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Updated: Jul 15, 2026

High-Density DNA and RNA microarrays - Photolithographic Synthesis, Hybridization and Preparation of Large Nucleic Acid Libraries
Published on: August 12, 2019
Total synthesis of multi-kilobase DNA sequences from oligonucleotides
Sarah J Reisinger1, Kedar G Patel, Daniel V Santi
1Department of Plant & Microbial Biology, University of California, Berkeley, Berkeley, California 94720, USA.
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 approximately 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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