A rapid polymerase chain reaction method for early screening of transgenic plants

Abstract

A polymerase chain reaction (PCR) approach using an air thermal cycler was developed to identify transgenic plants. The PCR amplification consisted of a pre-cycle denaturation (2 min at 94 °C), followed by 30 cycles of denaturation (10 sec at 94 °C), annealing (10 sec at 55 °C), and extension (1 min at 72 °C), and a post-cycle extension (7 min at 72 °C). The reaction could be completed in approximately 55 minutes. Denaturation and annealing times were found to be critical in obtaining repeatable amplification. This procedure was used to detect the neomycin phosphotransferase (NPT II) and [3-glucuronidase (GUS) genes from genomic DNA of transformed petunia (Petunia hybrida cv. Mitchell) plants and shining willow (Salix lucida Muhl.) calli. Both AmpliTaq and Pfu DNA polymerases were capable of amplifying the target genes, but had significant differences in sensitivity. transformation, Pfu