4 replies to this topic

[Swongel]

Hi everyone,

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.

Many thanks,

[bob1]

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.

[gonca]

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

[bob1]

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.

[ForEpi]

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).

Bram