title: A high-throughput genome-walking method and its use for cloning unknown flanking sequences.
abstract: We developed a PCR-based high-throughput genome-walking protocol. The novelty of this protocol is in the random introduction of unique walker primer binding sites into different regions of the genome efficiently by taking advantage of the rolling circle mode of DNA synthesis by Phi29 DNA polymerase after annealing the partially degenerate primers to the denatured genomic DNA. The inherent strand-displacement activity of the Phi29 DNA polymerase displaces the 5' ends of downstream strands and DNA synthesis continues, resulting in a large number of overlapping fragments that cover the whole genome with the unique walker adapter attached to the 5' end of all the genomic DNA fragments. The directional genome walking can be performed using a locus-specific primer and the walker primer and Phi29 DNA polymerase-amplified genomic DNA fragments as template. The locus-specific primer will determine the position and direction of the genome walk. Two rounds of successive PCR amplifications by locus-specific and walker primers and their corresponding nested primers effectively amplify the flanking DNA fragments. The desired PCR fragment can be either cloned or sequenced directly using another nested, locus-specific primer. We successfully used this protocol to isolate and sequence 5' flanking regions/promoters of selected plant genes.
authors.affiliation: International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi 110 067, India.
authors.forename: Palakolanu Sudhakar
authors.initials: PS
authors.lastname: Reddy
authors.forename: Srikrishna
authors.initials: S
authors.lastname: Mahanty
authors.forename: Tanushri
authors.initials: T
authors.lastname: Kaul
authors.forename: Suresh
authors.initials: S
authors.lastname: Nair
authors.forename: Sudhir K
authors.initials: SK
authors.lastname: Sopory
authors.forename: Malireddy K
authors.initials: MK
authors.lastname: Reddy
mesh_terms: D024506:5' Flanking Region; D001483:Base Sequence; D019663:Chenopodiaceae / Q000235:genetics; D016386:Chromosome Walking / Q000379:methods*; D003001:Cloning, Molecular; D004258:DNA Polymerase III; D017931:DNA Primers; D018745:Genome, Plant; D023281:Genomics / Q000379:methods; D008969:Molecular Sequence Data; D031782:Pennisetum / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*
chemical_list: D017931:DNA Primers; C065639:DNA polymerase A; D004258:DNA Polymerase III