PCR amplification of minimal gene expression cassette: an alternative, low cost and easy approach to ‘clean DNA’ transformation

We report here an alternative and easy approach to generate sufficient quantity of minimal gene expression cassette (promoter–open reading frame–terminator) DNA by PCR amplification using high fidelity DNA polymerases. Isolating the minimal gene cassette DNA in large quantities by restriction digestion and gel extraction for biolistic-mediated ‘clean DNA’ transformation, is not only a cumbersome and time-consuming process, but also laborious and expensive. The minimal cassette for GUS gene, generated from pCAMBIA1305.1, was used as a reporter system for bombardment of mature embryo-derived calluses of indica rice IR64. Transient GUS expression in the bombarded calluses showed no significant difference, in terms of GUS expression units, whether the GUS gene cassette was generated by restriction digestion or PCR amplification using high fidelity DNA polymerases. The results demonstrated that PCR amplification of the minimal gene cassette using high fidelity DNA polymerase is a reliable, low cost and easier approach to ‘clean DNA’ transformation.