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Johns Hopkins University v. 454 Life Sciences Corp.

United States District Court, D. Delaware

January 26, 2017

JOHNS HOPKINS UNIVERSITY, Plaintiff;
v.
454 LIFE SCIENCES CORPORATION, Defendant.

          Karen L. Pascale, James L. Higgins, YOUNG CONAWAYSTARGATT & TAYLOR LLP, Wilmington, DE Robert F. Altherr, .Jr., Joseph M. Skerpon, Christopher B. Roth, BANNER & WITCOFF, LTD., Washington, DC Attorneys for Plaintiff Johns Hopkins University.

          Kelly E. Farnan, Selena E. Molina, RICHARDS, LAYTON & FINGER, P.A., Wilmington, DE R. Danny Huntington, Sharon E. Crane, C. Nichole Gifford, Seth E. Cockrum, ROTHWELL, FIGG, ERNST & MANBECK P.C., Washington, DC Attorneys for Defendant 454 Life Sciences Corporation.

          MEMORANDUM OPINION

          STARK, U.S. DISTRICT JUDGE

         On November 6, 2013, Johns Hopkins University ("JHU" or "Plaintiff') filed this action pursuant to 35 U.S.C. § 146, seeking review of the Decision and Final Judgment of the Board of Patent Appeals and Interferences ("the Board") in Interference No. 105, 857 ("the Interference"). (See D.I. 1 at 1, 8; Statement of Admitted Facts ("SAF") D.I. 102-1 ¶ 1) The interfering applications are JHU's U.S. Patent Application No. 12/361, 690 (D.I. 39 Ex. 1) (JHU's "'690 application") and U.S. Patent Application No. 13/33, 240 (D.I. 39 Ex. 5) (454's "'240 application"), which was filed by Defendant 454 Life Sciences Corporation ("454" or "Defendant"). The Interference involves a single count ("Count"), [1] with the interfering subject matter represented by claim 1 of JHU's '690 application and claim 52 of 454's '240 application. (See D.I. 44 at 1; D.I. 45 at 1)

         Claim 1 of JHU's '690 application recites the following four-step method: A method for analyzing nucleic acid sequences comprising:

(a) generating a plurality of molecules of a fragment of deoxyribonucleic acid;
(b) delivering the plurality of molecules of the fragment of deoxyribonucleic acid into aqueous microreactors in a water-in-oil emulsion such that a plurality of aqueous microreactors comprise a single molecule of the fragment of deoxyribonucleic acid, a single bead capable of hybridizing the fragment of deoxyribonucleic acid, and reagents necessary to perform deoxyribonucleic acid amplification;
(c) amplifying the fragment of deoxyribonucleic acid in the microreactors to form amplified copies of said fragment of deoxyribonucleic acid bound to beads in the microreactors; [and]
(d) determining presence of amplified copies of said fragment of deoxyribonucleic acid bound to a bead.

(D.I. 44 at 2-3)[2]

         The Court held a claim construction hearing on June 9, 2015 and issued a memorandum opinion on claim construction on August 24, 2015. (D.I. .56) Thereafter, the parties filed summary judgment motions. On May 2, 2016, the Court denied all summary judgment motions, with the exception of JHU's motion for partial summary judgment that JHU's priority date with respect to the Count is no later than June 5, 2003, which the Court granted. (See generally D.I.; 97, 98)

         The Court held a bench trial on all remaining issues in June 2016. (See Transcript, D.I. 112, 113, 114 ("Tr.")) The parties later submitted post-trial briefing (D.I. 108, 110, 115, 118) and proposed findings of fact (D.I. 109, 111, 116, 117).

         Pursuant to Federal Rule of Civil Procedure 52(a), and after having considered the entire record in this case and the applicable law, the Court concludes that: (1) JHU has failed to prove that it is entitled to priority of invention, and (2) JHU has failed to prove that 454' s '240 application is invalid.

         The Court's findings of fact and conclusions of law are set forth in detail below.

         FINDINGS OF FACT

         This section contains the Court's findings of fact for issues raised by the parties during trial. Certain findings of fact are also provided in connection with the Court's conclusions of law.

