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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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Related in: MedlinePlus

Oligonucleotide amplification by circle-to-circle amplification.(a) Subpool-specific first-round primers determine which subpool is amplified in the respective well of the 96-well plate (as in Fig. 1c). Primers are orthogonal and complementary to only one subpool-specific barcode set of the library and parts of the common sequence (details in Supplementary Note 2) and therefore only hybridize to the strands of the respective subpool and do not bind to template strands of other subpools. The targeted template strands are cyclized via ligation into circular template strands. (b) A polymerase with high processivity and strand displacement capacity is added (Phi29, yellow). Initiating at the first-round primer, the polymerase synthesizes concatenated (chain-like) repeated copies of the circular template (green is complementary to red). Non-circularized template strands of other subpools are not amplified. Under optimal conditions, we observed up to 10,000 concatenated copies of each template molecule for one RCA (quantitative real-time PCR experiments, data not shown). However, this amplification rate by itself is not sufficient for most applications, as each library contains only attomoles (10−18 moles) per oligonucleotide sequence and only a small aliquot (for example, 1/1,000) of the library is used as a template for each amplification. (c) A second-round primer (blue dotted line) is hybridized to the intervening region of the first concatemer and the resulting double-stranded recognition site (orange) is digested with a restriction enzyme (HindIII) into monomers. A heat-inactivation step is necessary to inactivate the restriction enzyme for the next steps and is performed after most of the enzymatic reactions (details in Methods). The cut fragments of the second-round primer dissociate during this step. On cooling, an excess of the second-round primer hybridizes to the cut monomeric units and colocalizes the ends for a second ligation and RCA step. This second circular template has the reverse complementary sequence of the first circular template. (d) A third-round primer is annealed to the second concatemer (red again). Restriction digest, re-ligation and RCA are repeated to yield the final concatemer (e). One or two nicking primers are hybridized to this concatemer. A double-nicking reaction excises the intervening region from the target sequences (green). The nicking enzymes cut outside of their recognition sites; therefore, the entire intervening sequence can be removed to yield the (green) sequences of interest of a given subpool. Until this point, the entire process can be performed as a one-pot reaction without any intermediary workup steps. The excess of nicking primers, the intervening sequences, residual undigested concatemers and the excess of primers and enzymes can be removed using a final anion exchange chromatography step (see Methods).
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f2: Oligonucleotide amplification by circle-to-circle amplification.(a) Subpool-specific first-round primers determine which subpool is amplified in the respective well of the 96-well plate (as in Fig. 1c). Primers are orthogonal and complementary to only one subpool-specific barcode set of the library and parts of the common sequence (details in Supplementary Note 2) and therefore only hybridize to the strands of the respective subpool and do not bind to template strands of other subpools. The targeted template strands are cyclized via ligation into circular template strands. (b) A polymerase with high processivity and strand displacement capacity is added (Phi29, yellow). Initiating at the first-round primer, the polymerase synthesizes concatenated (chain-like) repeated copies of the circular template (green is complementary to red). Non-circularized template strands of other subpools are not amplified. Under optimal conditions, we observed up to 10,000 concatenated copies of each template molecule for one RCA (quantitative real-time PCR experiments, data not shown). However, this amplification rate by itself is not sufficient for most applications, as each library contains only attomoles (10−18 moles) per oligonucleotide sequence and only a small aliquot (for example, 1/1,000) of the library is used as a template for each amplification. (c) A second-round primer (blue dotted line) is hybridized to the intervening region of the first concatemer and the resulting double-stranded recognition site (orange) is digested with a restriction enzyme (HindIII) into monomers. A heat-inactivation step is necessary to inactivate the restriction enzyme for the next steps and is performed after most of the enzymatic reactions (details in Methods). The cut fragments of the second-round primer dissociate during this step. On cooling, an excess of the second-round primer hybridizes to the cut monomeric units and colocalizes the ends for a second ligation and RCA step. This second circular template has the reverse complementary sequence of the first circular template. (d) A third-round primer is annealed to the second concatemer (red again). Restriction digest, re-ligation and RCA are repeated to yield the final concatemer (e). One or two nicking primers are hybridized to this concatemer. A double-nicking reaction excises the intervening region from the target sequences (green). The nicking enzymes cut outside of their recognition sites; therefore, the entire intervening sequence can be removed to yield the (green) sequences of interest of a given subpool. Until this point, the entire process can be performed as a one-pot reaction without any intermediary workup steps. The excess of nicking primers, the intervening sequences, residual undigested concatemers and the excess of primers and enzymes can be removed using a final anion exchange chromatography step (see Methods).

Mentions: To enable the selective amplification of oligonucleotides, we extended the reverse complement of each target sequence (red poly-N sequences in Fig. 2a) with two nicking sites (blue), a restriction site (orange) and one or two orthogonal subpool-specific barcodes of 10 nucleotides (nt) each (black; detailed discussion of barcode designs in Supplementary Note 1, Supplementary Fig. 1 and sequences in Supplementary Note 2). Whereas the nicking and restriction sites are shared by all strands of the library, barcodes are subpool-specific. The ‘intervening region' (dark blue) comprises all non-target sequences and is removed after the final amplification (Fig. 2e).


