PCR uses cycles of heating and cooling to denature the strands, calling for special thermostable DNA polymerases. In a cell, during replication, Helicase unwinds the DNA without the requirement of heat. Can Helicase be used instead of the heat cycling to achieve single stranded dna? Would that eliminate the need for heat cycling, and hence special thermostable polymerases?
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2$\begingroup$ The temperature is cycled because we want to control the reaction. We control the reaction to multiply the DNA to the levels required. Thirty to forty cycles are usually good enough. You can't control helicase in the same way. So, no, helicase cannot be used instead. $\endgroup$– SANBI samplesCommented Nov 17, 2015 at 19:00
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2 Answers
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Edited after clarifications in question,
Let's start with normal functions of both enzymes. Helicases separates DNA strands while polymerase synthesize DNA strands as shown in the following figure. (Image Source: Wikimedia Commons)
Watch this animation, it will clear your doubts about function. However, RNA polymerase does have both activities.
In cells, helicase action is required to separate DNA strands. After that only polymerase can act. During PCR, separation of strands is done by increasing the temperature. This process is called DNA melting. This temperature is generally very high, around $90^oC$ to $100^oC$ depending on your sequence. At this temperature many proteins will not function, hence you need thermostable polymarase. About 40 years ago, such an enzyme was discovered from extremophiles (Chien et al 1976). Now it is commercially available as Taq polymerase. These enzymes are recombinant proteins with a few mutations which have also increased its yield and stability (Villbrandt et al 1997).
Now coming to your main question (Which I think I understood correctly this time).
The concept you are asking about has already been successfully tested in RNA detection. Researchers have used "Isothermal reverse transcription thermophilic helicase-dependent amplification"(Goldmeyer et al 2007). They used not only helicase but also reverse transcriptase (because they were detecting RNA). The following image (taken from the paper) shows the main steps in this process. Circles indicate DNA polymerase, squares indicate reverse transcriptase, and triangles indicate helicase.
Quoting from the paper (bullet points are mine),
Arrows indicate specific primers, circles indicate DNA polymerase, squares indicate reverse transcriptase, and triangles indicate helicase. The first-strand cDNA is first synthesized by a reverse transcriptase
- (steps 1 and 2). The RNA-DNA hybrids from the reverse transcription are then separated by UvrD helicases generating single stranded (ss) RNA and DNA templates
- (steps 3 and 4). The ssRNA enters next round of RT reaction
- (step 5-a) generating more first strand cDNA. The ssDNA was converted into double-stranded DNA by the DNA polymerase
- (step 5-b) and amplified concurrently in the tHDA reaction
- (steps 6 through 9). This process repeats itself to achieve exponential amplification of the RNA target sequence.
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1$\begingroup$ I think you misunderstood the question: it wasn’t asking whether a helicase could substitute a polymerase in the PCR, but whether it could substitute the heat cycling. $\endgroup$ Commented Nov 17, 2015 at 13:03
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$\begingroup$ Thank you for the answer, but Konrad Rudolph is right.I meant to ask if the heat cycles could be avoided altogether. $\endgroup$ Commented Nov 17, 2015 at 13:17
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$\begingroup$ Ah, ok. I will edit answer accordingly. Now wording is better and with more clarity. $\endgroup$– DexterCommented Nov 17, 2015 at 13:35
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$\begingroup$ CubeMaker, @KonradRudolph, check now. I have edited answer. I guess this is what you are looking for. $\endgroup$– DexterCommented Nov 17, 2015 at 13:46
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Isothermal DNA amplification technologies have been developed. You do not need thermal cycling to amplify DNA fragments by this method.
