sequences for the letters were as follows: Outer 5' -GC GAGCACAGAATTAATACGA

CT-3', Inner 5' -CGCGGATCCGAATTAATACGACTCACTATAGG-3'`

PCR amplification of entire UGT gene

 Elucidation of the complete gene sequence for liver UGT from catfish by RLM-

RACE (via partial sequence overlap) enabled the design of gene specific primers which

are complementary to the gene itself as well as the untranslated region. The primers used

are shown in Table 5-6. All primers complementary to the untranslated region (UTR)

were designed with the help of Primer3 software, except for the pair of primers that were

complementary to the exact start and end of the gene (LIVUGTF1 and LIVUGTR1

respectively).


Table 5-6. Primers used for amplifying liver and intestinal UGT gene

GSP ID Sequence (5' 3') Start position

UTR Fl CTGCTTCCTCTAGACGTAATTAGAAAC 40
UTR F2 CTCACATTCCTCCTCCTTCTTTTT 76
UTR R1 GAAC GT GGT GAT GAGAACACTATAACT + 121
UTR R2 TAGTGACATCATAACAACCGTAACTGC + 190
LIVUGT Fl ATGCCTCGTCTTCTTGCAGCTCTCTGT 1
LIVUGT R1 TCACTCCTTTTTGCTCTTCTGAGCCCT 1568

 Due to the length of the amplicon (~1.6kb), Super Taq Plus polymerase (Ambion

Inc) was employed. This enzyme results in higher yields with amplicons >1kb. In

addition, this enzyme mixture has a proof-reading ability, which will be important for

future expression of the gene, as well as providing greater fidelity and processivity than

ordinary thermos Taq DNA polymerase. An extension temperature of 680C and an

extension time of 1.75 min were used for this PCR. Different combinations of UTR