“…The reaction mixture contained 1.5 µl of 10 × PCR buffer containing MgCl 2 , 1.5 µl of 2 mM dNTP mixture, 1.5 µl of 2 µM primers (total volume for each primer), 0.07 µl of Taq DNA polymerase (5 U/µl) (all Roche Diagnostics GmbH, Mannheim, Germany), and 100-150 ng of template DNA. For the analysis of Ppd-A1, Ppd-D1, and the presence of the Vrn-A1, Vrn-B1, and Vrn-D1 alleles, protocols from Fu et al (2005), Beales et al (2007), Milec et al (2012), and Nishida et al (2012) were used. The alleles were differentiated according to fragment length and dominant markers.…”