High fidelity PCR - confusion (Jul/10/2005 )
Can anybody help me with some questions concerning picking the best enzyme for high fidelity PCR. The problem is that when browsing through manufacturer's websites, it's really hard to get clear information.
I checked for invitrogen's "Platinum pfx" and it states to have a fidelity of more than 26 times that of regular taq.
When checking for Stratagene's "PfuUltra" they state to have an error rate of 18 times lower that regular taq.
Invitrogen used 2 assays to determine fidelity (check this site browse to page 7) and stratagene uses one assay (check right here for the description of their assay)
One of Invitrogen's asays is the same stratagene uses, but stratagene claims that the error rate of PfuUltra is lower than that of Platinum pfx (table 1 on this page). Also, invitrogen claims that pfu polymerase has an error rate of 14 times lower than taq, whereas stratagene claims PfuUltra is 300% better that pfu, which would result in about 42 times lower error rate than taq (instead of the mentioned 18). I know invitrogen used a different assay for Pfu than stratagene, but the difference between their 2 tests for the other enzymes is rather small (for the relative fidelity that is).
Anybody have any clue on what's going on? Can it be that when comparing the enzymes there not using optimal buffer conditions? Is an "error rate" something completely different that fidelity? (my logic is that a higher fidelity results in a lower error rate, so they would be inversely correlated).
Also, has anybody here ever tried "accuprime" enzymes of invitrogen? As I'm working with primers that are "optimized" for diffferent HIV subtypes (which have sequence differences up to 30% in the region I'm working on, so my primers will never be 100% correct matches for all subtypes), I get a lot of aspecific bands and was wondering if this "accuprime" thing really works (if so, it would help me optimising my pcr's).
A last question: I will have to do several round of site directed mutagenesis on 3 plasmids (6-8 kb in size). As PCR enzymes will always incorporate some errors, do you think I have to always sequence the entire plasmids? Or just after the last round of SDM? Or do you think just sequencing the "touched" regions will do it?
Thanks a lot for any helpful reply!
I'll try and help with your questions.
1/ As far as the manufacturers literature goes, while these studies are supposed to be generated in independant labs I think when something like this happens than ti would be fair to say the products are comparable. When choosing it then comes down to price and word of mouth with peoples experience using these products. I have never used platinum pfx but have not had too many problems with PfuUltra. This forum is a good way of getting a diverse range of opinions.
2/ Sorry haven't had any experience in using Accuprime.
3/ As far as the sequencing of site directed mutagenesis I sequence across the coding region of the gene of interest. I don't sequence the whole plasmid as I believe a mutation in the ORI or Amp resistence etc. the plasmid may be non-functional and therefore won't grow anyway if it does grow then it shouldn't be an issue then anyway.
Hope this helps,
Thanks a lot, your answer helps me out (I also "feared" that independent research was never really done on this matter).
As for the site directed mutagenesis, I know that when my ori or AmpR genes are mutated and non-functional the colonies will not grow, but I need a bigger part to be "intact". Let me explain: I am modyfying certain genes in plasmids, inserting restriction sites and getting others out, so that afterwards I can ligate other variants of these genes in there. Afterwards I have to transfect a cell line with the plasmid(s) to grow a virus containing the new gene. I know others have done similar things and they just sequenced the "touched" area, but as any mutation in a plasmid can give rise to an alternate amino acid insertion (or even worse, a stop codon) I'm not convinced just sequencing the touched area is enough.