Abstract
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 language | English |
---|---|
Pages (from-to) | 308-310 |
Number of pages | 3 |
Journal | Analytical Biochemistry |
Volume | 399 |
Issue number | 2 |
DOIs | |
Publication status | Published - 15 Apr 2010 |