Software routines that automate primer selection were written at the Stanford Genome Technology Center (SGTC) and were based on annotated sequence information from SGD. ORF and sequence data were updated from the site over a 3-year period.
At least 10% of the yeast genome is redundant, and early in the project
the primer picking program did not determine whether or not the targeting
primers were unique within the genome. A BLASTN
search used chose unique sequences was added where the primer picking criteria
cutoff was set at E=10 and the number of mismatches allowed = 0. Subsequently,
6.1% of the genes were not attempted in the deletion process because unique
primers could not be chosen in the 45bp regions flanking these ORFs.
Software routines that generated the bar-coding tags used in the above
construction of the deletion cassette were developed in collaboration with
Confirmation primers were picked using a modified version of the Whitehead
Institute PRIMER program. Primer lengths ranged from 17 to 35 nucleotides;
their Tm was 65 +2° C; they had 30-70% G/C content.
All primers were synthesized at the SGTC on an Automated Multiplex Oligonucleotide Synthesizer (A.M.O.S.) in 5-10 nM amounts in batches of 96 using standard phosphoamidite chemistry. The common portions of the deletion module primers were built on common existing primers contained in Core Porous Glass (CPG's).. Other than trityl readings, no other quality control was exercised on the primers. Greater than 99% of primers successfully generated the deletion cassette modules. The remaining < 1% were successful when resynthesized.
Deletion cassettes were amplified at Stanford and distributed for subsequent transformation of yeast along with the confirmation primers to the several labs participating in this project.
Primer Descriptions:UPTAG PRIMER:
The UPTAG primers are 74mers that contain (5'->3'): 18 bases of homology to the region upstream of the yeast open reading frame, including the AUG; a common tag priming site U1 (GATGTCCACGAGGTCTCT); a unique20 base "tag" sequence; common tag priming site U2 (CGTACGCTGCAGGTCGAC) which is homologous to a region 5' to the Kan gene in the kanMX4 module.
UP_45 and DOWN_45 PRIMERS:
UP_90 and DOWN_90 PRIMERS: