Scalable amplification of strand subsets from chip-synthesized oligonucleotide libraries

Abstract

Synthetic oligonucleotides are the main cost factor for studies in DNA nanotechnology, genetics and synthetic biology, which all require thousands of these at high quality. Inexpensive chip-synthesized oligonucleotide libraries can contain hundreds of thousands of distinct sequences, however only at sub-femtomole quantities per strand. Here we present a selective oligonucleotide amplification method, based on three rounds of rolling-circle amplification, that produces nanomole amounts of single-stranded oligonucleotides per millilitre reaction. In a multistep one-pot procedure, subsets of hundreds or thousands of single-stranded DNAs with different lengths can selectively be amplified and purified together. These oligonucleotides are used to fold several DNA nanostructures and as primary fluorescence in situ hybridization probes. The amplification cost is lower than other reported methods (typically around US$ 20 per nanomole total oligonucleotides produced) and is dominated by the use of commercial enzymes.

In Situ Super-Resolution Imaging of Genomic DNA with OligoSTORM and OligoDNA-PAINT

“…Once a library is obtained, Oligopaint probes can be generated from the library in a variety of ways, all of which produce products that are single-stranded; single-stranded products are preferable to those that can become double-stranded, as the latter can renature after the denaturation step and thus become unavailable to their genomic targets during the hybridization step. There are several strategies for producing Oligopaint probes, including PCR amplification followed by asymmetric nicking and gel purification [5, 7, 8], PCR amplification followed by exonuclease digestion [5], T7 amplification followed by reverse transcription [RT; 5, 6, 18, 19, 22, 23], and rolling-circle amplification followed by cleavage [5, 24]. Depending on the strategy used, the final oligos may carry both Mainstreet and Backstreet or just one.…”

Block Copolymer Micellization as a Protection Strategy for DNA Origami

“…[1][2][3][4][5][6][7] Within the field, the DNAo rigami method has become one of the most powerful techniques, providing ar oute to construct megadalton-sized nanostructures. [8][9][10][11][12] These can be decorated with many functional elements, [13,14] including (bio)molecules [15,16] or inorganic nanoparticles, [17][18][19][20] for potential applications in electronics, [21,22] photonics, [23] or biology and nanomedicine. [24][25][26][27][28][29] However,t he nucleases present in most bodily fluids rapidly degrade DNA-based structures,a nd most structures are not stable under the salt conditions found in common cell culture buffers.…”

Block Copolymer Micellization as a Protection Strategy for DNA Origami

“…DNA nanotechnology enables the self‐assembly of objects with programmable shapes and topologies using synthetic oligonucleotides . Within the field, the DNA origami method has become one of the most powerful techniques, providing a route to construct megadalton‐sized nanostructures . These can be decorated with many functional elements, including (bio)molecules or inorganic nanoparticles, for potential applications in electronics, photonics, or biology and nanomedicine .…”