Interference No. 102,572
subcloning DNA into M13 vectors, preparing single-stranded template and carrying out the
sequencing reactions. Rey also testified that he assisted in the sequencing of the heavy
and light chain cDNA's. Rey, ¶ 4 (CR-33).
(8) Holmes testified that he and Heyneker analyzed the sequences and found that
the entire coding region of the light chain was found in the cDNA insert of pK17G4 and that
portions of the nucleotide sequence of the heavy chain were found in two isolated
plasmids: pGamma298 and pGamma11. Holmes, ¶ 7.
(9) Holmes indicated that the plasmid, pKCEAInt 2, for direct expression of the anti-
CEA light chain gene was prepared from five DNA fragments, 1-5. According to Holmes,
11
fragment 1 was prepared by digesting pHGH207-1* with EcoRI, filling in and digesting
with BamHI. Following purification of this fragment, Holmes stated that he treated the DNA
with bacterial alkaline phosphatase (BAP). The large fragment (fragment 1) was purified
by PAGE. Holmes, ¶ 8 (CR-30). For fragment 2, Holmes testified that he digested
pK17G4 DNA with PstI, purified the fragment by PAGE, digested with AvaII and isolated
the 333 bp PstI-AvaII fragment by PAGE; he used the PstI-AvaII fragment and an
oligonucleotide primer in a primer repair reaction to introduce the initiation codon to the
light chain gene. Following the primer repair, Rey sequenced a PstI to AvaII DNA fragment
of the light chain. Rey, ¶ 5 (CR-34). Holmes indicated that he and Heyneker analyzed the
11The Cabilly et al. brief (page 11) alleges that the “filling in” was done with DNA
polymerase I large fragment . No one testified as to how the filling in was done. Meitzner,
549 F.2d at 782, 193 USPQ at 22.
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