How to make bar-coded deletions

Fig. 18. This diagram, which comes from the Saccharomyces Genome Deletion Project web site, summarizes the methods used to generate specific gene deletions marked by two specific sequence tags ("bar codes").

Two rounds of PCR are used in order to build up stretches of DNA of sufficiently length for specific homologous recombination with the genome at each end of the final PCR product, without requiring any single oligonucleotide to be too long for automated synthesis with reasonably high yield.

The initial uptag and downtag primers (74 nucleotides) consist of (i) 18 bp of homology to the upstream or downstream portions of a particular ORF (including the relevant AUG and stop codons), a common priming site U1 or D1, a unique 20 base "barcode" sequence, and a common priming site U2 or D2. The latter are homologous to sequences at the 5' and 3' ends of the kanamycin-resistance gene, kanMX4.

The Up_45 and Down_45 primers (45 nucleotides) contain the 45 bp directly upstream or downstream of the relevant ORF (including the start and stop codons, as above). In some cases, extra nucleotides homologous to the genomic sequences needed to be added for efficient transformation. This was done with additional primers containing upstream and downstream sequences, with partial overlap of the round 2 PCR product. See the link above for further details.