The ability for DNA polymerase to copy a long stretch of DNA is becoming increasingly important. Why? It has to do with the advances in our sequencing technologies. Our next generation sequencing (NGS) technology requires the DNA polymerase to copy a long stretch of DNA (sometimes up to 50kb) as NGS is churning out genetic codes at a lightening speed.
One of the most important functions of DNA polymerase lies at its proofreading activity. Without this activity, our cells might be doomed because there would be too many mistakes being introduced during cell division.
You see, a typical recombinant polymerase such as Taq, which you use for routine PCR, lacks certain functions. Taq is very useful for simple amplification applications like genotyping or checking for the presence of an insert, something that only requires a yes or no answer. However, in experiments such as cloning, single nucleotide polymorphism analysis and NGS applications, high fidelity amplification is the number one priority to ensure the success of the analysis. In this case, the DNA Taq polymerase might not do the trick as it has a high rate of copying error (close to one nucleotide per kilo base), the copying error is simply too great to manage. In addition, the DNA Taq polymerase lacks 3’-5’ exonuclease activity required to excise the mis-incorporated base.
High-fidelity DNA polymerases have several cool features to combat the problems discussed above. First, the high fidelity has a very strong preference for binding the correct verses the incorrect nucleotide precursor (triphosphate) during polymerization. Second, high fidelity DNA polymerases have the 3’-5’ exonuclease domain to remove the mis-placed nucleotides. These two enhanced features make sure that whatever DNA template information you wish to analyze is faithfully copied.
Be Highly Possessive!
Another way you can engineer a DNA polymerase to tackle long distance copying is to increase its ability to hang on to the DNA parental template. It turns out that a lot of DNA polymerases fall off the track after copying. Don’t worry; several companies have incorporated non-specific DNA binding domains into their formulation to combat this problem. This added domain acts like safety harnesses that tether the DNA polymerase to the template, reducing the likelihood that it will fall off its track.
In addition, it is also noted that these types of fusion DNA polymerases are highly suitable for work in difficult-to-deal samples that might contain certain biological inhibitors or DNA templates that are G-C rich. Therefore, it makes perfect sense to combine DNA polymerases that already have proofreading activity with this extra feature to increase their long-range PCR capabilities.
Be Highly Adaptive!
Given the fact that NGS applications can come from so many un-imaginable sources (all the places you can think of), another desirability of the DNA polymerase used in long range PCR is the ease of use. The formulation should be highly adaptable in many different applications as well as sample conditions. For example, there are kits that can be used to directly amplify from plant tissues and blood samples without prior extraction and purification. On the other hand, tailor-made DNA polymerases for certain specific applications are on the rise as well. For example, some polymerases are designed to incorporate modified or labeled nucleotides, making them ideal reagents for DNA methylation studies.
Winning Formulation Mix!
A good enzyme and correct formulation are a perfect combination. For example, a hot-start option can minimize non-specific amplification and increase efficiency. Additionally, certain formulations are specifically designed for hard to amplify G-C content-rich regions.
So the next time you design your PCR experiments, hopefully you will appreciate the choices of DNA polymerases and master mixes that you have. By choosing carefully, you can ensure the first step to a successful sequencing reaction.
If you are a researcher who is using fluorescent staining on your cells and tissue sections, you should seriously consider using tyramide reagents. Why? Well, because they can have exquisite sensitivity, are easy to use, versatile and there is an increasing range of fluorophores in the pipeline. As they rely on a peroxidase catalysed visualization […]
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