Protocols &Technical Information
Deletion Strategy -- Overview
We are using a PCR-based gene deletion strategy (Baudin et al., Nucl.
Acids Res. 21, 3329-3330, 1993 and Wach et al., Yeast 10, 1793-1808,
1994). Eight to ten different primers are required for each open reading
frame. There are two or three pairs of long oligonucleotides that constitute
the "Deletion Module Primers". These primers are used to generate
the deletion constructs for the yeast transformations. There are also
four confirmation primers that are used to check the correct integration
of the deletion module (see Confirmation
PCR Strategy).
Because of the large number of open reading frames involved, one of the
challenges of this project is the automation and coordination of primer
design and synthesis. The primers used in this project were designed using
simple computer algorithms: Lists of 96 open reading frames were obtained
from the Saccharomyces Genome Database, along with ORF location
coordinates and chromosomal sequence data. No attempts were made to edit
the list of open reading frames chosen for disruption.These data were
used as input for the primer-picking scripts. In addition, two unique
20 base pair tag sequences were selected from a pre-existing list and
assigned to each open reading frame. The output from the primer-picking
scripts are files containing the ORF names and the corresponding primer
sequence data which are formatted for use with the Automated Multiplex
Oligonucleotide Synthesizer (A.M.O.S.).
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, followed by the common tag priming site U2 (CGTACGCTGCAGGTCGAC). The common tag priming site U2 is homologous to a region 5' to the Kan gene in the kanMX4 module.
DOWNTAG PRIMER
The DOWNTAG primers are 74mers that contain (5'-->3'): 18 bases of homology to the region downstream of and including the stop codon of a particular open reading frame, common tag priming site D1 (CGGTGTCGGTCTCGTAG), a unique20 base "tag" sequence, followed by the common tag priming site D2 (ATC GAT GAA TTC GAG CTC G). The common tag priming site D2 is homologous to3' to the Kan gene in the kanMX4 module.
UP_45 and DOWN_45 PRIMERS
The upstream deletion primers (UP_45) are 45mers that were designed by
finding the 45 bases directlly upstream of each yeast open reading frame
(including the ATG).
The downstream deletion primers (DOWN_45) are 45 mers that 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.
Notes:
At least 10% of the yeast genome is redundant. Early in project, the primer picking program does not determine whether the targeting primers are unique within the genome. In the case of some open reading frames, particularly telomeric open reading frames, substantial upstream and downstream genome sequence redundancy exists. Currently, primer sequences are checked to ensure that they are unique within the genome. If sequences are not unique, longer targeting sequences may be used in the upstream and downstream primers.
SGD periodically makes revisions to its databases. These revisions might include changes in ORF designation, ORF nomeclature or ORF location. The ORF data for chromosomes were downloaded between October 1996 and July 2000. In some cases, ORFs may have been removed, added or corrected in SGD. Therefore, the user might notice some inconsistencies between names in our collection and the names and locations in SGD. If you are unable to locate information about an ORF, perform a BLAST search against SGD genomic data with the unique regions of the upstream and downstream primers to find the corresponding ORF in SGD.
For batches chr5_4, chr1_1, chr13_1, chr13_2 and chr13_3, see Deletion Module PCR Strategy early rounds
Primers for batches chr13_4, and chr5_4 were synthesized in a slightly different way. No yeast sequence was included in the UPTAG and DOWNTAG primers, and the UPSTREAM45 and DOWNSTREAM45 primers contain sequences complementary to tag priming site U2 and D2, respectively at their 3' ends. They also contain 55 bases of yeast sequence instead of 45.
last updated January 2002 amchu@cmgm.stanford.edu