FIGURE 1.

The three-step PCR technique used to generate linear double-stranded DNA with 500-bp-long homology extensions. In the first step, the upstream 500 bp and the downstream 500 bp of the target chromosomal gene were amplified independently. Each of the UP.R and DOWN.F primers had a short segment homologous to the respective region of the resistance marker. The second step generated the linear DNA from the two homologous 500-bp segments and the resistance marker. In the third step, the linear DNA was transformed into the expression strain and incorporated into the chromosome by homologous recombination.