Taq and fusion polymerase - not the same results, I don't understand (Sep/22/2008 )

Hello everyone,

I run 2 pcr :
- same dna template (yeast genomics dna)
- same primers
- same batch of dNTPs
- same batch of water

Only thing that I change is polymerase and pcr buffer, in one case i using phusion polymerase, and Taq polymerase, it's a new batch. In first case, with phusion, it works great, i have good bands on gel, but with Taq nothing, i don't have pcr products. Here are pcr programs that i use :

For phusion:
PCR Program
- Initial Denaturation: 98 ° C, 30sec, 1 cycle
- Number of cycles: 30
- Denaturation: 98 ° C, 30sec
- Annealing: 55 ° C, 30sec
- Extension : 72 ° C, 1 min
- Final extension: 72 ° c, 5min

for Taq:
PCR Program
- Initial Denaturation: 94 ° C, 30sec, 1 cycle
- Number of cycle: 30
- Denaturation: 94 ° C, 30sec
- Annealing: 55 ° C, 30sec
- Extension: 68 ° C, 2 min30
- Final extension: 68 ° c, 7min

There are differences, denaturation temperature, temperature and duration of extension, but I follow invitrogen's recommandions. For the phusion = extension t° 72 °C and 15-30sec per kb. And for Taq = t extension 68 ° C and 1 min per kb. The size of my PCR product is 2800pb.

I don't understand, but I'll run a pcr with these settings for Taq:
PCR Program
- Initial Denaturation: 98 ° C, 30sec, 1 cycle
- Number of cycle: 30
- Denaturation: 98 ° C, 30sec
- Annealing: 55 ° C , 30sec
- Elongation: 68 ° C, 1 min
- Final extension: 68 ° c, 7min
in bold the difference

I need some advices
Thanks a lot

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past 1kb, PCR amplification with Taq is difficult. At 2.8kb is are very near the limit that Taq can amplify (~3kb, although I have seen 4kb with Taq once). You will probably have to open the your PCR optimisation kit.

Lowering the extension temperature is a good idea. It help amplify longer PCR product. However if the extension temperature is decreased, the extension time has to be increased to compensate for the lower polymerase activity. So using Taq you are probably looking at 3min 30sec to 4min for a 2.8kb fragment.

But in any case, Taq has a higher error rate as it has no proof reading ability. So there is no point using Taq to amplify this 2.8kb fragment. The PCR product would be riddled with errors. Error rate is of taq is given to be somewhere between 2 x 10^-5 to 1.1 x 10^-4.

So the percentage of PCR products that are okay, give (Error rate 2x10^5, 2800bp, 30 cycles)

P(Good) = [1- (error rate*product lenght bp)]^ number of cycles
P(Good) ~ 17.7%

It would be better to stay with Phusion. The enzyme has higher processivity, faster and with it has dsDNA binding protein connected to it and thus less likely to fall off the template, reducing truncated product.

Yes... i know... I know I love Phusion.

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thank you for your reply

Sorry but i don't say in my previous post : I use the Platinum Taq DNA polymerase high fidelity, from Invitrogen (it's an enzyme mixture is composed of recombinant Taq DNA polymerase, Pyrococcus species GB-D polymerase, and Platinum Taq ® Antibody (1.2). Pyrococcus species GB-D polymerase possesses a proofreading ability by virtue of its 3 'to 5' exonuclease activity (3). Mixture of the proofreading enzyme with Taq DNA polymerase increases fidelity approximately six times over that of Taq DNA polymerase alone and allows amplification of simple and complex DNA templates over a wide range of target sizes. Targets 12-20 kb can be amplified with some optimization)

I use this enzyme, because I want to do TA cloning. And phusion don't add 3'-A overhangs. My goal is to clone my PCR product in ta vector (pCR2.1), then cut the insert from the TA vector.

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ah a different kettle of fish. A polymerase mix.

Hmm.. have you tried decreasing your annealing temperature? Increasing MgSO4 concentration, add KCl?, Increasing the extension temperature? Use more template?

If worse comes to worse, you can take the PCR product from Phusion and treat it with Taq and dATP for 15-20min at 72 Celsius. That would add the A tail.

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My first suspicion is that you need a longer initial denaturation. Most antibody-inactivated Taqs (as yours seems to be) need an initial denaturation at 94° for several minutes to activate the Taq. Check the manufacturer's recommendations. The hot start Taq I use needs 2 minutes.

In general, I'd also say all your cycling parameters are unnecessarily long. 30s for denaturation and 30s for annealing are 'old-fashioned' values. Most denaturation happens within a few seconds at 95°C, and annealing happens pretty rapidly as well. I use 15s for denaturation, 15s for annealing, and in fact, Taq is normally good for about 2 kb/min. The old 1kb/min guideline is a very, very generous estimate. With a 2.8kb product I'd use 1:30 extenstion.

Ginger

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