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

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.

Figure 1. Scalable gene synthesis platform schematic for OLS Pool 2

Pre-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.

Figure 2. Gene synthesis products

GFPmut3 was PCR assembled (a) from two different assembly subpools (GFP42 and GFP35) that were amplified from OLS Pool 1. Because the majority of the products were of the wrong size, we gel-purified the full-length assemblies and re-amplified them (b). Using the longer oligonucleotides in OLS Pool 2 we were able to develop a PCR assembly protocol that did not require gel-isolation, which we used to build three different fluorescent proteins (c). We then attempted to build 42 scFv regions that contained challenging GC-rich linkers. Of the 42 assemblies (d) 40 resulted in strong bands of the correct size. We gel isolated and re-amplified the two that did not assemble (7 and 24) resulting in bands of the correct size (see Supplementary Fig. 10b online). The antibody that corresponds to each number is given in Supplementary Table 3 online.

Figure 3. Characterization of products from OLS Pools 1 and 2

The percentage of fluorescent cells resulting from synthesis products derived from column-synthesized oligonucleotides (black), OLS Chip 1 subpools GFP43 and GFP35 (green) and the three fluorescent proteins produced on OLS Chip 2 with and without ErrASE treatment (blue, yellow, and orange) are shown (a). The error bars correspond to the range of replicates from separate ligations. The error rates (average bp of correct sequence per error) from various synthesis products are shown (b). Error bars show the expected Poisson error based on the number of errors found (±√n). Deletions of more than 2 consecutive bases are counted as a single error (no such errors were found in OLS Pool 1).