         A. Patent Applications at Issue

         1. Plaintiff JHU's ' 690 application, entitled "Method and Compositions for Detection and Enumeration of Genetic Variations, " was filed on January 29, 2009. (D.I. 22-1 Document 1) The named inventors are Devin Dressman, Hai Yan, Kenneth W. Kinzler, and Bert Vogelstein. (Id.)

         2. Defendant 454' s '240 application, entitled "Bead Emulsion Nucleic Acid Amplification, " was filed on February 23, 2011. (ATX 1001)[3] The named inventors are Gary Sarkis, Jan Berka, John Leamon, Kenton Lohman, Maithreyan Srinivasan, Yi-Ju Chen, Vinod Makhijani, Jonathan Romberg, Steve Lefkowitz, and Michael Weiner, (Id.) The '240 application issued as U.S. Patent No. 8, 748, 102 ("'102 patent) on June 10, 2014. (DTX 12)[4]

         3. The '240 application is a continuation of U.S. Patent Application No. 11/982, 095, filed on October 31, 2007 (ATX 1005), which is a continuation of U.S. Patent Application No. 10/767, 899 ("'899 application"), filed on January 28, 2004 (ATX 1007). The'899 application claims benefit to a number of provisional applications, including U.S. Provisional Patent Application No. 60/476, 592, filed June -6, -2003 ("'592 provisional" or "'592 application") (ATX 1013), and U.S, Provisional Patent Application No. 60/465, 071, filed April23, 2003 ("'071 provisional" or "'071 application") (ATX 1015).

         4. The contents of the'071 and'592 provisional applications are incorporated by reference in their entirety into the'240 application. C240 application at 1:3-6)

         5. The '240 application also incorporates "by reference co-pending U.S. Patent Application No. 10/767, 779 ("'779 application"), which issued as U.S. Patent No. 7, 323, 305 ("'305 patent") on January 29, 2008. (ATX 1001; DTX12;DTX 13) The '305 patent contains the entire disclosure of the '592 provisional. (Id.)

         B. Procedural History in the Patent Office

         6. The Board initially accorded JHU a priority date of July 5, 2003 and 454 apriority date of June 6, 2003 (the filing date of the '592 provisional), making JHU the junior party and 454 the senior party in the Interference. (See ATX 23 6 at 3:16-26)

         7. In the Interference, JHU filed a motion attacking 454's claim to the '592 provisional's filing date. (D.I. 23-4 at 7:6-13) The Board denied JHU's motion, ruling that JHU had failed to show that the '592 provisional did not disclose a reduction to practice within the scope of the Count. (Id.)

         8. In the Interference, 454 filed a motion to obtain the benefit of the '071 application. (ATX 236 at 8:11-16) The Board denied 454's motion, finding that 454 had failed to establish adequate written description support in the '071 application. (Id. at 15:24-26) Specifically, the Board found that "454 ha[d] not established (e.g., by citing to data or expert testimony)" that use of restriction enzymes recited in the '071 application "would generate two or more molecules of a specific DNA fragment, or more specifically, that an ordinary artisan would understand that to be the case, " as required to practice step (a) of the Count. (Id. at 14:13-18)

         9. During the priority stage, JHU submitted evidence of priority from the January to May 2003 time frame, (ATX 370 at 5 n. 1) However, the Board found that the "evidence cited by JHU does not sufficiently establish that JHU conceived of the subject matter of Count 1 in the January to May 2003 time frame?' (Id.) Specifically, the Board found that the evidence "[did] not adequately show JHU conceived of elements (a) and (b)" of the Count during that time frame. (Id.) The Board also found that "JHU offer[ed] insufficient non-inventor evidence (testimony or otherwise) to corroborate conception by the JHU inventors at that time." (Id.) As a result, the Board accorded JHU a June 5, 2003 conception date. (Id. at 7:16-22) The Board found that JHU reduced the invention to practice one month later, on July 5, 2003. (Id.)

