Automated low-redundancy large-scale DNA sequencing by primer walking
Low-redundancy automated DNA sequencing by primer walking is described. T7 DNA polymerase is used together with computer-selected walking primers and fluorescein-dATP as internal label to sequence large plasmids or cosmids directly on a standard DNA sequencer with an error rate below 1% up to 500 ba...
| Veröffentlicht in: | BioTechniques. - 1988. - 15(1993), 4 vom: 01. Okt., Seite 714-21 |
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| 1. Verfasser: | |
| Weitere Verfasser: | , , , , , , , , |
| Format: | Aufsatz |
| Sprache: | English |
| Veröffentlicht: |
1993
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| Zugriff auf das übergeordnete Werk: | BioTechniques |
| Schlagworte: | Comparative Study Journal Article DNA Primers Deoxyadenine Nucleotides Fluoresceins bacteriophage T7 induced DNA polymerase EC 2.7.7.- DNA-Directed DNA Polymerase EC 2.7.7.7 2'-deoxyadenosine triphosphate mehr... |
| Zusammenfassung: | Low-redundancy automated DNA sequencing by primer walking is described. T7 DNA polymerase is used together with computer-selected walking primers and fluorescein-dATP as internal label to sequence large plasmids or cosmids directly on a standard DNA sequencer with an error rate below 1% up to 500 bases (in the unedited raw data). The low error rate allows efficient sequencing with low (2-3 times) redundancy. Plasmid subclones covering 20 kb of a cosmid insert were sequenced with an overall redundancy of 2.7 in the course of the European community Saccharomyces cerevisiae genome sequencing project. Neighboring plasmid subclones were linked by direct cosmid sequencing. Sets of ten walking primers are synthesized on the EMBL multiple segmental DNA synthesizer at low costs and used for sequencing with greater than 95% efficiency. The accuracy of the directed approach is improved by simultaneous walking on both strands by designing two primers in opposite directions in the same starting region. One primer is used to confirm sequence data on the opposite strand, and the other primer to obtain new sequence data |
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| Beschreibung: | Date Completed 07.01.1994 Date Revised 23.11.2016 published: Print Citation Status MEDLINE |
| ISSN: | 1940-9818 |