Primers Used for PCR Amplification
| Amplified Fragment . | Primer Sequence . |
|---|---|
| Mismatch primers | |
| bp 493-643 (150 bp) | S 5′-AACCGCATCATCGTGGAGGAG AS 5′-TCAGCACCATGAGGTTCTGC |
| Sequence primers for genomic DNA | |
| intron 5-bp 743 (270 bp) | S 5′-TGTAAAACGACGGCCAGTAGGAGGTTCTGGCCTCTACT AS 5′-TCAGCACCATGAGGTTCTGC |
| Sequence primers for complementary DNA | |
| bp 448-703 (255 bp) | S 5′-TGTAAAACGACGGCCAGTCACCAAGAACATTCACGAGTCC AS 5′-GGATAACGCAGGCGATGTTGTCCC |
| Amplified Fragment . | Primer Sequence . |
|---|---|
| Mismatch primers | |
| bp 493-643 (150 bp) | S 5′-AACCGCATCATCGTGGAGGAG AS 5′-TCAGCACCATGAGGTTCTGC |
| Sequence primers for genomic DNA | |
| intron 5-bp 743 (270 bp) | S 5′-TGTAAAACGACGGCCAGTAGGAGGTTCTGGCCTCTACT AS 5′-TCAGCACCATGAGGTTCTGC |
| Sequence primers for complementary DNA | |
| bp 448-703 (255 bp) | S 5′-TGTAAAACGACGGCCAGTCACCAAGAACATTCACGAGTCC AS 5′-GGATAACGCAGGCGATGTTGTCCC |
Bold print indicates mismatch nucleotide to permit BanII cutting of wild-type product into 3 fragments of 105 bp + 22 bp + 23 bp, but mutated 514T product into 2 fragments of 105 bp + 45 bp. Italics indicate binding site for dye primers. Amplification conditions for genomic DNA in Air Thermo-cycler 1605 (Idaho Technology, Idaho Falls, ID): 50 rounds of 5 seconds at 95°C (denaturation), 10 seconds at 50°C (annealing), and 30 seconds at 72°C (extension), at full speed. Amplification conditions for cDNA in Air Thermocycler: 50 rounds of 5 seconds at 95°C, 30 seconds at 60°C, and 15 seconds at 72°C, at full speed.