         10. With respect to 454, the Board concluded that a preponderance of the evidence showed and corroborated the fact that the 454 inventors conceived of all elements of the Count before June 2003. (ATX 370 at 17:1-23:9) In particular, the Board found the combination of page 16 of the notebook of Dr. Jan Berka (one of the 454 inventors) (ATX 1094), evidence regarding experiments conducted between August and December 2002, evidence of experiments conducted in January and February 2003, a "Best Practices" document from February 2003 (ATX 1102; ATX 1103), and an invention disclosure form (ATX 1106 at 107-12), taken as a whole, established that the 454 inventors conceived the subject matter of the Count before JHU's earliest accorded date. (ATX 370 at 21:7-23:2)

         11. The Board held that "as long as the inventors conceived of performing [step] (a), i.e., generating a plurality of molecules of a particular fragment of DNA, along with [steps] (b) through (d), it [did] not matter whether the inventors failed 'to appreciate the value of step (a)' beyond its use to generate a control used in the method [as alleged by JHU]." (ATX 370 at 18:20-19:3) The Board further held that the "evidence establishe[d] that the inventors appreciated that [step] (a) took place, regardless of its 'value' in relation to benefits of the protocol in amplifying genomic template DNA." (Id. at 19:3-5)

         12. The Board awarded priority of invention to 454, finding that 454 conceived of the invention of the Count before JHU's conception and was also first to reduce the invention to practice. (ATX 370 at 23:8-9)

         C. The Court's Claim Constructions[5]

         13. The Court construed the term "generating a plurality of molecules of a fragment of deoxyribonucleic acid" in step (a) to mean "generating two or more of the same DNA fragment, not merely generating a plurality of DNA fragments overall." (D.I. 57 at 2) The Court's construction of step (a) does not require any particular method of generating two or more copies of the same DNA fragment. (Levy Tr. at 3 67:9-19)[6] However, the fragment formed in step (a) is a sequence that is ultimately amplified in the emulsion and sequenced. (Id. at 505:8-17)

         14. The Court determined that the following term required no further construction: "delivering the plurality of molecules of the fragment of deoxyribonucleic acid into aqueous microreactors in a water-in-oil emulsion such that a plurality of aqueous microreactors comprise a single molecule of the fragment of deoxyribonucleic acid, a single bead capable of hybridizing to the fragment of deoxyribonucleic acid, and regents necessary to perform deoxyribonucleic acid amplification." (D.I. 57 at 2) There is no significant distinction between delivering to a microreactor double-stranded DNA separately from a bead versus delivering single-stranded DNA pre-hybridized to ahead. (Levy Tr. at 405:8-24, 470:14-19; see also Tyagi Tr. at 116:13-15)

         15. The Court construed the terms "deoxyribonucleic acid" and "DNA" to mean "a nucleic acid molecule comprising deoxyribonueleotides." (D.I. 57 at 2) Single-stranded DNA is a nucleic acid molecule comprising deoxyribonucleotides. (Levy Tr. at 404:3-9)

         16. The Court construed the term "a single bead capable of hybridizing to the fragment" to mean "a single bead capable of binding to the fragment of deoxyribonucleic acid." (D.I. 57 at 2) The Court's construction of "a single bead capable of hybridizing to the fragment" only requires that a bead be capable of hybridizing to the DNA fragment. (Levy Tr. at 390:16-391:14) A bead that has bound to the fragment is capable of binding, and a bead that has not bound to a fragment may also be capable of binding. (Id.)

         17. The Count may be satisfied even if the fragment from step (a) is pre-hybridized to a bead before delivery to a microreactor. (Id. at 505:18-506:1) The sequence of the fragment formed in step (a) remains intact regardless of whether it is hybridized to the bead. (Id. at 391:24-393:14) Moreover, when the fragment of DNA hybridizes to the bead, it does not create a new molecule. (Id.) The DNA fragment and the bead retain their individual identity, even while hybridized. (Id. at 393:10-14, 459:19-460:4)

         18. Hybridization of the DNA fragment to the bead is through hydrogen bonding, which is a non-colalent, electrostatic interaction. (Id. at 392:16-393:9) Hydrogen bonding can b>e thought of like a sock sticking to a sweater when it is pulled out of a dryer. (Id.) Just because a sock is stuck to a sweater does not mean a new entity has formed. (Id.) Similarly, just because a DNA fragment has hybridized to a head does not mean a new molecule has formed. (Id.)

