general PCR question - (Dec/14/2008 )

I was wondering why we need Taq polymerase in PCR. Why can't scientists just use helicase to unwound and break the hydrogen bonds that form between the parental strands, which would, in effect, replace the heating. Then it would just be a simple matter from there to just use a primer (like in PCR) and use a regular DNA polymerase as there was no heating, so there was no need for a heat-resistant form of DNA polymerase. Of course, you would also need a steady supply of nucleotides, but then again, you need it in PCR. And then the process could repeat itself. Wouldn't this alternative PCR work? If no, why not? So why did scientists have to go through all that hassle just to find Taq polymerase - or a form of DNA polymerase that was heat-resistant?

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http://en.wikipedia.org/wiki/Helicase-depe...t_amplification

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Thanks for the speedy response, hobglobin
However...
the wikipedia article says that it "still requires a relatively large amount of DNA"... How?????
Isn't HDA the same as PCR except that you now have a regular DNA polymerase and you now have helicase, instead of heat, breaking the hydrogen bonds between the nitrogenous bases? So wouldn't the DNA strands be the same and the amount of DNA nucleotides used also be the same (because the Taq and regular DNA polymerases use the same amount of nucleotides)?????????????????????????

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Actually I'm not very familiar with it, never did a HDA. But I guess a more severe problem is the specificity of the reaction. Helicase and normal Taq are quite heat susceptible, but a specific bond between primer and template (and therefore the specific amplification later) needs a quite high temperature. If you do HDA with 30°C you'll get a big mixture of everything...
Here perhaps the mentioned large amount of the template DNA might help.

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The main reason the denaturation step takes as long as it does is the need to ensure a uniform temperature throughout the sample. The original Lightcycler PCR systems using glass capillaries usually have 0 second denaturation times as well as 0 second annealing times. The effect has been seen when comparing glass capillaries and ultrathin Lightcycler plastic plates (a coupe of papers in BioTechniques from earlier this year, where glass did 7 cycles and plastic did 6 in the same time). 35 cycles in ~30 minutes - how much faster do you really need?

I'd also ask about the speed of unwinding the DNA with helicase. Plus t he fact that you'd need it to act close to the ends of your template sequence. It really is simpler to just denature the whole lot and allow the huge excess of primers to work.

As for why go through the hassle to "find" a thermostable polymerase, this is a classic case of someone seeing a useful application for a pure research finding. Scientists didn't "decide" to search for a thermostable polymerase, Kary Mullis (or whoever originated the idea, if you believe the stories) figured out that thermostable polymerases such as that from Thermus aquaticus would be a better alternative to having to add Klenow each cycle. At least, that's how I remember it.



Oh, hobgoblin, "normal" Taq is heat-stable, of course, seeing as how it was purified from a thermophile. But you must have been thinking of "normal" E. coli DNA polymerase, right?

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Yes of course... careless mistake.

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Thanks!!!!
And hobglobin too!!!!!

I still have a couple of questions, however
1) How would the fact that the helicases act close to the ends of the template strand be a bad thing? (isn't it a good thing because at least it wouldn't get in the way of the reaction)?

2) So in HDA, would regular DNA polymerase work??????

But I do understand that a major factor that HDA is not used that much is that it takes a long time, right?
But why would you need to keep the temperature uniform? If so, why would it even be so hard (just put some equipment that has some in-built regulating temperature mechanism)? In the regular PCR, wouldn't the single-stranded DNA primers get denatured by the heating and cooling?

AND how is HDA (much) slower than PCR???????????????????????????? Why?
[Even though the helicase might unwound it slowly (as evident in DNA replication) it's not as if the DNA polymerase can elongate the growing DNA strand any faster (again refer to DNA replication), right?????]

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HDA is a technology covered by patents to a company, BioHelix. It is used for situations where a thermocycler is not readily available, such as field testing or point-of care diagnostics (as per their website).
Answers:
1. I'm not sure where you heard that helicase just acts near the ends of the template. My understanding is that the helicase moves along the DNA duplex, so it would always be reasonably close to the reaction site.
2. I presumed HDA would use "regular" DNA pol...

Speed of reaction: The length of reaction time is probably part of the reason why HDA isn't typically used. I mean, why do an experiment that takes 'n' hours to complete, when you can do the PCR in a shorter time?
Why keep the temperature uniform? Come on, wax, why do you think? It's an enzymatic reaction...
In PCR, the primers should be single-stranded anyway... are you meaning they might get degraded? If so, then no.
Speed of HDA vs PCR: no idea. What is the processivity of DNA pol compared to Taq, or any of the other thermophilic polymerases?

Remember, in PCR the template strands are completely separated, so the only thing slowing down the rate of elongation is the polymerase itself.

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So are you saying that the primer in regular PCR will not get degraded even with the constant heating and cooling??????????

Thanks swanny

The question still remains, however: why does HDA take longer than PCR?????????????

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I sure am. Why would it get degraded because of 94C? The covalent bond is stronger than that!


Like I said, you'll have to look up the kinetics of helicase and DNA polymerase from whatever mesophile you choose (shall we say E coli, just for argument's sake?), and compare that figure to the thermophilic polymerases. How many turns of DNA (I am guessing that's what is measured) does helicase unwind per second? How many nucleotides does each polymerase incorporate per second, what is the on/off rate for each enzyme etc etc?

Going back a step, what kind of gDNA does HDA use? Is it untreated genomic DNA, or is it digested/sheared in some way? The longer the DNA the lower the frequency of helicase opening the duplex near your primers, so the longer it will take to allow specific extension to start. Then again, how does the reaction stop? It's simple for PCR: after the first few rounds of extension, the specific product starts to significantly outnumber all other pieces of DNA, but I can't see how the equivalent size restriction happens with HDA.

It might end up being one of those pieces of interesting scientific thinking that never gets anywhere, or has only a very limited use.

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