Rapid in vitro production of single-stranded DNA

Nucleic Acids Res. 2019 Dec 16;47(22):11956-11962. doi: 10.1093/nar/gkz998.

Abstract

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • 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
  • Time Factors

Substances

  • DNA, Single-Stranded
  • Polymers
  • CRISPR-Associated Protein 9
  • Methanol