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ÓÉÓÚûÓÐÉúÎï±³¾°£¬ÏÖÐèÒª·ÒëÏÂÃæÄÚÈÝ£¬Å·Òë²»×¼£¬ÇëÐֵܽãÃÃÃǰï°ï棬·Ç³£¸Ðл£¡ DNA templates were prepared¡¡by the freeze-thaw method in a DNA extraction buffer(25). PCR was conducted for 40 cycles, with denaturation¡¡at 94¡ãC for 60 s, annealing for 30 s, and extension¡¡at 72¡ãC for 30s using the PROGRAM TEMP CONTROL¡¡SYSTEM (PC-800, ASTEC, Fukuoka). Here,¡¡considering that each Cl20 or C230 target sequence has¡¡a different homology score with the designed primers, a¡¡step down method was employed (26). For the Cl20¡¡genes, the annealing temperature was 61¡¯C in the first 10¡¡cycles followed by a step down to 59¡ãC in the next 15¡¡cycles, and 57¡ãC in the last 15 cycles, while for the C230¡¡genes, the annealing temperature was 59¡ãC in the first 10¡¡cycles, 57¡ãC in the next 15 cycles, and 55¡ãC in the last¡¡15 cycles. Aliquots (10~1) of the PCR products were¡¡analyzed by electrophoresis on a 1.0% agarose gel stained¡¡with 0.5 pg/ml ethidium bromide. |
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