TY - JOUR
T1 - Optimization of single strand DNA incorporation reaction by Moloney murine leukaemia virus reverse transcriptase
AU - Ohtsubo, Yoshiyuki
AU - Sasaki, Haruna
AU - Nagata, Yuji
AU - Tsuda, Masataka
N1 - Funding Information:
This work was supported by Grants-in-Aid from the Ministry of Education, Culture, Sports, Science, and Technology, Japan (15H04471 and 17H03781) and by a grant from the Institute for Fermentation, Osaka (IFO).
Publisher Copyright:
© The Author(s) 2018. Published by Oxford University Press on behalf of Kazusa DNA Research Institute.
PY - 2018/10/1
Y1 - 2018/10/1
N2 - 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.
AB - 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.
KW - Adaptor ligation
KW - DNA polymerase
KW - Reverse transcriptase
KW - Template-independent DNA addition
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U2 - 10.1093/dnares/dsy018
DO - 10.1093/dnares/dsy018
M3 - Article
C2 - 29897438
AN - SCOPUS:85055075061
SN - 1340-2838
VL - 25
SP - 477
EP - 487
JO - DNA Research
JF - DNA Research
IS - 5
ER -