PCR primers help - (Dec/29/2011 )
I have a forward primer and a 1:1 mix of 2 types of reverse primers. Both the reverse primers start in the same place, only a few bases are different between them and that they are mixed in 1:1 ratio.
If I use these primers on genomic DNA, will they amplify a product especially since the reverse is a 1:1 mix of 2 different primer sequences? Has anyone heard of others doing this type of approach? Thanks.
Yes, you will amplify either of the matching sequences.
Phage, thanks for the reply. Do you have any articles that describe this approach? I would like to learn more about it.
google for 'degenerate pcr' or 'wobbles' there are lots of information available.
You can think of this as a kind of multiplex PCR with primers that just happen to be quite similar.
I use this kind of PCR for determining HLA B27 status of patients. Here, I use two reverse primers to amplify either of two variant of HLA DR gene, which is used as an internal control. The reference is:
Tonks S, Marsh SGE, Bunce M, Bodmer JG. Molecular typing for HLA class I using ARMS-PCR: further developments following the 12th International Histocompatibility Workshop. Tissue Antigens. 1999; 53:175-83.
I hope this helps.