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Rapid in vitro production of single-stranded DNA

Minev, Dionis ; Guerra, Richard ; Kishi, Jocelyn Y ; Smith, Cory ; Krieg, Elisha ; Said, Khaled ; Hornick, Amanda ; Sasaki, Hiroshi M ; Filsinger, Gabriel ; Beliveau, Brian J ; Yin, Peng ; Church, George M ; Shih, William M

Nucleic acids research, 2019-12, Vol.47 (22), p.11956-11962 [Periódico revisado por pares]

England: Oxford University Press

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Título:
Rapid in vitro production of single-stranded DNA
Autor: Minev, Dionis ; Guerra, Richard ; Kishi, Jocelyn Y ; Smith, Cory ; Krieg, Elisha ; Said, Khaled ; Hornick, Amanda ; Sasaki, Hiroshi M ; Filsinger, Gabriel ; Beliveau, Brian J ; Yin, Peng ; Church, George M ; Shih, William M
Assuntos: Base Sequence ; CRISPR-Associated Protein 9 - metabolism ; DNA Repair - drug effects ; DNA, Single-Stranded - chemical synthesis ; DNA, Single-Stranded - chemistry ; Gene Targeting - methods ; HEK293 Cells ; Humans ; Methanol - chemistry ; Methanol - pharmacology ; Mutagenesis, Site-Directed - methods ; Polymerase Chain Reaction - methods ; Polymers - chemistry ; Synthetic Biology and Bioengineering ; Time Factors
É parte de: Nucleic acids research, 2019-12, Vol.47 (22), p.11956-11962
Notas: The authors wish it to be known that, in their opinion, the first and second author should be regarded as Joint First Authors.
The authors wish it to be known that, in their opinion, the third and fourth author should be regarded as Joint Second Authors.
Descrição: There is increasing demand for single-stranded DNA (ssDNA) of lengths >200 nucleotides (nt) in synthetic biology, biological imaging and bionanotechnology. Existing methods to produce high-purity long ssDNA face limitations in scalability, complexity of protocol steps and/or yield. We present a rapid, high-yielding and user-friendly method for in vitro production of high-purity ssDNA with lengths up to at least seven kilobases. Polymerase chain reaction (PCR) with a forward primer bearing a methanol-responsive polymer generates a tagged amplicon that enables selective precipitation of the modified strand under denaturing conditions. We demonstrate that ssDNA is recoverable in ∼40-50 min (time after PCR) with >70% yield with respect to the input PCR amplicon, or up to 70 pmol per 100 μl PCR reaction. We demonstrate that the recovered ssDNA can be used for CRISPR/Cas9 homology directed repair in human cells, DNA-origami folding and fluorescent in-situ hybridization.
Editor: England: Oxford University Press
Idioma: Inglês

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Rapid in vitro production of single-stranded DNA

Minev, Dionis ; Guerra, Richard ; Kishi, Jocelyn Y ; Smith, Cory ; Krieg, Elisha ; Said, Khaled ; Hornick, Amanda ; Sasaki, Hiroshi M ; Filsinger, Gabriel ; Beliveau, Brian J ; Yin, Peng ; Church, George M ; Shih, William M

England: Oxford University Press

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