| Â | Â | Â | Amplimer | Â | Fingerprint |
|---|
Primers | Sequences | PCR conditions | size(bp) | Endonuclease | sizes(bp) |
|---|
|  |  | 95°C 10 min |  |  | 519 |
DF1 | 5'gggagctgggacatgg | Hot Sart, | Â | Â | Â |
|  | ac | 95°C - 45 sec/ | 1.1 kb |
PpumI
| 316 |
|  |  | 64°C - 2 min |  |  |  |
| Â | Â | 40 cycles, | Â | Â | Â |
DR2 | 5'ctggaccacatcagga | 72°C 5 min. |  |  | 265 |
| Â | gaca | Â | Â | Â | Â |
- Total RNA (3 μg) was denatured together with oligo-dT primer (10 pmol) for 15 min at 68°C. After 5 min incubation on ice, poly-adenosine (poly-A) RNA was reverse-transcribed at 42°C for 90 min in RT solution (50 mM Tris-HCl, pH 8.3; 40 mM potassium chloride; 8 mM magnesium chloride; 0.5 mM each dNTP; 225 ug/ml bovine serum albumin; 5 mM dithiothreitol; 20 units Rnasin and 200 U superscript (Gibco-BRL, Gaithersberg, MD, USA)). The cDNA was incubated at 95°C for 5 min to inactivate the reverse transcriptase, and served as the template for PCR amplification. Sequence information in the genomic clones have been used to generate the PCR primers. The most suitable PCR amplification conditions were determined for each set of primers by on line primer design program (primer3). PCR was performed after adding 80 ul of PCR mixture (50 mM each dNTP; 50 pmol of each sense and the antisense primer; 2.5 units of Taq polymerase (Perkin-Elmer, USA).
