of each outer primer 1 u taq polymerase roche molecular biochemicals laval canada and 5 10 u03bcl nucleic acid sample for the first round pcr the denaturation annealing and elongation temperatures and times used were 95 u00b0 c for 30 s 42 u00b0 c for 30 s and 78 u00b0 c for 2 m respectively for 42 cycles after the final cycle the products were kept at 78 u00b0 c for 10 m one u03bcl of the pcr product was subjected to a second round pcr with the set of inner primers denaturation annealing and elongation temperatures and times used were 95 u00b0 c for 30 s 47 u00b0 c for 30 s and 78 u00b0 c for 1 m respectively for 42 cycles followed by a single incubation at 78 u00b0 c for 10 m after the first and second round pcr 10 u03bcl of pcr product was analyzed by gel electrophoresis using 1 5 agarose stained with ethidium bromide visualized under ultraviolet light and photographed visible bands with appropriate size were cut and sequenced using the inner primers dna landmarks st jean sur le richelieu canada sequence homology search was performed using the blast program of the nih web site samples negative for the appropriate band by pcr.