Deletion Strategy -- Introduction
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
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.).
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.
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.
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 email@example.com