Gene walking using sequential hybrid primer polymerase chain reaction

Michael Martin-Harris, Paul Bartley, Alexander Morley

    Research output: Contribution to journalArticlepeer-review

    5 Citations (Scopus)


    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.

    Original languageEnglish
    Pages (from-to)308-310
    Number of pages3
    JournalAnalytical Biochemistry
    Issue number2
    Publication statusPublished - 15 Apr 2010


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