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 walker primers...
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| Main Authors: | , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
Taylor & Francis Group
2002-10-01
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| Series: | BioTechniques |
| Online Access: | https://www.future-science.com/doi/10.2144/02334st07 |
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| Summary: | 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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| ISSN: | 0736-6205 1940-9818 |