Isothermal Approaches to DNA Amplification.

I’ve already discussed PCR on this blog before. PCR is an adiabatic process that takes place at varying temperatures, which brings with it the requirement for accurate heating and cooling equipment to maintain and alter temperatures to predetermined set points, arguably, this makes precision PCR equipment extremely expensive.

Could there been an alternative method for amplification that could do away with complicated equipment?
The people who came up with HDA (Helicase Dependent Amplification) answered that question with an emphatic yes, and the process is isothermal.

So, how exactly does this method work?

As with PCR, HDA is also based on in-vitro DNA replication. Here, a primer, a polymerase and a helicase are employed.
The helicase unwinds the DNA template enzymatically, a direct consequence of which is that DNA needn’t be denatured by heating to a high temperature. The primers can then bind to the template and the polymerase in the reaction mixture can then bind extend the primers, and produce a duplicate copy. So you can put in one double stranded template, primers complementary to your target sequence at the extension start site, and a polymerase, and just set the temperature to the optimum level for polymerase activity. Ta-dah.

Schematic diagram of HDA. Two complementary DNA strands are shown as two lines: the thick one is the top strand and the thin one is the bottom strand. 1: A helicase (black triangle) separates the two complementary DNA strands, which are bound by SSB (grey circles). 2: Primers (lines with arrow heads) hybridize to the target region on the ssDNA template. 3: A DNA polymerase (squares with mosaic patterns) extends the primers hybridized on the template DNA. 4: Amplified products enter the next round of amplification. From reference cited below.

The system, when reported in 2004, was able to easily achieve a million-fold amplification. This is the relevant paper.

There you go, that is all from me on HDA. The paper has examples which show the method in action successfully amplifying template DNA, which makes it a potentially suitable method for the development of diagnostics.

There is also a related method called Recombinase Polymerase Amplification, which is also isothermal, and you may read more about it here.

The difference between HDA and RPA lies in the fact that the latter uses a recombinase, which is involved in exchanging segments. A brief visual summary of the method follows.

RPA

RPA

The fact that both processes can be carried out at 37’C means that they can be carried out in a bog-standard incubator instead of in one of those really expensive PCR thermocyclers (however, that might be beginning to change, too, with a $10 cycler that someone built in the USA).

That is all from me in this post. Happy reading.

Ankur.

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