Error removal in microchip-synthesized DNA using immobilized MutS

The development of economical de novo gene synthesis methods using microchip-synthesized oligonucleotides has been limited by their high error rates. In this study, a low-cost, effective and improved-throughput (up to 32 oligos per run) error-removal method using an immobilized cellulose column containing the mismatch binding protein MutS was produced to generate high-quality DNA from oligos, particularly microchip-synthesized oligonucleotides. Error-containing DNA in the initial material was specifically retained on the MutS-immobilized cellulose column (MICC), and error-depleted DNA in the eluate was collected for downstream gene assembly. Significantly, this method improved a population of synthetic enhanced green fluorescent protein (720 bp) clones from 0.93% to 83.22%, corresponding to a decrease in the error frequency of synthetic gene from 11.44/kb to 0.46/kb. In addition, a parallel multiplex MICC error-removal strategy was also evaluated in assembling 11 genes encoding ∼21 kb of DNA from 893 oligos. The error frequency was reduced by 21.59-fold (from 14.25/kb to 0.66/kb), resulting in a 24.48-fold increase in the percentage of error-free assembled fragments (from 3.23% to 79.07%). Furthermore, the standard MICC error-removal process could be completed within 1.5 h at a cost as low as $0.374 per MICC.


CBM3-TaqMutS-EGFP (tMutS)
The E. coli MutS gene was amplified from pET32-muts (a gift from Dr. Tianyin Zhong) using the primers EcoMutS-NheI-F and EcoMutS-SacI-R (Supplementary Table S1) and sub-cloned into the NheI and SacI sites of the pET-21c vector to generate the pEcoMutS plasmid. Then, the fusion gene segments of CBM3 (GenBank: HF912725.1) and EGFP (GenBank:ACX42327.1), which were amplified using the primers CBM3-EGFP-SacI-F and CBM3-EGFP-NotI-R (Supplementary   Table S1) and the pCG plasmid (46) Table S1) and cloned into the Sal I and Not I sites of the pET-21c vector to generate the pTaqMutS plasmid. Then, the fusion gene segments of CBM3 and EGFP, which was amplified from the pCG plasmid using the primers CBM3-EGFP-NdeI-F and CBM3-EGFP-SalI-R, was inserted into the

Expression and purification of the eMutS and tMutS proteins
The constructed MutS fusion protein expression plasmids were independently transformed into E.

Oligonucleotide design and re-annealing reactions
To evaluate the specific mismatched DNA binding properties of EcoMutS-CBM3-EGFP (eMutS) and CBM3-TaqMutS-EGFP (tMutS), 10 oligos were designed and obtained from Sangon Biotech Co (Shanghai, China) (Supplementary Table S2). These oligos (A1~A5 and B1~B5) can form all possible single-nucleotide mismatches when two oligos are dissociated and re-annealed to form heteroduplex DNA (Supplementary Table S3). Equimolar concentrations of the two complementary single-stranded oligos at a final concentration of 2 µM were dissolved in annealing buffer containing 10 mM Tris-HCl (pH 7.6), 50 mM NaCl and 1 mM EDTA. Then, the duplexes were re-annealed by slowly cooling from 100 °C to 25 °C in a water bath. Fourteen different types of duplexes were used in these studies, the sequences of which are presented in Supplementary Table S3.

MutS and DNA binding
To  Table S2, Seq A5 and B5) was set as the control.

Evaluation of the binding properties of the MICCs to error-containing oligos
The binding ability of MICC to error-containing oligos was evaluated. An additional two oligos were synthesized by Sangon Biotech Co. (Shanghai, China) (Supplementary Table S2)，which formed a 54 bp homoduplex (homoduplex-54) after a re-annealing reaction (Supplementary Table   S3). This homoduplex was used to produce error-free duplex. The results were analyzed using Tanon Gis v4.0 software, and the quantity of the unbound 54 bp homoduplex was calculated using Tanon Gis v4.0 software using the 20 bp DNA ladder marker (TaKaRa) as a standard.

EGFP oligonucleotide pool design
The design of the EGFP oligos is shown in Supplementary Figure S3. Briefly, the codon of EGFP gene (720 bp) was first optimized based on E. coli codon usage, and the restriction enzyme sites were removed using DNAWorks (54). Then, the optimized sequence was cut into three similar length fragments (~260 bp) containing approximately 30 bp of overlap between the neighboring fragments. Next, the fragments were further cut into a set of oligos using Gene2Oligo (55 respectively, and the c1-3 subpools contained the antisense strand of EGFP Fra 1, Fra 2 and Fra 3, respectively. Thus, the a1 and c1 subpools, the a2 and c2 subpools and the a3 and c3 subpools could be used to assemble EGFP Fra 1, Fra 2 and Fra 3, respectively (Supplementary Figure S3).
These fragments, which contained approximately 30 bp overlap between the consecutive fragments, could be assembled into the full length EGFP gene via overlap extension PCR.

sMMO and Epo oligonucleotide pool design
The codons of the target genes were all optimized (sMMO gene cluster: E. coli; Epo A, B and C: Streptomyces coelicolor), and the unnecessary restriction endonuclease sites were removed using DNAWorks (54 Figures S5 and S6). The oligos of each subpool were ligated to form from one to three 286-456 bp length fragments or genes from the consecutive adjacent complementary pairs of oligos. There was no gap between the consecutive oligos; thus, they could be assembled into fragments via either ligation-mediated assembly or polymerization-mediated assembly (PCA, LCR or the combination of these two methods). These fragments, which contained approximately 30 bp overlaps between neighboring fragments, were assembled into the corresponding full-length genes. To simplify the subsequent processes, a pair of common primer sequences containing the Bbs I restriction site were designed and added to the corresponding oligos during synthesis (Supplementary Figure S6).