Possible mutations in primers? - (Mar/01/2007 )
Our lab has been having problems with cloning lately...we're doing basic cloning with PCR amplification of antigens of interest with restriction sites in the primers for directional cloning...we then digest the vector and PCR product with RE and ligate and transform into E.coli DH5a, followed by subcloning.
Our tests for expression of the protein of interest were negative, so we sequenced our constructs, and found that their were **frameshift addition/deletion mutations** specifically in the region that corresponded to the primer region.
We have found this in 7 different constructs (5 sets of primers ordered on one day; the other two were on separate days)....to add to our confusion, some of the same primers have been used to successfully clone in and get expression of the antigen of interest...we have used high fidelity Pfu for all of our cloning...the largest insert size was 1.1 kb, and sthe smallest was 500bp...
We can't imagine that these are random mutations since they are all specifically ONLY in the primer region...suggesting the primers may be wrong? or that we had a mix of correct and incorrect primers? on the other hand, we also don't understand how so many different primers could have mistakes.
We are wondering if anyone else has had similar problems. In the meantime we are ordering new primers and restarting the process, and will be sequencing our products after the first cloning step before subcloning. Thanks for any info!
Primers often have point deletions or shortened 5' ends. These are normally a small fraction of the primers, but definitely not negligible. My suspicion is that the expression of this gene is toxic, and that you are selecting for clones which have point mutations in the promoter region to reduce or eliminate expression.
we did have a similar problem some time back. There was an error in the primer. So had to start from scratch, and finally we had the right sequence.
This can happen if you have very long primers.
phage 434 could be on the money, I have had 2 completely different constructs turn up with mutations on the start codon (ATG), I think this happened because the protein was not well tolerated in E. coli. This also happened after I used a hi-fi PCR system...
Hi,
Had the same prob, the company synthesizing primer said it's a prominent problem if the primer length is very large, i.e. >50 mer. They recommended PAGE purification to remove these.
http://www.protocol-online.org/forums/inde...showtopic=25022
Anyway, I think it's best to pick more colonies to screen and sequence and look for the right one, if you're not into PAGE purification. When I screened 10 colonies, 7 out of 10 were what I wanted. It's not so bad.
Bests