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.
Highlights
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
We use an oligonucleotide library as a template that can be produced on a microchip either by inkjet printing[16,17] or by other techniques[18]
One reason is that the final concentration of the oligonucleotide copies in a rolling-circle amplification (RCA) can be B15 times higher than in PCR29
Summary
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. The price for standard column-synthesized oligonucleotides had steadily decreased in the last decades but has stabilized at around US$ 0.10 per base and can still dominate the cost of certain studies or make them prohibitively expensive Examples for such application include DNA origami[1,2,3]; single-stranded tile[4] or brick structures[5]; multiplexed PCR and targeted sequencing[6]; gene and genome synthesis[7,8,9,10]; multiplexed genome engineering[11,12]; and fluorescence in situ hybridization (FISH)[13,14,15]. Strand displacement amplification could be an interesting alternative to PCR as oligonucleotides are directly produced, but so far has been limited to nanogram amounts[21] or a fourfold amplification after chip synthesis[22] Another method was recently presented to amplify virtually error-free oligonucleotides with exactly controlled stoichiometry[23]. Our process is more economical than any of the previously published amplification methods
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