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

Schmidt TL, Beliveau BJ, Uca YO, Theilmann M, Da Cruz F, Wu CT, Shih WM - Nat Commun (2015)

Bottom Line: Inexpensive chip-synthesized oligonucleotide libraries can contain hundreds of thousands of distinct sequences, however only at sub-femtomole quantities per strand.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Cancer Biology, Dana-Farber Cancer Institute, 450 Brookline Avenue, Boston, Massachusetts 02215, 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.

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Oligonucleotide quality.PAGE analysis of three different subpools (A, B and C) synthesized by enzymes (enz) or else chemically (syn). Lane 1: crude product of final double-nicking reaction. Bands marked in green are staple strands, bands marked in blue are undesired concatemers (c), the intervening sequence (i) and the excess of two nicking primers (n) and can be removed well using HPLC purification (lane 2). Lanes 2, 4 and 6 are HPLC-purified c2ca reactions. Lanes 3, 5 and 7 contain unpurified synthetic oligonucleotides from a commercial vendor. M is a size marker.
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f3: Oligonucleotide quality.PAGE analysis of three different subpools (A, B and C) synthesized by enzymes (enz) or else chemically (syn). Lane 1: crude product of final double-nicking reaction. Bands marked in green are staple strands, bands marked in blue are undesired concatemers (c), the intervening sequence (i) and the excess of two nicking primers (n) and can be removed well using HPLC purification (lane 2). Lanes 2, 4 and 6 are HPLC-purified c2ca reactions. Lanes 3, 5 and 7 contain unpurified synthetic oligonucleotides from a commercial vendor. M is a size marker.

Mentions: As in the original report of c2ca24, we achieved about a billionfold amplification from three rounds. We detected no crosstalk between subpools by denaturing polyacrylamide gel electrophoresis (PAGE). Only gel bands of oligonucleotides present in the respective subpool could be seen; different lengths present only in other subpools were never observed. Control experiments with wrong or no primer sequences showed no detectable amplification. Lane 1 in Fig. 3 shows a typical result after the final double-nicking step. Incompletely cut concatemers (c) need to be removed for many applications, for example, to prevent multimerization or misfolding of structures in DNA origami. For this, preparative denaturing PAGE would be the standard method but is a very labour-intensive and lossy process, especially when products are of different lengths. Instead, we developed an automatable anion exchange high-performance liquid chromatography (HPLC) purification strategy (details in Methods). We recovered mixed length oligonucleotides with length between 25 and 55 nt for folding DNA nanostructures (Fig. 3, lane 1 before purification, lane 2 after purification) or 70-mers for FISH studies, with only traces of nicking primers and intervening sequences and no detectable concatemers. Our enzymatically produced oligonucleotides contained fewer truncated sequences compared with conventional synthetic oligonucleotides.


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

Schmidt TL, Beliveau BJ, Uca YO, Theilmann M, Da Cruz F, Wu CT, Shih WM - Nat Commun (2015)

Oligonucleotide quality.PAGE analysis of three different subpools (A, B and C) synthesized by enzymes (enz) or else chemically (syn). Lane 1: crude product of final double-nicking reaction. Bands marked in green are staple strands, bands marked in blue are undesired concatemers (c), the intervening sequence (i) and the excess of two nicking primers (n) and can be removed well using HPLC purification (lane 2). Lanes 2, 4 and 6 are HPLC-purified c2ca reactions. Lanes 3, 5 and 7 contain unpurified synthetic oligonucleotides from a commercial vendor. M is a size marker.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4660042&req=5

f3: Oligonucleotide quality.PAGE analysis of three different subpools (A, B and C) synthesized by enzymes (enz) or else chemically (syn). Lane 1: crude product of final double-nicking reaction. Bands marked in green are staple strands, bands marked in blue are undesired concatemers (c), the intervening sequence (i) and the excess of two nicking primers (n) and can be removed well using HPLC purification (lane 2). Lanes 2, 4 and 6 are HPLC-purified c2ca reactions. Lanes 3, 5 and 7 contain unpurified synthetic oligonucleotides from a commercial vendor. M is a size marker.
Mentions: As in the original report of c2ca24, we achieved about a billionfold amplification from three rounds. We detected no crosstalk between subpools by denaturing polyacrylamide gel electrophoresis (PAGE). Only gel bands of oligonucleotides present in the respective subpool could be seen; different lengths present only in other subpools were never observed. Control experiments with wrong or no primer sequences showed no detectable amplification. Lane 1 in Fig. 3 shows a typical result after the final double-nicking step. Incompletely cut concatemers (c) need to be removed for many applications, for example, to prevent multimerization or misfolding of structures in DNA origami. For this, preparative denaturing PAGE would be the standard method but is a very labour-intensive and lossy process, especially when products are of different lengths. Instead, we developed an automatable anion exchange high-performance liquid chromatography (HPLC) purification strategy (details in Methods). We recovered mixed length oligonucleotides with length between 25 and 55 nt for folding DNA nanostructures (Fig. 3, lane 1 before purification, lane 2 after purification) or 70-mers for FISH studies, with only traces of nicking primers and intervening sequences and no detectable concatemers. Our enzymatically produced oligonucleotides contained fewer truncated sequences compared with conventional synthetic oligonucleotides.

Bottom Line: Inexpensive chip-synthesized oligonucleotide libraries can contain hundreds of thousands of distinct sequences, however only at sub-femtomole quantities per strand.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Cancer Biology, Dana-Farber Cancer Institute, 450 Brookline Avenue, Boston, Massachusetts 02215, 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.

Show MeSH