The polymerase chain reaction (PCR)-based genome walking method has been extensively used to isolate
unknown flanking sequences, whereas nonspecific products are always inevitable. To resolve these problems,
we developed a new strategy to isolate the unknown flanking sequences by combining A-T linker
adapter PCR with inverse PCR (I–PCR) or thermal asymmetric interlaced PCR (TAIL–PCR). The result
showed that this method can be efficiently achieved with the flanking sequence from the Arabidopsis
mutant and papain gene. Our study provides researchers with an additional method for determining
genomic DNA flanking sequences to identify the target band from bulk of bands and to eliminate the
cloning step for sequencing.