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Oligonucleotide Details

Primer choice:

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 Affymetrix.

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.

Synthesis:

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.

DNTAG PRIMER:
The DNTAG primers are 74mers that contain (5'-->3'): 18 bases of homology to the region downstream of and including the stop codon of th open reading frame, a common tag priming site D1 (CGGTGTCGGTCTCGTAG), a unique20 base "tag" sequence, followed by the common tag priming site D2 (ATCGATGAATTCGAGCTCG) which is homologous to3' to the Kan gene in the kanMX4 module.

UP_45 and DOWN_45 PRIMERS:
UP_45 were designed by finding the 45 bases directly upstream of each yeast open reading frame, including the ATG.
DOWN_45 were designed by finding the 45 bases directly downstream of each yeast open reading frame, including the stop codon.

UP_90 and DOWN_90 PRIMERS:
UP_90 and DOWN_90 are 63 bp primers were designed to extend the flanking genomic ends of the cassettes to 90bp of homology from 45 bp. Less than 10% of the deletion cassettes were made with these additional primers.