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Nucleic Acids Research 2005 33(6):e58; doi:10.1093/nar/gni058
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Published online 30 March 2005

© The Author 2005. Published by Oxford University Press. All rights reserved
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Methods Online

Removal of mismatched bases from synthetic genes by enzymatic mismatch cleavage

Markus Fuhrmann*, Wolfgang Oertel1, Peter Berthold2 and Peter Hegemann2

Universität Regensburg, Kompetenzzentrum für Fluoreszente Bioanalytik Josef-Engert-Strasse 9, 93053 Regensburg, Germany 1Universität Regensburg, Lehrstuhl für Genetik Universitätsstrasse 31, 93053 Regensburg, Germany 2Universität Regensburg, Lehrstuhl für Biochemie I Universitätsstrasse 31, 93053 Regensburg, Germany

*To whom correspondence should be addressed. Tel: +49 941 943 5013; Fax: +49 941 943 5018; Email: markus.fuhrmann{at}vkl.uni-regensburg.de

Received February 18, 2005. Accepted March 8, 2005.

The success of long polynucleotide de novo synthesis is largely dependent on the quality and purity of the oligonucleotides used. Generally, the primary product of any synthesis reaction is directly cloned, and clones with correct products have to be identified. In this study, a novel strategy has been established for removing undesired sequence variants from primary gene synthesis products. Single base-pair mismatches, insertions and deletions were cleaved with specific endonucleases. Three different enzymes—T7 endonuclease I, T4 endonuclease VII and Escherichia coli endonuclease V—have been tested. As a model, a synthetic polynucleotide encoding the bacterial chloramphenicol-acetyltransferase (cat) was synthesized using different methods for one step polynucleotide synthesis based on ligation of oligonucleotides. The influence of enzymatic mismatch cleavage (EMC) as an error correction step on the frequency of correct products was analyzed by functional cloning of the synthetic cat and comparing the error rate with that of untreated products. Significant reduction of all mutation types was observed. Statistical analysis revealed that the T4 and E.coli endonucleases reduced the occurrence of mutations in cloned synthetic gene products. The EMC treatment was successful especially in the removal of deletions and insertions from the primary ligation products.


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