Allele-Specific PCR Assay

DNA samples were subjected to PCR amplification in reaction volumes of 50 μL containing 1×PCR buffer，5 mmol/L MgCl2，200 μmol/L each dNTP，and 0.1 ng template genomic DNA.Each mixture was overlayed with 2 drops of Nujol mineral oil.Samples were preheated in the thermal cycler for 10 min at 95℃.As soon as the block temperature reached 95℃，2.5 units Taq DNA polymerase（Boehringer Mannheim）and 5 pmol each of four primers（BTOX5P1，5'-CGGTGTGGTACACCTGATCTGGTCCGGTTC-3'；JET1372，5'-GATAAACTCTTCCGTTCCGACTTGC-3'；197M，5'-GAAATTATGACGATGATTGGAAAGA-3'；197N，5'-TATTTATTGTCGGTACTATAAAACC-3'were added to the tubes under the mineral oil.Following preheating，30 PCR cycles were carried out，each cycle including denaturation for 1 min，94℃；annealing 1 min，65℃；and extension 1 min，72℃.PCR products were finally extended for 7 min at 72℃.Five microliters of each PCR product was analyzed by 2%agarose gel electrophoresis in 0.5×TBE.(https://www.daowen.com)