PCR - Long PCR with short primers (Apr/18/2005 )
I would like to perform a long-PCR (about 5.5 kb) with 6 nucleotide long primers. The primers are most likely too short, but I have heard about a technique were you can use primers extended with nonsense nucleotides. Does anyone have an idea what I could do, or have a reference I would be happy
A primer length of 6 bp is very short, in fact so short that I suspect that you will never be able to get a specific product, let alone a 5.5kb product, due to the relative frequency of bases in the genome.
I think that if you try to extend the primers with nonsense nucleotides, you will end up with a situation similar to RAPD profiling, where a random oligo is used in PCR to generate a "unique" profile (lots of issues there, but I won't go into it) and you never get a single band. i.e your primer of 6 bp is so short that randomly, the sequence will turn up in the genome every 4 to the power of 6 bases (4096 bp). Adding random nucleotides to this will only lower the chance of it repeating, and will still not necessarily include your gene of interest.
Long PCR can also be problematic in that you need specific polymerases to amplify anything over about 2 kb (taq falls off about there), as well as a proofreader, or the error rate of amplification increases with length.
Conclusions... design longer primers.