Journal
BIOTECHNIQUES
Volume 33, Issue 4, Pages 830-+Publisher
EATON PUBLISHING CO
DOI: 10.2144/02334st07
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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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