this case it is recommended to avoid CpG dinucleotides within your primer sequence in order to avoid amplification bias due to differences in annealing temperatures of the primers depending on the methylation status of the template, but even in this cases it is normally accepted to include, at most, one CpG dinucleotide within your BS primers and if this is the case you will need to design primers to amplify the template in both scenarios (methylated or unmethylated).
To find the region that you want to amplify you need to know which gene (or original sequence) you are trying to analyze. If you only have the MSP primers sequence (already desinged to amplify bisulfite converted DNA) it will be more difficult, since you will need to determine the primer sequence as if it was designed to amplify normal DNA. If the later is your case i will use blast and try to identify the region that the MSP primers will amplify, it will not be that simple though.
Once you have identified the region you wish to analyze it will be quite simple to obtain the BS primers. Most people use http://www.urogene.org/methprimer/ to desing primers for DNA methylation analysis, it works quite good. You will just need to input your sequence and the plataform will make the rest.
Hope this helps.
Thank you, Mauricio de la Rosa, for all these information actually I've tried using blast but it could not identify the region, I used also the unmethylated primer to identify it in blast but also nothing appeared and the primers used by different papers so it must be well known, is there any instructions that could help me please?
A normal Blast won´t work in this case, due to the bisulfite treatment and consquent replacement of Cs with Ts (Us).
Check this tread:
https://www.researchgate.net/post/Where_or_how_can_I_blast_BSP_primers_of_fish
Unfortunately the Blastmeth site (http://medgen.ugent.be/MethBLAST/) is currently not working, in the web page they mention other software that you could use, although you´ll need to install it on a computer.
I fully agree on the above answers. If you chose to do MSP, you could/should consider to use modified primers and probes (like SuPrimers, EasyBeacons, MGB TaqMan and/or HydrolEasy probes) for your reaction, as the affinity of those are higher than standard primers and probes.
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