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 amplification by circle-to-circle amplification.(a) Subpool-specific first-round primers determine which subpool is amplified in the respective well of the 96-well plate (as in Fig. 1c). Primers are orthogonal and complementary to only one subpool-specific barcode set of the library and parts of the common sequence (details in Supplementary Note 2) and therefore only hybridize to the strands of the respective subpool and do not bind to template strands of other subpools. The targeted template strands are cyclized via ligation into circular template strands. (b) A polymerase with high processivity and strand displacement capacity is added (Phi29, yellow). Initiating at the first-round primer, the polymerase synthesizes concatenated (chain-like) repeated copies of the circular template (green is complementary to red). Non-circularized template strands of other subpools are not amplified. Under optimal conditions, we observed up to 10,000 concatenated copies of each template molecule for one RCA (quantitative real-time PCR experiments, data not shown). However, this amplification rate by itself is not sufficient for most applications, as each library contains only attomoles (10−18 moles) per oligonucleotide sequence and only a small aliquot (for example, 1/1,000) of the library is used as a template for each amplification. (c) A second-round primer (blue dotted line) is hybridized to the intervening region of the first concatemer and the resulting double-stranded recognition site (orange) is digested with a restriction enzyme (HindIII) into monomers. A heat-inactivation step is necessary to inactivate the restriction enzyme for the next steps and is performed after most of the enzymatic reactions (details in Methods). The cut fragments of the second-round primer dissociate during this step. On cooling, an excess of the second-round primer hybridizes to the cut monomeric units and colocalizes the ends for a second ligation and RCA step. This second circular template has the reverse complementary sequence of the first circular template. (d) A third-round primer is annealed to the second concatemer (red again). Restriction digest, re-ligation and RCA are repeated to yield the final concatemer (e). One or two nicking primers are hybridized to this concatemer. A double-nicking reaction excises the intervening region from the target sequences (green). The nicking enzymes cut outside of their recognition sites; therefore, the entire intervening sequence can be removed to yield the (green) sequences of interest of a given subpool. Until this point, the entire process can be performed as a one-pot reaction without any intermediary workup steps. The excess of nicking primers, the intervening sequences, residual undigested concatemers and the excess of primers and enzymes can be removed using a final anion exchange chromatography step (see Methods).
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Related In: Results  -  Collection

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f2: Oligonucleotide amplification by circle-to-circle amplification.(a) Subpool-specific first-round primers determine which subpool is amplified in the respective well of the 96-well plate (as in Fig. 1c). Primers are orthogonal and complementary to only one subpool-specific barcode set of the library and parts of the common sequence (details in Supplementary Note 2) and therefore only hybridize to the strands of the respective subpool and do not bind to template strands of other subpools. The targeted template strands are cyclized via ligation into circular template strands. (b) A polymerase with high processivity and strand displacement capacity is added (Phi29, yellow). Initiating at the first-round primer, the polymerase synthesizes concatenated (chain-like) repeated copies of the circular template (green is complementary to red). Non-circularized template strands of other subpools are not amplified. Under optimal conditions, we observed up to 10,000 concatenated copies of each template molecule for one RCA (quantitative real-time PCR experiments, data not shown). However, this amplification rate by itself is not sufficient for most applications, as each library contains only attomoles (10−18 moles) per oligonucleotide sequence and only a small aliquot (for example, 1/1,000) of the library is used as a template for each amplification. (c) A second-round primer (blue dotted line) is hybridized to the intervening region of the first concatemer and the resulting double-stranded recognition site (orange) is digested with a restriction enzyme (HindIII) into monomers. A heat-inactivation step is necessary to inactivate the restriction enzyme for the next steps and is performed after most of the enzymatic reactions (details in Methods). The cut fragments of the second-round primer dissociate during this step. On cooling, an excess of the second-round primer hybridizes to the cut monomeric units and colocalizes the ends for a second ligation and RCA step. This second circular template has the reverse complementary sequence of the first circular template. (d) A third-round primer is annealed to the second concatemer (red again). Restriction digest, re-ligation and RCA are repeated to yield the final concatemer (e). One or two nicking primers are hybridized to this concatemer. A double-nicking reaction excises the intervening region from the target sequences (green). The nicking enzymes cut outside of their recognition sites; therefore, the entire intervening sequence can be removed to yield the (green) sequences of interest of a given subpool. Until this point, the entire process can be performed as a one-pot reaction without any intermediary workup steps. The excess of nicking primers, the intervening sequences, residual undigested concatemers and the excess of primers and enzymes can be removed using a final anion exchange chromatography step (see Methods).
Mentions: To enable the selective amplification of oligonucleotides, we extended the reverse complement of each target sequence (red poly-N sequences in Fig. 2a) with two nicking sites (blue), a restriction site (orange) and one or two orthogonal subpool-specific barcodes of 10 nucleotides (nt) each (black; detailed discussion of barcode designs in Supplementary Note 1, Supplementary Fig. 1 and sequences in Supplementary Note 2). Whereas the nicking and restriction sites are shared by all strands of the library, barcodes are subpool-specific. The ‘intervening region' (dark blue) comprises all non-target sequences and is removed after the final amplification (Fig. 2e).

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
Related in: MedlinePlus