the first strand cdna was synthesized using mmlv reverse transcriptase clontech and each 5 u2032 cds primer and 3 u2032 cds primer provided in smart u2122 race cdna amplification kit clontech respectively to carry out race gene specific primers fig1 were designed based on sequences of chicken gst u03b1 class transcripts cgsta1 cgsta2 cgsta3 and cgsta cl3 to perform 5 u2032 and 3 u2032 race fragments were amplified in pcr reactions with both universal primer mix upm long primer 5 u2032 ctaatacgactcactatagggcaagcagtggtatcaacgcagagt 3 u2032 seq id no 7 and short primer 5 u2032 ctaatacgactcactatagg gc 3 u2032 seq id no 8 and nested universal primers nup 5 u2032 aagcagtggtatcaacgcagagt 3 u2032 seq id no 9 provided in smart u2122 race cdna amplification kit clontech the following pcr profile was performed with advantage 2 pcr kit clontech 2 minutes at 94 u00b0 c followed by 34 cycles for first reaction and 25 cycles for nested pcr consisting of 30 seconds at 94 u00b0 c 30 seconds at an optimal annealing temperature 58 68 u00b0 c and 8 minutes extension time at 72 u00b0 c pcr products were subcloned in ta cloning vector pdrive qiagen and transformed into chemically competent e coli dh5 u03b1 invitrogen the presence of race fragments within the clones was confirmed by colony pcr.