Methods

 

PCR components:

PCR amplifications were performed in 30-μL volumes containing 1X PCR buffer (ABM Inc.), 1.5 mM MgCl2 (ABM Inc.), 2.5 mM dNTP, 0.5 mM each primer, 1.0 unit of Taq polymerase (ABM Inc.), and 1–2 μL (50 ng DNA) of template DNA. The 5′ ends of forward primer were labeled with fluorophores (FAM and HEX).

PCR conditions:

    Temp. (ºC) Time
Initial denaturation   95 5 mins
  Denaturation 95 30 sec
               35 cycle Annealing 55-60  30 sec
  Elongation 72 45 sec
Final Elongation   72 7 mins

 

Primers:

Locus Forward primer (5'-3') Reverse primer (5'-3')
BH142 TACAGCTGTTCCACCCACTC CTCCCCTCTCCCCTAGTCAC
BH217 GGAGGGAGGTTCTTGGTCTC AGAGGGATGTGGTGGAGTTG
BH278 CCTCCTTGCGCTTCTGAC AGGAGGCGGAAAGGAGAAAG
BH302 GAGATGCGACGATTCTCCTC GATTCGCGTTCAATGGTTCT
BH399 ATTCTTCGCACGATTCTTCG TCATGGAGGTGGTCAACAAA
BH476 AGAAGAAGCTCTTCGCGTTG CAGATCCGCGTAGGTCATTT
BH806 GACGTGGGTGAGGAGGATTA GTTGGAGAGTTCGGGGGTAT
BH808 TGACAGGTTGCTCCTTACCC TTGGAGTCGTTGGACATTGA