Limits...
Scalable gene synthesis by selective amplification of DNA pools from high-fidelity microchips.

Kosuri S, Eroshenko N, Leproust EM, Super M, Way J, Li JB, Church GM - Nat. Biotechnol. (2010)

Bottom Line: Currently, the reliance on column-synthesized oligonucleotides as a source of DNA limits further cost reductions in gene synthesis.Oligonucleotides from DNA microchips can reduce costs by at least an order of magnitude, yet efforts to scale their use have been largely unsuccessful owing to the high error rates and complexity of the oligonucleotide mixtures.These assemblies were performed from a complex background containing 13,000 oligonucleotides encoding ∼2.5 megabases of DNA, which is at least 50 times larger than in previously published attempts.

View Article: PubMed Central - PubMed

Affiliation: Wyss Institute for Biologically Inspired Engineering, Boston, Massachusetts, USA. sri.kosuri@wyss.harvard.edu

ABSTRACT
Development of cheap, high-throughput and reliable gene synthesis methods will broadly stimulate progress in biology and biotechnology. Currently, the reliance on column-synthesized oligonucleotides as a source of DNA limits further cost reductions in gene synthesis. Oligonucleotides from DNA microchips can reduce costs by at least an order of magnitude, yet efforts to scale their use have been largely unsuccessful owing to the high error rates and complexity of the oligonucleotide mixtures. Here we use high-fidelity DNA microchips, selective oligonucleotide pool amplification, optimized gene assembly protocols and enzymatic error correction to develop a method for highly parallel gene synthesis. We tested our approach by assembling 47 genes, including 42 challenging therapeutic antibody sequences, encoding a total of ∼35 kilobase pairs of DNA. These assemblies were performed from a complex background containing 13,000 oligonucleotides encoding ∼2.5 megabases of DNA, which is at least 50 times larger than in previously published attempts.

Show MeSH
Scalable gene synthesis platform schematic for OLS Pool 2Pre-designed oligonucleotides (no distinction is made between dsDNA and ssDNA in the figure) are synthesized on a DNA microchip (a) and then cleaved to make a pool of oligonucleotides (b). Plate-specific primer sequences (yellow or brown) are used to amplify separate Plate Subpools (c) (only two are shown), which contain DNA to assemble different genes (only three are shown for each plate subpool). Assembly specific sequences (shades of blue) are used to amplify assembly subpools (d) that contain only the DNA required to make a single gene. The primer sequences are cleaved (e) using either Type IIS restriction enzymes (resulting in dsDNA) or by DpnII/USER/γ exonuclease processing (producing ssDNA). Construction primers (shown as white and black sites flanking the full assembly) are then used in an assembly PCR reaction to build a gene from each assembly subpool (f). Depending on the downstream application the assembled products are then cloned either before or after an enzymatic error correction step.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3139991&req=5

Figure 1: Scalable gene synthesis platform schematic for OLS Pool 2Pre-designed oligonucleotides (no distinction is made between dsDNA and ssDNA in the figure) are synthesized on a DNA microchip (a) and then cleaved to make a pool of oligonucleotides (b). Plate-specific primer sequences (yellow or brown) are used to amplify separate Plate Subpools (c) (only two are shown), which contain DNA to assemble different genes (only three are shown for each plate subpool). Assembly specific sequences (shades of blue) are used to amplify assembly subpools (d) that contain only the DNA required to make a single gene. The primer sequences are cleaved (e) using either Type IIS restriction enzymes (resulting in dsDNA) or by DpnII/USER/γ exonuclease processing (producing ssDNA). Construction primers (shown as white and black sites flanking the full assembly) are then used in an assembly PCR reaction to build a gene from each assembly subpool (f). Depending on the downstream application the assembled products are then cloned either before or after an enzymatic error correction step.

Mentions: We used these OLS pools to test if they would provide a better starting point for more scalable DNA microchip-based gene synthesis methods. We designed two OLS pools (OLS Pools 1 & 2) of different lengths, each containing ~13,000 130mer or 200mer oligonucleotides respectively. Figure 1 is a general schematic of our methods for utilizing OLS pools in a gene synthesis platform. Briefly, we designed oligonucleotides that were then printed on DNA microchips and recovered as a mixed pool of oligonucleotides (OLS Pool). Next, we took advantage of the long oligonucleotide lengths to independently PCR amplify and process only those oligonucleotides required for a given gene assembly. For the 200mer OLS Pool 2, we first amplified a “plate subpool” that contained DNA to construct up to 96 genes, and then amplified individual “assembly subpools” to separate the oligonucleotides for an individual gene. For the 130mer OLS Pool 1, we directly amplified into assembly subpools, foregoing the plate subpool step. Next, the primers used for these amplification steps were removed by either Type IIS restriction endonucleases to form double-stranded DNA (dsDNA) fragments (OLS Pool 2), or a combination of enzymatic steps to form single-stranded DNA (ssDNA) fragments (OLS Pool 1).


