DNA Synthesis of the PhiX Genomic DNA library from Deoxynucleotide Diphosphate Substrates

DNA synthesis is an essential process necessary for the survival of living organisms. This biochemical event is important for self-maintenance and is even required to pass down genetic information to future offspring. Though this process has been well characterized and studied, very little evidence is provided for how this process emerged, especially in a pre-biotic world where enzymatic processes had yet to exist. In this work we investigated how the DNA polymerase selectively chose the deoxynucleotide triphosphate (dNTP) as its naturally occurring substrate to make copies of its DNA. We studied DNA synthesis with a more prebiotically relevant substrate, that being the deoxynucleotide diphosphate (dNDP). Previous work has already shown the various thermophilic and mesophilic DNA polymerase can utilize dNDPs to perform DNA synthesis with a short DNA template (~100 bps), but it has yet to be discovered if this substrate will produce full length product when provided a longer DNA template (500 bps) with a higher sequence diversity DNA library. A single triphosphate was replaced by a diphosphate substate in DNA polymerase synthesis to see whether it were possible to amplify the 500 bps PhiX genomic DNA library. Here showed that when replacing guanosine triphosphate with guanosine diphosphate (dGDP), full length product is produced using Taq DNA polymerase. Vent(-exo) DNA polymerase can also produce full length product with dGDP, but when performing this same replacement experiment with any other diphosphate, no full-length product can be visualized for both polymerases. What this means is that these DNA polymerases have difficulty fully reading down the template strand when length of template and diversity of nucleotide context is increased with dNDP synthesis. This counteract the idea a dNDP to be prebiotically reliant, but one must consider the template context as the increase diversity in nucleotides may not have existed in pre-biotic times. Further experiments on sequence context is necessary to better understand how it is the that diphosphates can be utilized by DNA polymerases