Biotinylated primers - (Dec/10/2012 )
In preparation for a methylation analysis using pyrosequencing, I have designed forward and reverse primers. My forward primer will be biotinylated. Now, I understand that I also have to design a sequencing primer. My question is, will it not be possible to use my reverse primer as a sequencing primer rather than design a new primer for sequencing?
Would be great to get your comments on this.
I think it should be OK, but it will be a lot more expensive to use than a regular primer. I don't know how the biotinylation will affect migration in the sequencing capillary though.
Hi, sorry I dont have idea regarding the answer of your question but I have a question to you. I will start a similar study and would like to ask how you designed your primers and how are they gonna be biotinylated? what sort of analysis you plan to do? I would appreciate your reply. Thanks
You can buy biotinylated primers from most of the large providers. Primers can be designed manually, but there are many free programs/websites that offer primer design. The best known of these are Primer3 and Primer-Blast at the NCBI.
It's best to use nested sequencing primers for pyrosequencing as you will increase the specificity of your assay (remember you're only dealing with 3 instead of 4 bases).