PCR without the PCR machine!
Sounds like fiction? Guess not. It is a distinct possibility now. This innovation is likely to revolutionize field applications of PCR and further expand its commercial potential.
For those of you, who are unfamiliar with PCR: PCR stands for Polymerase Chain Reaction. Since 1983, when the idea of PCR was first conceived by Kary Mullis, it has grown to be the method of choice for economically amplifying fragments of DNA or RNA over a billion times with the help of polymerases- enzymes that make more copies of nucleic acids such as DNA and RNA. Due to its ability to make more of the same from very less of the starting material, PCR is the backbone of both basic-science labs and application-oriented labs such as biotech and forensics. The expiration of the original PCR patent is bound to bring in more innovations and cheaper methods from competing players in the multi-million dollar PCR industry!
Watch the following movie, which explains the molecular basis of the reaction in layman’s terms. In essence the two strands of DNA act as molds for making more DNA molecules that contain the same coded sequence information.
http://www.youtube.com/watch?v=_YgXcJ4n-kQ
Until recently, the PCR machine was an indispensable part of PCR reaction as it drives the temperature cycles in the PCR. Recently, scientists have successfully attempted to replace the PCR machine with helicases to achieve amplification of DNA. Currently PCR uses three different temperatures that cycle multiple times to accomplish amplification. The first step of denaturation melts the DNA by increasing the temperature to 94°C. In nature however, molecular machines called helicases carry out DNA melting. Helicases melt double helical DNA by using chemical energy provided as ATP. They hydrolyze ATP and utilize the energy released for mechano-chemical cycles that help them to physically separate DNA strands in a stepwise manner. Inside living cells, helicase reactions support a variety of DNA and RNA transactions.
There were a couple of challenges that had to be overcome to ensure specific and successful amplification of DNA with the help of helicases. Initially the process used a mesophilic (optimal temperature if neither too hot or too cold; typically between 30-37°C) version of a DNA helicase called UvrD from Escherichia coli (EMBO Reports, 2004, 5: 795-800). High temperatures increase the specificity of the annealing step in PCR. Therefore, in the next step, specificity was increased by using a thermophilic (high-temperature loving; typically between 50-72°C) version of UvrD helicase from Thermoanaerobacter tengcongensis (Journal of Biological Chemistry, 2005, 280: 28952-58). However the challenge of amplifying long stretches of DNA remained. Technically termed processivity, which is nothing but how long an enzyme stays and does its work on a molecule of DNA before falling off, this was another hurdle to be overcome. The longer an enzyme stays on the DNA without falling off, the longer it is likely to keep doing its job on the molecule. In this case, a highly processive helicase could melt long stretches of DNA and therefore help in amplifying long templates of DNA. This would increase the practical value of the technique.
The processivity issue was recently addressed by fusing the helicase to a DNA polymerase (Gene, 2008, 420: 17-22). Whereas the helicase alone could amplify only short substrates, its fusion to DNA polymerase (DNA polymerase by itself is so processive that it can copy the entire genome of E. coli, which is more than 4 million base pairs without falling off) made it much more processive. The helicase-DNA polymerase fusion can efficiently amplify DNA fragments upto couple of thousand base pairs length, which brings it into the realm of practical use.
Further improvements in processivity and specificity should see the technique finding wider use in biology laboratories. The technique christened Helicase-dependent amplification of DNA (HDA) would find applications in diagnostics and environmental monitoring by driving down costs and increasing its accessibility for applications in the field. HDA should be a huge benefit for people who do not want to routinely do PCR, to those who are not experts and also for whom investing in a PCR machine is not worth it. Along with the ability to detect amplified DNA by non-electrophoretic methods, HDA should make the PCR process user-friendlier. It would also increase the application potential of PCR-based techniques where there is a shortage of skilled personnel, especially in poorer nations. In the words of HDA’s original proponents, “the development of simple portable DNA diagnostic devices to be used in the field and at the point-of-care” should be around the corner.