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DNA polymerases for PCR-sequencing


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11 replies to this topic

#1 mray

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Posted 04 August 2009 - 05:32 PM

Hi,

I'm trying to PCR amplify specific loci from human genomic DNA from a solid tumor for the purpose of mutation detection (by sequencing). I was wondering, can I use Taq for this purpose, or should I use a high-fidelity polymerase? If so, is there a particular one that you recommend?

Thanks!

#2 leelee

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Posted 04 August 2009 - 07:09 PM

Definitely use a high fidelity enzyme. My favourite (and imho the best) is Phusion from NEB.

#3 phage434

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Posted 05 August 2009 - 05:19 AM

I disagree. Taq is just fine, and probably easier to use. You don't care about the fidelity of the PCR reaction in this case -- single bases can be wrong on individual copies with zero effect on the sequencing results. You only care about the statistical average, which will be just fine. Taq is cheap, robust, and probably the ideal enzyme for this application.

#4 ivanbio

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Posted 05 August 2009 - 07:22 AM

I agree with phage434. Standard Taq only makes an error every 10,000 bp or so, so unless you are amplifying very large fragments you should have no problems using regular Taq.

Ivan
Carlsbad, CA

#5 leelee

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Posted 06 August 2009 - 12:33 AM

What about if your error occurs very early on in the PCR? Wouldn't it then make up a big enough proportion of the amplicons to cause a problem?

#6 phage434

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Posted 06 August 2009 - 05:40 PM

If you are doing pcr on single molecules, that could be a concern. Almost any normal PCR starts with millions or billions of molecules of template.

#7 leelee

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Posted 06 August 2009 - 08:38 PM

That makes sense.
So for what applications do you really need high fidelity for then? Only those with a very few copies of template?

#8 phage434

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Posted 07 August 2009 - 03:11 AM

Any cloning reaction needs high fidelity. Sequencing samples the AVERAGE base composition at each position. Cloning selects a single molecule, and gives a sequence which is a duplicate of that single molecule. So, a population average can have the correct sequence, while any individual molecule is wrong in some position.

#9 mray

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Posted 07 August 2009 - 01:25 PM

ok...but one cannot directly sequence the PCR reaction, right? I was told to PCR amplify, clone in e.coli (TA cloning) and then sequence the clone...so in such a scenario a hi-fi polymerase is advisable??

#10 phage434

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Posted 07 August 2009 - 05:33 PM

No, it is routine to sequence PCR reaction results directly, if one is interested in knowing the sequence of the original sample (as opposed to knowing that you have a correct clone of that sequence).

#11 HomeBrew

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Posted 07 August 2009 - 08:44 PM

I agree with phage434 on all points.

#12 leelee

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Posted 09 August 2009 - 03:41 AM

Thanks phage434!




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