         D. Person of Ordinary Skill in the Art

19. One of ordinary skill in the art at the pertinent time would have had around four years of research experience, a Master's degree or PhD. in molecular biology or other related fields such as genetics or biochemistry, and would have been familiar with polymerase chain reaction ("PCR"). (Tyagi Tr. at 45:25-46:7; Levy Tr. at 353:7-354:4) A person of ordinary skill in the art would have been aware of emulsion PCR as a general concept. (Levy Tr. at 353:25-354:4 (stating that at time of invention "there were a couple of very high profile papers that describe[d] using emulsions to either do PCR, in the case of the Hollinger paper, or to use emulsions to encapsulate other things"); ATX 1099; ATX 1097 at 4)

         E. JHU's Witnesses

         20. JHU called just a single witness to testify live at trial. Dr. Sanjay Tyagi obtained a B.S. from the University of Rajasthan in India, two M.S. degrees in biology from the Jawaharlal Nehru University in New Delhi, and a Ph.D. from the University of Maryland. (Tyagi Tr. at 42:16-21; PTX 001)

         21. Since 1987, Dr. Tyagi has worked at the Public Health Associate Institute, which currently is a part of Rutgers University, initially as Associate Professor and currently as a full Professor. (Id. at 42:24-43:2) Dr. Tyagi has 29 years of postdoctoral experience in the field of nucleic acids, molecular biology, and cell biology research. (Id. at 43:5-44:7)

         22. JHU called several other witnesses to testify by reading some or all of the declaration(s) these witnesses submitted as part of the Interference proceedings. (See Tr. at 164:21-175:3; ATX 2024, 2034, 2051, 2079, 2080)

         23. JHU presented testimony from six witnesses by declaration:

a. Dr. Bert Vogelstein:is a professor of oncology at Johns Hopkins University School of Medicine, Baltimore, MD, and a Howard Hughes Medical Institute investigator. (ATX 2034¶2) Dr. Vogelstein supervised the work of Dr. Devin Dressman as a post-doctoral fellow in his and Dr. Kenneth Kinzler's laboratory beginning at the end of January 2003 and continuing beyond July 5, 2003. (/c/. ¶ 3)
b. Dr. Devin Dressman currently works in research and development for Life Technologies Corporation in Beverly, MA. (ATX 2051¶ 2) He is in the "Ion Torrent" division at Life Technologies, which deals with a sequencing system that utilizes bead emulsion amplification for sequencing sample preparation. (Id.) Dr. Dressman worked as a post-doctoral fellow in the laboratory of Drs. Kenneth Kinzler and Bert Vogelstein at Johns Hopkins University beginning at the end of January 2003 and continuing beyond July 5, 2003. (Ztf.'¶ 3)
c. Dr. Hai Yan worked with Dr. Dressman between January 2003 and July 5, 2003 on bead emulsion amplification projects. (Id.)
d. Dr. Kenneth Kinzler ran a laboratory with Dr. Vogelstein at Johns Hopkins University, as discussed above. (ATX 2034 ¶ 3)
e. Ms. Leslie Meszler held the position of Core Manager at the Cell Imaging Core at Johns Hopkins University in 2003. (ATX 2029 ¶ 1) She was responsible for managing the day-to-day operations of the Cell Imaging Core during 2003. (Id.) One of her responsibilities included tracking scientists' use of a flow cytometer. (Id., ¶¶ 3-5) She testified (by declaration) that the flow cytometer was used by Dr. Dressman during the week of April 20, 2003. (Id. ¶ 6)
f. Mr. Jason Briody is an Associate at Jones Dykstra & Associates, a specialized services company that provides computer forensics, electronic data discovery, litigation support, training, and computer security services for commercial and governmental clients. (ATX 2081 at 6) Mr. Briody is primarily responsible for planning and technically executing electronic discovery projects and performing computer forensic analysis. (Id.) Mr. Briody analyzed files given to him by Johns Hopkins University for authentication purposes related to this litigation. (Id. at 4-5)

