Optimization of single strand DNA incorporation reaction by Moloneymurine leukaemia virus reverse transcriptase
| Autor: | Masataka Tsuda, Yuji Nagata, Yoshiyuki Ohtsubo, Haruna Sasaki |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2018 |
| Předmět: |
0301 basic medicine
DNA Replication Sequence analysis DNA polymerase DNA Single-Stranded Substrate Specificity 03 medical and health sciences chemistry.chemical_compound 0302 clinical medicine reverse transcriptase Genetics Molecular Biology Gene biology Base Sequence Oligonucleotide Nucleic acid sequence DNA replication RNA-Directed DNA Polymerase General Medicine Sequence Analysis DNA Full Papers Molecular biology Reverse transcriptase 030104 developmental biology chemistry template-independent DNA addition biology.protein adaptor ligation Moloney murine leukemia virus 030217 neurology & neurosurgery DNA |
| Zdroj: | DNA Research: An International Journal for Rapid Publication of Reports on Genes and Genomes |
| ISSN: | 1340-2838 |
| Popis: | In this study, we investigated CIS reaction (clamping-mediated incorporation of single-stranded DNA with concomitant DNA syntheses) of Moloney murine leukaemia virus reverse transcriptase (MMLV-RT), and established a set of conditions with which single-stranded DNA is ligated to a G-tailed model substrate DNA at efficiencies close to 100%. Prior to the CIS reaction, a target blunt-end DNA was 3′ G-tailed by MMLV-RT in the presence of a tailing enhancer, deoxycytidine. In the CIS reaction, the G-tail reacted with a single-stranded DNA carrying a stretch of Cs on its 3′ end (termed as GAO for guide adaptor oligonucleotide), and MMLV-RT performed DNA polymerization, starting from the 3′ overhang, using the GAO as a template. We could append a given nucleotide sequence of as long as 72 nucleotides, which would be sufficient for various NGS-sequencing platforms. The high efficiency and the unique features of this MMLV-RT activity that enables the labelling of each DNA molecule with a unique degenerate sequence as a molecular identifier has many potential uses in biotechnology. |
| Databáze: | OpenAIRE |
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