Single-stranded RNA as primers of terminal deoxynucleotidyl transferase for template-independent DNA polymerization

Applications of Terminal Deoxynucleotidyl Transferase Enzyme in Biotechnology

“…Another classical application of TdT in molecular biology is the terminal end labelling of DNA at the 3' end. [24] This has allowed for the functionalization of DNA with different nucleotide analogues (Scheme 1 and Scheme 2) such as fluorescent tagged nucleotides (6,14) as chemical reporters and chemically modified nucleotides to increase nuclease resistance. [12] Researchers have demonstrated the incorporation of redox reporters onto both DNA and RNA oligonucleotides at the 3' end.…”

“…[5] RNA based sequences have also been demonstrated as initiators but the catalytic efficiency of incorporation of both dNTPs (1-4) and rNTPs (10-13) onto RNA based initiators was lower when compared to ssDNA initiators incorporating dNTPs (as seen by the lower observed sizes in gels. [6][7][8][9] TdT plays an important role in immunology in the development of lymphocytes. As a key enzyme in V(D)J recombination (the process by which lymphocytes alter and shuffle DNA for increasing the diversity of T-cell receptors and immunoglobulins [10] ) where it is responsible for incorporating random nucleotides onto one strand in a 5' to 3' direction.…”

Semiarbitrary qPCR for Sensitive Detection of Circulating miRNA via Terminal Deoxynucleotidyl Transferase-Assisted RNA-Primed DNA Polymerization

“…Previous literature has shown that terminal deoxynucleotidyl transferase (TdT) could modify a single DNA base onto the 3′-hydroxy terminus (3′-OH) of miRNAs for array detection. − Our previous work also studied the extension performance with ssRNA as primers. Our important finding is that four mixed nucleotides can be randomly added to the miRNA 3′ end to form an RNA–DNA chimera sequence (RDCS), and the extension efficiency is higher than any single nucleotides, which may be related to the conformational change of the active center of TdT upon binding to the base and the divalent metal cation. , However, the chimera sequence contained a known target miRNA sequence and an unknown, arbitrarily long DNA sequence. To the best of our knowledge, when only a few sequences are known in the long target nucleic acid fragments (less than 25 nucleotides), PCR amplification cannot be achieved by existing methods.…”