Directional genome walking using PCR
We describe here a PCR-based "directional genome walking" protocol. The basic procedure for the amplification consists of two rounds of PCR. A primary PCR was performed, on the genomic DNA using a biotinylated primer specific to a known sequence in the genome along with four universal walk...
| Veröffentlicht in: | BioTechniques. - 1993. - 33(2002), 4 vom: 14. Okt., Seite 830-2, 834 |
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| Weitere Verfasser: | , , , |
| Format: | Aufsatz |
| Sprache: | English |
| Veröffentlicht: |
2002
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| Zugriff auf das übergeordnete Werk: | BioTechniques |
| Schlagworte: | Technical Report Journal Article DNA Primers DNA, Plant |
| Zusammenfassung: | We describe here a PCR-based "directional genome walking" protocol. The basic procedure for the amplification consists of two rounds of PCR. A primary PCR was performed, on the genomic DNA using a biotinylated primer specific to a known sequence in the genome along with four universal walker primers that were designed with partial degeneracy. The biotinylated primary PCR products were immobilized on streptavidin-linked paramagnetic beads. This step removed all nonspecific amplification products, and the purified template was used for the second PCR using a nested primer and the walker primer-2 to increase specificity. This technique is potentially useful for cloning promoter regions and has been successfully used to isolate 5'-flanking genomic regions of many cDNA clones previously isolated by us |
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| Beschreibung: | Date Completed 26.03.2003 Date Revised 28.09.2018 published: Print Citation Status MEDLINE |
| ISSN: | 0736-6205 |