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.

Figure 1.

MeRPy-PCR overview and recovery yields for strands 1 (untagged) and 2 (initially tagged) of different amplicon lengths. (A) Production of the polymer-tagged primer. A 5′ acrydite modified primer is polymerized with acrylamide and sodium acrylate (ratio 99:1) to form a long linear DNA-tagged polymer. (B) MeRPy-PCR procedure following standard PCR guidelines. (C) (1.) Recovery of strand 1 under alkaline denaturing conditions and methanol precipitation. (2.) Recovery of strand 2, after treatment with UDG and DMEDA followed by a methanol precipitation. (D) Recovery yield for strand 1 and 2 of various lengths. Bar graphs denoting the recovery yield (%). Strand recovery yield was determined by the absolute recovered strand yield (pmol) relative to MeRPy-PCR input (pmol). Data are shown as mean ± STD (N = 3). (E) Gel electrophoresis of MeRPy-PCR derived ssDNA. Left, denaturing polyacrylamide gel with L – 20 bp Ladder, C – 200mer control from Integrated DNA Technologies (IDT). Middle and right, native agarose gels with L – 1 kb Ladder, C–750mer control from IDT. MeRPy-PCR derived and commercial ssDNAs were loaded with normalized mass amounts for each gel lane in (E).

Figure 2.

Applications using ssDNA of various lengths. (A) Genome editing in human cells using CRISPR/Cas9. (left) A genomically integrated GFP coding sequence is disrupted by the insertion of a stop codon and a 68-bp genomic fragment from the AAVS1 locus. Restoration of the GFP sequence by HDR with a ssDNA donor sequence results in GFP+ cells that can be quantified by FACS. (right) Bar graph depicting HDR efficiencies induced by MeRPy-PCR derived ssDNAs of different lengths versus a 200mer chemically synthesized strand from IDT. Data are shown as mean ± STD (N = 3). (B) ssDNA scaffold was generated via MeRPy-PCR from the phage genome, p7308 and used in the folding of a 30 nm DNA origami barrel and a 20 nm rectangle. Agarose gel electrophoresis shows the 1 kb Ladder (L), purified scaffold strand (S) alongside the folded barrel and 20 nm rectangle structures (F). TEM depicts the folded DNA origami barrel (left) and a 20 nm rectangle (right). Scale bars denote 100 and 50 nm for small insert. (C) A library comprising 42 000 probe sequences designed to tile along an 8.4 Mbp region of Human Chromosome 8 was amplified from a small amount of template using MeRPy-PCR with a Cy3-labeled reverse primer and subsequent recovery of fluor-tagged strand 1 library. The generated fluor-labeled ssDNA library was validated insitu on fixed human metaphase spreads and interphase cells. Scale bars denote 20 μm (zoom of metaphase spread scale bar denotes 5 μm).