         F. 454's Witnesses

         24. Dr. Matthew Levy testified live at trial. The Court found him to be credible and persuasive on every material point.

         25. Dr. Levy earned a B.S. in biochemistry and a M.S. in chemistry, both from the University of California San Diego, and a Ph.D. in molecular biology from the University of Texas. (DTX 32; Levy Tr. at 347:18-348:7) Dr. Levy is an Associate Professor of biochemistry at the Albert Einstein College of Medicine. (DTX 32; Levy Tr. at 348:10-17) Dr. Levy teaches a variety of classes, including biochemistry, immunology, and chemical biology. (Levy Tr. at 348:19-21) A major focus of his research is developing nucleic acid-based therapeutics and diagnostics. (Id. at 349:3-7) He is familiar with genomic DNA isolation, DNA sequencing, and emulsion PCR. (Id. at 349:14-350:1) Dr. Levy has personally conducted emulsion PCR and has experience making emulsions for emulsion PCR. (Id. at 349:18-21)

         26. Dr. Gary J. Sarkis is one of the 454 inventors. Dr. Sarkis received a B.S. in microbiology and an M.S. in biochemistry and molecular genetics, both from the University of Pittsburgh. He also received a Ph.D. in molecular, cellular, and developmental biology from the University of Pittsburgh in 1997. He was a postdoctoral research fellow at Yale University from 1997 to 2002 and was employed at 454 as a research scientist, section leader (sample preparation), training manager, and customer support manager from 2002 to 2006. (Sarkis Tr. at 177:11-15, 178:5-179:4) He testified live at trial.

         27. Janna Lanza Thompson received a B.S. in biology from the University of Vermont and a M.S. in cell and molecular biology from Central Connecticut State University. She was employed at 454 as a research associate from 2001 to 2006. (Lanza Tr. at 278:3-279:2) • She testified live at trial.

         28. Dr. Alex de Winter received a B.A. in chemistry from Amherst College and a Ph.D. in chemistry from Stanford University. He was employed at 454 as a research scientist from 2001 to 2004. (de Winter Tr. at 322:22-324:9) He testified live at trial.

         29. Dr. Jan Berka is one of the 454 inventors. He received a B.S. and M.S. in molecular biology and genetics and a Ph.D. in molecular biology and genetics, all from Masaryk University, Brno, Czech Republic. He was a postdoctoral research fellow at the Barnett Institute at Northeastern University in Boston from 1992 to 1996. Dr. Berka was employed at 454 as a senior scientist and director from 2000-2006.. (ATX 1113 ¶¶ 2-3) Dr. Berka testified by declaration.

         30. Dr. Maithreyan Srinivasan is one of the 454 inventors. Dr. Srinivasan has a Ph.D. in biochemistry. He was employed at 454 as a project leader in the Protein Sciences group from 2000-2007. (ATX 1116 ¶¶ 2-3) He testified by declaration.

         31. Mr. Keith McDade received a B.S. in molecular biology from the University of Connecticut and a M.S. in computer science from the University of New Haven. He was employed at 454 as a research associate from 2000 to 2006. (ATX 1124 ¶¶ 2-3) He testified by declaration.

         32. Dr. John Leamon is one of the 454 inventors. Dr. Leamon received a B.A. in zoology and a Ph.D. in physiology from the University of Connecticut. He was a postdoctoral research fellow at the Yale School of Medicine from 1999-2001. Dr. Leamon was employed at 454 in various positions ranging from research scientist to group leader from 2001-2007. (ATX 1114 ¶¶ 2-3) He testified by declaration.

         33. Dr. Louis Ferland received a B.S. in biochemistry from Universite Laval and a Ph.D. in experimental medicine from McGill University. He was a postdoctoral research fellow at the Salk Institute, Regulatory Biology Department from 1986-1989; and at the Institut Pasteur, Department de Genetique Moleculaire du Development from 1989-1991. He was employed at 454 in various positions ranging from technical writer to manager of documentation from 2001-2012. (ATX 1118 ¶¶ 2-3) He testified by declaration.

