On the assumption that the hygromycin B phosphotransferase gene (hph) was integrated randomly into the genome of A95, total genomic DNA of the transformants was amplified with the primer pair hphRforw (5'-ATGCCTGAACTCACCGCGAC-3') and hphRrev (5'-CTATTCCTTTGCCCTCGGACGAG-3').
The coding sequence (CDS) of poswo1 in transformants was amplified using the primers Po-F1 and Po-R1.
The plasmid DNA from the transconjugants and transformants was amplified by five multiplex and three simplex PCRs using 18 pairs of primers that recognized Inc replicons FIA, FIB, FIC, HI1, HI2, I1-I, L/M, N, P, W, T, A/C, K, B/O, X, Y, F, and FIIA.
For it, RNA from paromomycin-resistant transformants was reverse transcribed, and products were amplified by PCR using aphVIII specific primers.
In all transformants, PCR fragments of the expected sizes were amplified and, thus, the genomic integration of the artificial gene constructs could be confirmed by PCR.
Inserts were amplified by colony PCR of transformants using vector-specific primers (p-topo2.1).
In each co-transformation experiment, fragments of the expected sizes of 212 bp, 479 bp, 343 bp, and 343 bp were amplified from some of the paromomycin-resistant transformants.
These genes had been amplified in original transformants, but they were completely eliminated by BA.
For analysis of the Δ tku70 transformants, the 3.4 kb tku70 probe was amplified with primers E/K (Table 1) and genomic DNA was digested with EcoRI.
