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Error correction of microchip synthesized genes using Surveyor nuclease.

Abstract

The development of economical and high-throughput gene synthesis technology has been hampered by the high occurrence of errors in the synthesized products, which requires expensive labor and time to correct. Here, we describe an error correction reaction (ECR), which employs Surveyor, a mismatch-specific DNA endonuclease, to remove errors from synthetic genes. In ECR reactions, errors are revealed as mismatches by re-annealing of the synthetic gene products. Mismatches are recognized and excised by a combination of mismatch-specific endonuclease and 3'→5' exonuclease activities in the reaction mixture. Finally, overlap extension polymerase chain reaction (OE-PCR) re-assembles the resulting fragments into intact genes. The process can be iterated for increased fidelity. With two iterations, we were able to reduce errors in synthetic genes by >16-fold, yielding a final error rate of ∼1 in 8700  bp.

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  • Authors

    Saaem I, Ma S, Quan J, Tian J

    Institution

    Department of Biomedical Engineering, Duke University, Durham, NC27708, USA.

    Source

    Nucleic acids research 40:3 2012 Feb pg e23

    MeSH

    Base Pair Mismatch
    Endodeoxyribonucleases
    Endonucleases
    Genes, Synthetic
    Oligonucleotide Array Sequence Analysis
    Polymerase Chain Reaction

    Pub Type(s)

    Journal Article
    Research Support, N.I.H., Extramural

    Language

    eng

    PubMed ID

    22127863