Protein-mediated error correction for de novo DNA synthesis.
The availability of inexpensive, on demand synthetic DNA has enabled numerous powerful applications in biotechnology, in turn driving considerable present interest in the de novo synthesis of increasingly longer DNA constructs. The synthesis of DNA from oligonucleotides into products even as large as small viral genomes has been accomplished. Despite such achievements, the costs and time required to generate such long constructs has, to date, precluded gene-length (and longer) DNA synthesis from being an everyday research tool in the same manner as PCR and DNA sequencing. A critical barrier to low-cost, high-throughput de novo DNA synthesis is the frequency at which errors pervade the final product. Here, we employ a DNA mismatch-binding protein, MutS (from Thermus aquaticus) to remove failure products from synthetic genes. This method reduced errors by >15-fold relative to conventional gene synthesis techniques, yielding DNA with one error per 10 000 base pairs. The approach is general, scalable and can be iterated multiple times for greater fidelity. Reductions in both costs and time required are demonstrated for the synthesis of a 2.5 kb gene.
Carr
Peter A
PA
Center for Bits and Atoms, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139, USA.
Park
Jason S
JS
Lee
Yoon-Jae
YJ
Yu
Tiffany
T
Zhang
Shuguang
S
Jacobson
Joseph M
JM
D001426:Bacterial Proteins / Q000378:metabolism; D003001:Cloning, Molecular; D004247:DNA / Q000096:biosynthesis* / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005434:Flow Cytometry; D005798:Genes, Bacterial; D005813:Genes, Synthetic; D049452:Green Fluorescent Proteins / Q000235:genetics; D016133:Polymerase Chain Reaction; D017422:Sequence Analysis, DNA
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D049452:Green Fluorescent Proteins; D004247:DNA