Chemical synthesis of the mouse mitochondrial genome

Journal name:
Nature Methods
Volume:
7,
Pages:
901–903
Year published:
DOI:
doi:10.1038/nmeth.1515
Received
Accepted
Published online

We describe a one-step, isothermal assembly method for synthesizing DNA molecules from overlapping oligonucleotides. The method cycles between in vitro recombination and amplification until the desired length is reached. As a demonstration of its simplicity and robustness, we synthesized the entire 16.3-kilobase mouse mitochondrial genome from 600 overlapping 60-mers.

At a glance

Figures

  1. Schematic demonstrating assembly of the synthetic mouse mitochondrial genome.
    Figure 1: Schematic demonstrating assembly of the synthetic mouse mitochondrial genome.

    The 60-base oligos (red lines) were assembled in groups of eight into seventy-five 284-bp cassettes (red arrows). These were joined in sets of five to produce fifteen 1.2-kb assemblies (blue arrows) and then again in sets of five to produce three 5.6-kb assemblies (green arrows). These three fragments were recombined into a complete 16.5-kb genome (orange arrow), which includes a 221-bp repeat. NotI restriction sites (N, black lines) were designed to release the 284-bp cassettes from the pUC19 vector (gray lines).

  2. Summary of results for obtaining the 75 sequence-verified first-stage assemblies.
    Figure 2: Summary of results for obtaining the 75 sequence-verified first-stage assemblies.

    (a,b) The 75 reactions of eight oligos each were pooled and transformed into E. coli (a) or individually transformed (b). The number of correct clones obtained for each segment (1–75) is shown.

Accession codes

Referenced accessions

GenBank/EMBL/DDBJ

References

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Author information

Affiliations

  1. The J. Craig Venter Institute, Rockville, Maryland, USA.

    • Daniel G Gibson &
    • Chuck Merryman
  2. The J. Craig Venter Institute, San Diego, California, USA.

    • Hamilton O Smith &
    • Clyde A Hutchison III

Contributions

D.G.G. and C.M. designed research, performed research, analyzed data and wrote the paper. H.O.S., C.A.H. III and J.C.V. designed research and analyzed data.

Competing financial interests

J.C.V. is chief executive officer and co-chief scientific officer of Synthetic Genomics, Inc (SGI). H.O.S. is co-chief scientific officer and a member of the board of directors of SGI. C.A.H. III is chairman of the SGI Scientific Advisory Board. J.C.V., H.O.S. and C.A.H. III hold SGI stock.

Corresponding authors

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Supplementary information

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  1. Supplementary Text and Figures (1M)

    Supplementary Figures 1–6, Supplementary Tables 1–15, Supplementary Note 1

Additional data