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Review
. 2014 May;11(5):499-507.
doi: 10.1038/nmeth.2918.

Large-scale de novo DNA synthesis: technologies and applications

Sriram Kosuri  1 , George M Church  2
Affiliations
Review

Large-scale de novo DNA synthesis: technologies and applications

Sriram Kosuri et al. Nat Methods. 2014 May.

Abstract

For over 60 years, the synthetic production of new DNA sequences has helped researchers understand and engineer biology. Here we summarize methods and caveats for the de novo synthesis of DNA, with particular emphasis on recent technologies that allow for large-scale and low-cost production. In addition, we discuss emerging applications enabled by large-scale de novo DNA constructs, as well as the challenges and opportunities that lie ahead.

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Conflict of interest statement

S.K. and G.M.C. own stock in and are on the Scientific Advisory Board of Gen9, a company that sells synthetic genes. G.M.C. is on the Board of Directors of Sigma-Aldrich and the Scientific Advisory Board of Cambrian Genomics, both companies that sell synthetic genes or oligos.

Figures

Figure 1
Figure 1. Lengths and costs of different oligo and gene synthesis technologies.
Commercial oligo synthesis from traditional vendors (pink) and array-based technologies (brown) are plotted according to commonly available length scales and price points. Costs of gene synthesis from commercial providers for cloned, sequence-verified genes (dark green) and unpurified DNA assemblies (light green) are shown, as are costs of gene synthesis from oligo pools (blue) derived from academic reports,.
Figure 2
Figure 2. Phosphoramidite chemistry.
The four-step synthetic oligo synthesis is the most commonly used chemistry for the production of DNA oligos.
Figure 3
Figure 3. Different strategies for dealing with microarray oligo complexities.
Top, Kosuri et al. use amplification of barcoded subpools by PCR (thus eliminating background complexity), remove the barcode sequences and then assemble the genes. Bottom, Quan et al. use a custom synthesizer to print oligos needed for any assembly into separate micropatterned wells. Leveraging the spatial separation that enables microarray synthesis, they then amplify and assemble these genes within the microwells themselves.
Figure 4
Figure 4. Comparison of reported error rates from error-correction techniques.
The error rates are included along with the indicated oligo source and error-correction methodology. When starting error rates were unreported, we estimated the error rates on the basis of the oligo sources and assembly method. Open circles denote starting error rates; filled circles denote error rates of assembled genes (two filled circles denote error rates before and after error correction). ssDNA, single-stranded DNA; dsDNA, double-stranded DNA; Column, column-synthesized oligos; Array, microarray-based oligo pools; Hyb, oligo hybridization–based error correction; Seq, NGS-based error correction; Lig, high-temperature ligation/hybridization–based error correction; Nuclease, nuclease-based error correction.
See this image and copyright information in PMC

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