6 replies to this topic

[lab_microbe]

My supervisor asked me to order polimerase with high fidelity because she will be doing some experiment with molecular  genotyping of some bacterial strains, so I was thinking of ordering a Hot Start Taq polimerase, but while reading additional info, I came across a picture where it says that it improves amplification of targets that require hot start. Now, I am confused, can I use it for amplification of any kind of target gene, or only "special"? And what is so special about this genes- they higher GC-content (so it is needed a higher temperature to disrupt the strands), or?

Thank you in advance!

[ivanbio]

Do not worry about the statement "improves amplification of targets that require hot start". All that means is that because you need a hot start step to activate the Taq enzyme you are likely also helping denature DNA that has strong stability, like high GC-content DNA. In other words, going through a hot start step (3 to 15 minutes at around 94oC to activate your Taq, depending on the brand of Taq you get) creates an environment (high temperature for a few minutes) that helps in the amplification of high-stability DNAs.

Your hot start Taq will work just fine with most templates; it just so happens that a hot start step also leads to DNA denaturation of otherwise hard to amplify DNAs.

[lab_microbe]

ivanbio, on 06 January 2011 - 02:22 PM, said:

Do not worry about the statement "improves amplification of targets that require hot start". All that means is that because you need a hot start step to activate the Taq enzyme you are likely also helping denature DNA that has strong stability, like high GC-content DNA. In other words, going through a hot start step (3 to 15 minutes at around 94oC to activate your Taq, depending on the brand of Taq you get) creates an environment (high temperature for a few minutes) that helps in the amplification of high-stability DNAs.

Your hot start Taq will work just fine with most templates; it just so happens that a hot start step also leads to DNA denaturation of otherwise hard to amplify DNAs.

thank you

[genom38]

Hello,

Have you ever search information about pfu DNA polymerase? As far as I know it polymerizes DNA more accurately than Taq polymerase.

[Adrian K]

hi, just for sharing:
high fidelity polymerase is not the same with hot start polymerase...
She might mean pfu polymerase which have proof reading capabilities.
high fidelity taq usually is a blend of both pfu and taq together like:

high fidelity taq: Platinum® Taq DNA Polymerase High Fidelity
high fidelity taq with hot start: AccuPrime™ Taq DNA Polymerase High Fidelity

make sure which one suits your application the best.

just my 2 cents.

[lab_microbe]

Eventually, when I saw all the enhancement (yield, sensitivity and specificity) features that are gained by hot start polymerase, my mind thought it also has high fidelity.

So to make it clear: hot start polymerase is suitable for small yield templates with/out high GC-content and when we have primer-dimer issues, and Pfu is best for high fidelity, because it has the proof-reading activity.

Thank you

[ElHo]

There also are some polymerases with both hot start and high fidelity!