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Optimization of single strand DNA incorporation reaction by Moloneymurine leukaemia virus reverse transcriptase
- Source :
- DNA Research: An International Journal for Rapid Publication of Reports on Genes and Genomes
- Publication Year :
- 2018
- Publisher :
- Oxford University Press, 2018.
-
Abstract
- 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.
- Subjects :
- 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
Subjects
Details
- Language :
- English
- ISSN :
- 13402838
- Volume :
- 25
- Issue :
- 5
- Database :
- OpenAIRE
- Journal :
- DNA Research
- Accession number :
- edsair.doi.dedup.....b0963526910ba3d118bfdb165ffe01eb