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 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.
Mishra
R N
RN
International Centre for Genetic Engineering and Biotechnology, New Delhi, India.
Singla-Pareek
S L
SL
Nair
S
S
Sopory
S K
SK
Reddy
M K
MK
D001483:Base Sequence; D016386:Chromosome Walking; D017931:DNA Primers; D018744:DNA, Plant / Q000737:chemistry*; D018745:Genome, Plant; D021141:Nucleic Acid Amplification Techniques; D016133:Polymerase Chain Reaction / Q000379:methods*
D017931:DNA Primers; D018744:DNA, Plant