Template independent synthesis of nucleic acid libraries

Abstract

Though, directed evolution/In vitro evolution has greatly enhanced the applicability of natural biomolecules, there is still a big void in synthetic biology, which could be filled only when we are able to make novel/synthetic functional biomolecules. Terminal deoxyribonucleotidyl transferase (TdT) is the only known DNA polymerase, which can add deoxyribonucleotides without the requirement of a DNA template. Here, we are introducing the concept of Template-Independent Synthesis of Nucleic Acids (TISNA), where we have exploited the property of terminal deoxyribonucleotidyl transferase to add deoxyribonucleotides to the 3' end of an oligonucleotide for the generation of de novo libraries of ssDNA, dsDNA coding sequences and RNA. We are able to generate libraries that have diversity not only in sequence but also in length in a single library itself. The length of double stranded random gene libraries generated using this approach ranges from 200 base pairs to 10 kilobase pairs. The ability to make random nucleic acid libraries from scratch (independent of any template information) in the laboratory could open up new avenues and holds promise for the pharmaceutical and biotechnological sectors.

Applications of Terminal Deoxynucleotidyl Transferase Enzyme in Biotechnology

“…They demonstrated that the ratio of nucleotide concentration to initiator concentration can affect the size distribution of the products. It is worth noting that upon introducing a mix of dNTPs, the size distributions of the resultant polynucleotides broaden significantly, observed as smears in native gels [21,22] …”

“…It is worth noting that upon introducing a mix of dNTPs, the size distributions of the resultant polynucleotides broaden significantly, observed as smears in native gels. [21,22]…”