Rapid in vitro production of single-stranded DNA
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.
Document Details
- Document Type
- Pub Defense Publication
- Publication Date
- Nov 12, 2019
- Source ID
- 10.1093/nar/gkz998
Entities
People
- Amanda Hornick
- Brian J Beliveau
- Cory Smith
- Dionis Minev
- Elisha Krieg
- Gabriel Filsinger
- George M. Church
- Hiroshi M. Sasaki
- Jocelyn Y. Kishi
- Khaled Said
- Peng Yin
- Richard Guerra
- William M. Shih
Organizations
- Damon Runyon Cancer Research Foundation
- Dana–Farber Cancer Institute
- Harvard Medical School
- Harvard University
- National Institutes of Health
- National Sleep Foundation
- Office of Naval Research
- Uehara Memorial Foundation