Rapid and efficient isolation of unknown flanking DNA sequences adjacent to known regions is important for molecular biology research.For this purpose, several PCR-based methods have been reported, including inverse PCR (Uchiyama and Watanabe, 2006), ligation-mediated PCR (Yan et al., 2003;Ballester et al., 2005;Wang et al., 2007;Trinh et al., 2012) and randomly primed PCR (Liu and Whittier, 1995;Liu et al., 1995;Antal et al., 2004;Liu and Chen, 2007;Reddy et al., 2008;Wanget al., 2011).