Gene walking using sequential hybrid primer polymerase chain reaction.
We developed a simple and robust method for removing nonspecific amplification produced during gene walking with a gene-specific primer and a degenerate primer. The primary walking polymerase chain reaction (PCR) was followed by two or three PCR rounds, each incorporating a low concentration of a reverse hybrid primer, where the 3' end was bound to a target sequence generated in the preceding PCR round and the 5' end was a new sequence that generated a target sequence for the next PCR round. The low concentration of the hybrid primer and the extent of amplicon stem-loop formation inhibited nonspecific amplification and enabled successful walking along three genes.
Martin-Harris
Michael H
MH
Department of Hematology and Genetic Pathology, Flinders University and Medical Centre, Bedford Park, South Australia 5042, Australia.
Bartley
Paul A
PA
Morley
Alexander A
AA
D025601:Adenomatous Polyposis Coli Protein / Q000235:genetics; D019313:BRCA1 Protein / Q000235:genetics; D001483:Base Sequence; D016386:Chromosome Walking / Q000379:methods*; D003598:Cytoskeletal Proteins / Q000235:genetics; D017931:DNA Primers / Q000737:chemistry*; D005136:Eye Proteins / Q000235:genetics; D006023:Glycoproteins / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D000099179:Myocilin
D025601:Adenomatous Polyposis Coli Protein; D019313:BRCA1 Protein; D003598:Cytoskeletal Proteins; D017931:DNA Primers; D005136:Eye Proteins; D006023:Glycoproteins; D000099179:Myocilin