I was just thinking why we could not replace the taq polymerase with Bst polymerase used in LAMP and run our PCR ?
Dear Vinaykumar,
Bst DNA polymerase are commonly used in LAMP because they have strong strand displacement activity (required in isothermal techniques). However most of this polymerase are really unstable above 70°C, so it is impossible to use them in a pre-denaturation steps where temperatures are close to 90 °C.
Best wishes,
Marc
Thanks Marc but what id I use the regular PCR primers and then run the entire process at 70oC ?
Well for each PCR cycle you need to Denature the DNA. This first step of every cycle requires high temperature to denature the dsDNA. This temperature usually goes between 92-94°C.
If you just run the PCR at 70°C to maintain the stability of the Bst polymerase I highly doubt you will get any PCR product, as you don't denature DNA at 70°C (i think it is established that all DNA denatures at 90 - 95°C). You would just be annealing and extending nothing.
Best wishes
Marc
Vinaykumar,
I have never used Bst before, but after reading up on LAMP for my own research, I don't see why you can't use it. If you add betaine to the reaction (as you would in LAMP), the DNA should be a mixture of ss and ds at 70C, because betaine hase the effect of strongly reducing the melting temperature of the DNA. I can't imagine that your reaction would be as robust as it might be with Taq, but I would think you should still see some amplification using standard primers (especially if they are long enough to anneal nicely at 70C).
Hmm the point on primers annealing at 70C is well taken ! Thanks Aric and Marc
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