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Nat Commun. 2015 Nov 16;6:8634. doi: 10.1038/ncomms9634.

Scalable amplification of strand subsets from chip-synthesized oligonucleotide libraries.

Author information

1
Department of Cancer Biology, Dana-Farber Cancer Institute, 450 Brookline Avenue, Boston, Massachusetts 02215, USA.
2
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 240 Longwood Avenue, Boston, Massachusetts 02115, USA.
3
Wyss Institute for Biologically Inspired Engineering at Harvard, 3 Blackfan Circle, Boston, Massachusetts 02115, USA.
4
Center for Advancing Electronics Dresden (cfaed), Technische Universität Dresden, 01062, Dresden, Germany.
5
Department of Genetics, Harvard Medical School, 77 Avenue Louis Pasteur, Boston, Massachusetts 02115, USA.

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.

PMID:
26567534
PMCID:
PMC4660042
DOI:
10.1038/ncomms9634
[Indexed for MEDLINE]
Free PMC Article

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