Fuhrmann, Markus and Oertel, Wolfgang and Berthold, Peter and Hegemann, Peter (2005) Removal of mismatched bases from synthetic genes by enzymatic mismatch cleavage. NUCLEIC ACIDS RESEARCH, 33 (6): e58. ISSN 0305-1048,
Full text not available from this repository. (Request a copy)Abstract
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.
| Item Type: | Article |
|---|---|
| Uncontrolled Keywords: | ESCHERICHIA-COLI; DEOXYINOSINE 3-ENDONUCLEASE; DNA; ENDONUCLEASE; RECOGNITION; MUTATIONS; SPECIFICITY; PROTEINS; |
| Subjects: | 500 Science > 570 Life sciences |
| Divisions: | Biology, Preclinical Medicine > Institut für Biochemie, Genetik und Mikrobiologie Biology, Preclinical Medicine > Institut für Biochemie, Genetik und Mikrobiologie > Lehrstuhl für Biochemie I |
| Depositing User: | Dr. Gernot Deinzer |
| Date Deposited: | 08 Jun 2021 13:27 |
| Last Modified: | 08 Jun 2021 13:27 |
| URI: | https://pred.uni-regensburg.de/id/eprint/36784 |
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