Interference 102,728
DPAP A site; (5) sequence the clone; (6) reinsert the mutagenized BamHI
fragment into pC1/1 as disclosed in Brake 1.
13. Brake argued that other techniques for making the n=0 construct
disclosed in the ‘325 Application were also known by those skilled in the art by January,
1983. Paper No. 15, p. 12.
14. Brake relied on the following declaration testimony of Dr. Tekamp-Olson
(para. 5b) for support:
b. An alternative technique, which also would have been within the
level of ordinary skill in January 1983 would have been to digest the disclosed
vector in Brake 1 (e.g., pAB112) with the restriction endonucleases EcoRI and
Hind III. Doing this would have isolated a fragment containing the "-factor
leader and promoter, including the DPAP A site. This fragment would then be
treated to a limited digestion with Bal 31 to remove the DPAP A site, e.g., to
Serine 81 in the "-factor fragment. This would result in a blunt fragment, the "-
factor promoter/leader fragment shortened on both the promoter and on the
processing site of the leader. This fragment would be digested with Bgl II to
generate a Bgl II sticky end at the promoter end of the fragment and gel isolated.
The EGF-fragment is generated by HgaI digestion of an EcoRI EGF
fragment as described on p. 13, lines 3-6. This fragment was ligated to a blunt-
Hga linker as follows (assumes screening for a Bal 31 fragment which includes
Ser81 coding sequence):
CTAGATAAAAAGA
GATCTATTTTCTTTGAG as well as the Hga-Sal linker disclosed on p. 13,
lines 14, 15. This EGF fragment would be gel isolated.
The Bgl II-blunt "-factor promoter fragment, the blunt Sal EGF fragment
and pAB112, digested with Bgl II and Sal I would be ligated together and clones
screened for the appropriate insert. One would then screen for clones in which
the DPAP A site had been removed by screening with an oligonucleotide which
spanned the Lys-Arg of the "-factor and the N-terminus of the EGF gene. This,
combined with the teaching in Brake 1 would easily have led one of ordinary skill
in the art in January 1983 to generate the spacerless construct of the Count.
11
Page: Previous 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 Next
Last modified: November 3, 2007