Figure 1

Principle of ccd B-gene replacement strategy. The schematic representation of the ccd B-gene replacement strategy follows three simple steps, two PCR reactions and a direct transformation of the second PCR product. Primers F1 and R1 are used in a gene-specific amplification in the first PCR round. Gene-specific regions of primers are indicated by thin, green arrows, whereas the introduced overhang regions are coloured in orange. In the second PCR, the product from the first PCR is used as a megaprimer in linear plasmid amplification. The overhang regions of the product bind to the desired insertion sites in the vector that flank the ccd B gene. ccd B-gene for negative selection becomes replaced during amplification of the plasmid. The R2 primer can be used for exponential amplification. Complementary primer-binding regions in the vector are marked in the same colours as primers. Finally, the parental vector and the product from the second PCR can be transformed directly to competent cells. Those cells that take up the paternal vector will be subjected to un-repairable chromosomal damage caused by a toxin encoded by the ccd B-gene, and will ultimately die. Positive clones, however, will survive since the negative selection marker gene has been replaced with the gene of interest. Generally, input DNA, such as genomic DNA and vector, is coloured in grey to differentiate it from the black-coloured product DNA. Thin and fat arrows differentiate primers and open reading frames, respectively.