How to use Bst polymerase for PCR? - (Sep/19/2005 )

Hi all

Bst polymerase large fragment is used for cycle sequencing & whole genome amplification but I am interested in only using it for normal PCR, amplifying a specific target & use the PCR product for fragment analysis.

However, it is a mesophilic enzyme.

Is there anybody who uses Bst pol for normal PCR?

Thanks in advance!

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Why do you want to do this? You could try using additives like betain or proline to increase the thermostability and reduce the melting temp of the DNA.

Daniel

DNA sequencing

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Thanks Daniel!
I had been working on STR fragment analysis using Genescan & because TaqGold polymerase slips easily during amplification of the dinucleotide repeats, I keep getting stutter peaks. My assay requires these stutter peaks to be completely eliminated & therefore, needs a polymerase that doesn't slip...ie. Bst Pol, large fragment...
Do you have any other suggestions to eliminate these stutters? I have tried optimising Mg concentrations, annealing temperature etc with TaqGold but to no avail...

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Try lowering your extension temperature. I found with regions of very low GC content, it was impossible to amplify across the regions with "normal" extension temperatures. I use 64C for extension when I encounter those regions. You may have luck in reducing the stuttering with this technique.

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There was a paper many years ago looking at the cause of PCR stuttering and from memory it was linked to processivity (ie low processivity high stutter). There are a few thermostable polymerases around with better processivity than Taq (I think Takara sells one). These might be worth trying.

I also second phage' comment about the reducing extension temp. This is particularly important if you have AT rich repeat.

Daniel

DNA sequencing services

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The NEB/Finnzyme Phusion enzyme is highly processive and claims to "hold on" more tightly to the strand. I have no experience with it, but they write good spec sheets. Perhaps it's worth a try.

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Thanks all! I took a look at the Phusion enzyme & it certainly looks good! Will give it a try. Will also try the lowered extension temp in the meantime with my TaqGold.

By the way Daniel, I saw your reply in my previous post.

'PCR slippage is actually linked to low polymerase processivity, although low fidelity will contribute somewhat to the problem. If you think about it this makes sense because in slippage the 3' end of the growing strand is perfectly paired with a looped out region.'

Can you elaborate more on why slippage can cause the 3' end to pair with the looped out region? Or is there any literature on this?

Thanks again!

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Have a look at this paper

Taq DNA polymerase slippage mutation rates measured by PCR and quasi-likelihood analysis: (CA/GT)n and (A/T)n microsatellites

Daniel

DNA sequencing programs

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Thanks Daniel! Is certainly useful as it describes exactly the problems I have! Though the mathematical part is rather difficult for me to grasp...

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