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Total synthesis of multi-kilobase DNA sequences from oligonucleotides

Nature Protocols volume 1, pages 2596–2603 (2006)Cite this article

Abstract

A method for synthesizing DNA from 40-mer oligonucleotides, which we used to generate a 32-kb DNA fragment, is explained. DNA sequences are synthesized as ∼500 bp fragments (synthons) in a two-step PCR reaction and cloned using ligation-independent cloning (LIC). Synthons are then assembled into longer full-length sequences in a stepwise manner. By initially synthesizing smaller fragments (synthons), the number of clones sequenced is low compared with synthesizing complete multi-kilobase DNA sequences in a single step. LIC eliminates the need for purification of fragments before cloning, making the process amenable to high-throughput operation and automation. Type IIs restriction enzymes allow seamless assembly of synthons without placing restrictions on the sequence being synthesized. Synthetic fragments are assembled in pairs to generate the final construct using vectors that allow selection of desired clones with two unique antibiotic resistance markers, and this eliminates the need for purification of fragments after digestion with restriction endonucleases.

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Figure 1: Schematic of the procedure for synthesizing DNA fragments from oligonucleotides, showing time estimates for each step.
Figure 2: Synthesis and cloning of fragments from oligonucleotides.
Figure 3: Preparation of the UDG vector.
Figure 4: LBS with type IIs restriction enzymes BbsI and BsaI using double antibiotic selection of the desired ligation products.
Figure 5: Type IIs restriction endonucleases are used to assemble contiguous sequences in-frame.
Figure 6: Dendrogram of the plan for a three-cycle, eight-fragment LBS synthesis of a 3,408 bp PKS module.

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Authors and Affiliations

  1. Department of Plant & Microbial Biology, University of California, Berkeley, Berkeley, 94720, California, USA

    Sarah J Reisinger

  2. Department of Chemical Engineering, Stauffer III, Stanford University, Stanford, 94305, California, USA

    Kedar G Patel

  3. Department of Pharmaceutical Chemistry, University of California, San Francisco, 94158, California

    Daniel V Santi

Authors
  1. Sarah J Reisinger
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  2. Kedar G Patel
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  3. Daniel V Santi
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Correspondence to Daniel V Santi.

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Reisinger, S., Patel, K. & Santi, D. Total synthesis of multi-kilobase DNA sequences from oligonucleotides. Nat Protoc 1, 2596–2603 (2006). https://doi.org/10.1038/nprot.2006.426

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