In Vitro Cell. Dev. Bio. 32:73-74A Contributed paper # P-1030. World Congress on In Vitro Biology, San Francisco, CA, June 22-27, 1996.

Polymerase chain reaction (PCR) has been employed to identify transgenic plants early in the selective cultivation process, when there are too many candidates and not enough purified DNA available to perform Southern blot tests. We developed a rapid PCR approach making use of an Air Thermo-cyclerÆ, Model 1605 (Idaho Technology Inc.). The thermo-cycler was set to sequentially run the following protocols: (1) pre-cycle denaturation at 94C for 2 min, (2) 30 cycles of denaturation (10 s at 94 C), annealing (10 s at 55C), and extension (1 min at 72C), and (3) post-cycle extension at 72 C for 7 min. The whole process could be completed in approximately 55 minutes. The procedure was used to amplify NPT II and GUS genes from genomic DNA of transformed petunia (Petunia hybrida cv. Mitchell) plants and shining willow (Salix lucida Muhl.) calli. PCR reaction was performed in a 10 microleter of mixture containing 2 microleters of rapid cycling 5X buffer (Idaho Technology Inc.), 0.2 mM of each dNTP, 0.5 micromoles of each 20 base-pair primer, 0.2 units of AmpliTaq DNA polymerase, and 50 ng template DNA. The reactions were also performed in 10 microliter mixtures containing 1 microleter of Pfu 10X buffer #2 (Stratagene), 500 mg/ml BSA, 0.2 mM each dNTP, 0.5 mM each 20 base-pair primer, 0.4 units of Pfu DNA polymerase, and 10 ng template DNA. The NTP II and GUS genes were specifically amplified using either AmpliTaq or Pfu polymerase.