The helicase dependent amplification
About the helicase dependent amplification
Lee Kyoung Jun
khsslb95@naver.com
Introduction
Helicase is the enzyme that can denature the double strand structure of DNA into two single strands. It is crucially needed during DNA replication due to its denaturing function. Then the question arose. Why isn't it used for amplification even though it requires denaturing process of the double strand as well? PCR is the most widely used method in vitro DNA amplification.however, PCR has heating phase to break the hydrogen bonds between double strand DNA so that it requires thermal cycler which is expensive and power hungry. Therefore, I want to briefly introduce the denaturing method in HDA(Helicase dependent amplification), which is based on function of helicase.
Denaturing phase of HDA
As helicase can unwind the double stranded DNA enzymatically, it is called isothermal amplification. In this process the HDA more special enzyme to make it real. The key component of the reagent is Uvrd, MutL, SSB, and ATP. Uvrd is the one of the helicase which was discovered in E coli so that it is the main enzyme in this process. And for the processivity of Uvrd, the DNA repair enzyme MutL, which helps Uvrd to be recruited on the DNA, is necessary. MutL activates Uvrd performance by more than 10times. SSB is short of single strand binding DNA. It binds to the denatured strand of DNA to stabilize it so that SSB protein prevent the immediate annealing of two single stranded DNA after denaturing happened. Lastly, ATP is required to both Uvrd and MutL performance. As you can see below figure, absence of each of key component is critical for denaturation process.
Electrophoresis of HDA products amplified from plasmid DNA. A two-step HDA reaction, with a 1 h incubation at 37°C, was performed in the presence of all components (lane 1) including a pUC19-derived plasmid DNA (0.035 pmol), primer-1224 (10 pmol) and primer-1233 (10 pmol), UvrD helicase (100 ng), MutL (400 ng), T4 gene 32 protein (4.5 μg), ATP (0.15 μmol) and exo− Klenow polymerase (5 U). HDA products in the absence of UvrD helicase (lane 2), accessory protein MutL (lane 3), T4 gene 32 protein (lane 4) or ATP (lane 5) are shown. M: 100-bp DNA ladder.
Benefits and limits
There are two big advantages that HDA offers. Firstly it has a simple reaction scheme, in which a target sequence can be amplified by two flanking primers, similar to PCR. Reminding of that the other isothermal DNA amplification techniques have more complicated reaction mechanisms, HDA seems way efficient method among those isothermal DNA amplification techniques when it comes to simplicity. Secondly, As mentioned above, HDA is a isothermal technique so that it doesn't require thermal cycler which is expensive and power hungry. Contrary to the fact that seeral isothermal DNA amplification method still have high temperature phase, like SDA, HDA is true isothermal whcih means it doesn't need high temperature phase.
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.
But there are several critical disadvantages exist in this method. Firstly, as it is still in its development stage, so the ratio of key components and the research about what enzymes synergitically work well are not clearly found yet. So the processivity of Uvrd and efficiency of denaturing speed is low. Secondly, since HDA requires a lot of enzymes, it needs the ready made kit of reagent which is way more expensive than reagent of PCR. Actually, these two disadvantages are main reasons that researchers still use PCR method to amplify the DNA instead of HDA.
Improvement and Prospect
The first short, which is about ideal ratio and component of reagent can be overcome by an amount of research. Since this method is develpoed in 2007, so it now remains at its development stage. Further amount of research will find the ideal ratio and components of reagent so it will be matter of time. Second short is the cost of the reagent. This is basical problem because the low productivity of each component of reagent is higering the cost of the reagent. This should be improved by excogitating the efficient method of producing the key enzymes and components of HDA. If These shorts are overcome, It is clear that HDA has its own adcantages compare to other method. Then someday HDA is expected to be widley used and might replace the PCR method.
Reference
Figures and the information about HDA
