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Error correction of microchip synthesized genes using Surveyor nuclease.
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.
Department of Biomedical Engineering, Duke University, Durham, NC27708, USA., ,
MeSHBase Pair Mismatch
Oligonucleotide Array Sequence Analysis
Polymerase Chain Reaction
Pub Type(s)Journal Article
Research Support, N.I.H., Extramural