Interference No. 102,572
therefrom were stamped onto agar plates and allowed to grow. Holmes, ¶ 5 (CR-29).
(5) Rey, a research assistant at Genentech reporting to Heyneker, testified that he
began work on the project in July 1982 when he received microliter dishes with cultures
containing cDNA from the hybridoma cell line CEA.66-E3. He transferred these cultures to
agar plates and allowed them to grow and later transferred the colonies to nitrocellulose
filters, layered them onto agar plates and allowed them to grow. Once grown, he lysed the
colonies on the filters and treated them for subsequent probing. Rey, ¶ 3 (CR-33).
(6) Holmes used oligonucleotides from the Organic Chemistry Department at
Genentech to prepare light and heavy chain oligonucleotide probes to hybridize with the
filters. After exposure to X-ray film, he picked several colonies which hybridized to the light
or heavy chain oligonucleotide probes, characterized the colonies by Pst restriction
9
endonuclease digestion and fractionation by [sodium dodecylsulfate(SDS)]
10
polyacrylamide [gel] electrophoresis (PAGE) . Several colonies which hybridized to the
heavy chain probe were also digested with both PstI and NcoI and analyzed by PAGE.
Holmes subcloned these DNA sequences into M13 vectors. Holmes, ¶ 6, ¶ 7 (CR-30).
(7) Rey testified that he assisted in the sequencing of the heavy chain cDNA by
9Restriction digestion is a process involving the use of enzymes which recognize
different DNA sequences and cleave the DNA backbone at the site recognized forming
either blunt or stick ends.
10SDS-PAGE stands for sodium dodecylsulfate-polyacrylamide gel
electrophoresis which is a technique which separates various species of proteins or
polynucleotides of different sizes in an electric field.
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