Scalable gene synthesis by selective amplification of DNA pools from high-fidelity microchips.

Kosuri S, Eroshenko N, Leproust EM, Super M, Way J, Li JB, Church GM - Nat. Biotechnol. (2010)

Scalable gene synthesis platform schematic for OLS Pool 2Pre-designed oligonucleotides (no distinction is made between dsDNA and ssDNA in the figure) are synthesized on a DNA microchip (a) and then cleaved to make a pool of oligonucleotides (b). Plate-specific primer sequences (yellow or brown) are used to amplify separate Plate Subpools (c) (only two are shown), which contain DNA to assemble different genes (only three are shown for each plate subpool). Assembly specific sequences (shades of blue) are used to amplify assembly subpools (d) that contain only the DNA required to make a single gene. The primer sequences are cleaved (e) using either Type IIS restriction enzymes (resulting in dsDNA) or by DpnII/USER/γ exonuclease processing (producing ssDNA). Construction primers (shown as white and black sites flanking the full assembly) are then used in an assembly PCR reaction to build a gene from each assembly subpool (f). Depending on the downstream application the assembled products are then cloned either before or after an enzymatic error correction step.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3139991&req=5

Figure 1: Scalable gene synthesis platform schematic for OLS Pool 2Pre-designed oligonucleotides (no distinction is made between dsDNA and ssDNA in the figure) are synthesized on a DNA microchip (a) and then cleaved to make a pool of oligonucleotides (b). Plate-specific primer sequences (yellow or brown) are used to amplify separate Plate Subpools (c) (only two are shown), which contain DNA to assemble different genes (only three are shown for each plate subpool). Assembly specific sequences (shades of blue) are used to amplify assembly subpools (d) that contain only the DNA required to make a single gene. The primer sequences are cleaved (e) using either Type IIS restriction enzymes (resulting in dsDNA) or by DpnII/USER/γ exonuclease processing (producing ssDNA). Construction primers (shown as white and black sites flanking the full assembly) are then used in an assembly PCR reaction to build a gene from each assembly subpool (f). Depending on the downstream application the assembled products are then cloned either before or after an enzymatic error correction step.
Mentions: We used these OLS pools to test if they would provide a better starting point for more scalable DNA microchip-based gene synthesis methods. We designed two OLS pools (OLS Pools 1 & 2) of different lengths, each containing ~13,000 130mer or 200mer oligonucleotides respectively. Figure 1 is a general schematic of our methods for utilizing OLS pools in a gene synthesis platform. Briefly, we designed oligonucleotides that were then printed on DNA microchips and recovered as a mixed pool of oligonucleotides (OLS Pool). Next, we took advantage of the long oligonucleotide lengths to independently PCR amplify and process only those oligonucleotides required for a given gene assembly. For the 200mer OLS Pool 2, we first amplified a “plate subpool” that contained DNA to construct up to 96 genes, and then amplified individual “assembly subpools” to separate the oligonucleotides for an individual gene. For the 130mer OLS Pool 1, we directly amplified into assembly subpools, foregoing the plate subpool step. Next, the primers used for these amplification steps were removed by either Type IIS restriction endonucleases to form double-stranded DNA (dsDNA) fragments (OLS Pool 2), or a combination of enzymatic steps to form single-stranded DNA (ssDNA) fragments (OLS Pool 1).

Bottom Line: Currently, the reliance on column-synthesized oligonucleotides as a source of DNA limits further cost reductions in gene synthesis.Oligonucleotides from DNA microchips can reduce costs by at least an order of magnitude, yet efforts to scale their use have been largely unsuccessful owing to the high error rates and complexity of the oligonucleotide mixtures.These assemblies were performed from a complex background containing 13,000 oligonucleotides encoding ∼2.5 megabases of DNA, which is at least 50 times larger than in previously published attempts.

View Article: PubMed Central - PubMed

Affiliation: Wyss Institute for Biologically Inspired Engineering, Boston, Massachusetts, USA. sri.kosuri@wyss.harvard.edu

ABSTRACT
Development of cheap, high-throughput and reliable gene synthesis methods will broadly stimulate progress in biology and biotechnology. Currently, the reliance on column-synthesized oligonucleotides as a source of DNA limits further cost reductions in gene synthesis. Oligonucleotides from DNA microchips can reduce costs by at least an order of magnitude, yet efforts to scale their use have been largely unsuccessful owing to the high error rates and complexity of the oligonucleotide mixtures. Here we use high-fidelity DNA microchips, selective oligonucleotide pool amplification, optimized gene assembly protocols and enzymatic error correction to develop a method for highly parallel gene synthesis. We tested our approach by assembling 47 genes, including 42 challenging therapeutic antibody sequences, encoding a total of ∼35 kilobase pairs of DNA. These assemblies were performed from a complex background containing 13,000 oligonucleotides encoding ∼2.5 megabases of DNA, which is at least 50 times larger than in previously published attempts.

Show MeSH