| Anchor
Spots |
 |
Hybridization filters are gridded with the incorporation
of a Caenorhabditis briggsea BAC clone (RPCI-94 1A1) in the
corner wells of each of the six "fields" that comprise
an entire high-density filter, resulting in 24 pairs of "anchor
spots". When the anchor spots are available on the hybridization
filter, an additional anchor-spot probe can be included in
each hybridization bottle. These spots are intended to be
an aid in the lining up a grid to the phosphoimager image
of the hybridized filter, when using "ArrayVision"
software. These spots are also helpful for matching a transparent
"grid overlay" to an autorad image of filters with
low background [clone] intensity. The anchor spots have been
generated by spotting the C. briggsae clone in duplicate using
a diagonal vector direction pattern (see below) different
from the eight patterns used for the actual library clones
on the filter. This reduces the time spent aligning the templates
on the various filter images encountered and allows for rapid
scoring of filters with minimal background. We have sequenced
the T7-end of RPCI-94 1A1 clone and designed a 3'-overlapping
oligonucleotide (overgo) probe comprised of two 24 base oligonucleotides
with an 8 bp overlap. The sequences of these oligonucleotides
are described below. The overgo probe is labeled by adding
radioactive nucleotide monophosphates to the 3'-recessed ends,
as described below.
Control pattern:
The insert-vector junction sequence of control clone RPCI-94
1A1 (T7 end). The vector sequence (the first 30 nucleotides
including the "G" at position 30 belonging to the
GAATTC cloning site) is followed by the insert sequence:
TGTGCNTCCTNTGCGGATTCTGNNTCGACAAGAATTCCGGCAAAATCGAAAATCACATGG
GTGACAGGAGAAAATGACATCTNGAAAATGAAATTTGTCAACGAACTGAAATCTGAGAAT
TTACAGATAGAAGTAGATGAACACCGCCAGAAAATAAAAAAGCCAAACAAATCCAATAAA
ATAAATTATTAATTTAAAATTAAGGAATGTACACTTCGACATACAATAAAAAAAAAGAAG
TTGAGATTTTCAGAGTGATACAGAAATAAAAGGAGAAGGCATGATTTTCAAGCATGAAGT
GAACACATTTTTTTAATTATGAAAATCTGAAAAATGAGATTCTGACATAAAGAGAGAGGG
CGATATAATAATAAATGTGTGATTATTCGGGATTAAAAAGAGTACATTAACGTTGAAATA
TTCTGAGCAGACAATTTTCAGATTAATTGACATGGAACATTTCGAACTCGCGCGGTTTTT
GTTGCCAAATTCCGAGATCTTGGCGACGAAGCCACATGATGAATGACTTTTTGGCTTNCA
GAGTAGTCCAGACATCCAGCTTTCATTTCTTTCGTCCGTCATGGACTCGTCTAGAGGAGT
TTTGGNTAGGAAACCAGGTCTNGAAAAATTTACAAAAATTCAAAGAAAAACCTTTNAAGA
CATATGCTCGAAATNCTCCTTNCCNNTCAATTTGCCTAACCNATTTCTTGNCCCAATTCT
CCNANGGGTTCTTAANATTNCCNTTTCCNNNCCTNTTCCCTTTTTNTTGGAACNTTTCCC
TCTNNAAATNNTTTNATANNCTTTTTNAANNTGTCCNTTTCNCCAANATTCCAATTNTTN
NTTTCTCCNNTNTCCGNGCCANANNNTGNCNTGGTTTGTNCAACATTCTTCCCGGTTGGC
NNTCNTNCAATTATTTNCNTGNNTTACCCCTGTAATNGGAGCTNGTNGGTTGGATTGNAN
NTANCNCNGGTNGTNTCNCGCTTTTNGTNAATTCTNACNGANAATTGTTTTGTCNGCCCT
The sequences of the two oligos are:
ALIAS 5'-3' SEQUENCE
RPCI941a1-OVF GTTGCCAAATTCCGAGATCTTGGC
RPCI941a1-OVR ATCATGTGGCTTCGTCGCCAAGAT
The complete overgo sequence:
GTTGCCAAATTCCGAGATCTTGGCGACGAAGCCACATGAT
Overgo probes
24mer
5'-------------------------3'
(8 bp)
3'----------------------5'
| 24mer
|
Klenow, 32-P-dATP, dCTP
|
|
V
5'------------------------****************
(8 bp)
******************-----------------------5'
ds 40mer
|
|
V
Screen BAC filters
|