         34. Mr. William Airman received a B.S. in biology from Guilford College. He was employed at 454 from 2001 to 2013, holding several positions ranging from research assistant to senior customer support specialist. (ATX 1120 ¶¶ 2-3) He testified by declaration.

         G. 454's Conception of the Invention by December 2002

         35. Dr. Sarkis testified at trial, and previously submitted a declaration to the Board, indicating that he recalls discussing the idea for a method for analyzing nucleic acid sequences using bead PCR emulsion amplification with Dr. Berka - which is reflected in Dr. Berka's laboratory notebook dated June 7, 2002. (Sarkis Tr. at 180:8-183:5; ATX 1115 ¶16; ATX 1094 atl6;ATX1113'¶18)

         36. On June 7, 2002, Dr. Berka recorded in his lab notebook the notes of a conversation with Dr. Sarkis about the idea of PCR in water droplets in oil (water-in-oil emulsion) as individual microreactors that would contain a single effective copy of a sequence of DNA, a capture bead, and enough PCR reaction solution to produce amplified amounts of individual DNA fragments for massively parallel sequencing. (Sarkis Tr. at 180:8-183:5; ATX 111.5 ¶ 17; ATX 1094 atl6; ATX 1113 ¶ 18)

         37. That same page of Dr. Berka's notebook (page 16) also contains a drawing that depicts the concept of a PCR reaction occurring from a single bead and a single starting DNA fragment within the individual microreactors. (Sarkis Tr. at 182:7-23; ATX 1115 ¶¶ 17-20; ATX 1094 at 16; ATX 1113 ¶ 19) The drawing also shows the single-stranded DNA fragment attached to a primer on the capture bead and the resulting double-stranded DNA that would be present after the amplification process had begun. (ATX 1094 at 16) To the right of the drawing of the microreactor, Dr. Berka noted that "isolated bead bound primer extension" would occur "inside of the individual bead reactors." (Sarkis Tr. at 184:4-13; ATX 1115¶ 19; ATX 1094 at 16; ATX 1113 ¶ 20)

         38. Dr. Maithreyan Srinivasan testified by declaration that he recalled discussing the idea for a method of analyzing nucleic acid sequences using bead emulsion amplification with Dr. Berka. (Srinivasan Tr. at 267:25-268:13; ATX 1116 ¶¶ 9-15) This idea, also recorded on page 16 of Dr. Berka's notebook (ATX 1094), was contemporaneously witnessed and signed by Dr. Srinivasan on the same day it was written down by Dr. Berka: June 7, 2002. (Id.)

         39. Dr. Leamon testified by declaration about experiments he performed in August 2002 to improve the stabilization of emulsions for bead emulsion PCR. Although Dr. Leamon was able to successfully form the emulsions, after a number of cycles the emulsions were breaking down or "crashing." (Leamon Tr. at 307:2-308:24; ATX 1096 at 110-12, 123; ATX 1114¶¶21-24)

         40. On December 11, 2002, Dr. Leamon proposed the use of restriction endonuclease enzymes, known as "4-cutters, " instead of DNase I, to digest DNA fragments. (Leamon Tr. at 308:25-309:17; ATX 1097 at 1; ATX 1114 ¶¶ 25-26) The '592 provisional specifically discloses using Sau31, Mspl, and TaqI, which are 4-cutters. (Levy Tr. at 368:24-369:10; ATX 1013 at 11:24-26) Dr. Leamon recognized using 4-cutters would make DNA template with at least two copies of a fragment of DNA. (ATX 1114 ¶ 25) Dr. Leamon suggested using as many 4-cutters as prudent, perhaps 4 or 5, ligating adaptors onto each pool, and then hybridizing the DNA template onto the beads. (Id.)

         41. On December 19, 2002, Drs. Leamon, Sarkis, and Berka attended a lecture at Yale University given by Jennifer Ong. (Sarkis Tr. at 186:18-187:22; ATX 1105 at 9.3; Leamon Tr. at 309:18-25; ATX 1095 at 69; ATX 1114¶27; Berka Tr. at 303:1-4; ATX 1113 ¶ 23) Dr. Ongis one of the co-authors of a well-known journal article entitled, "Directed evolution of polymerase function by compartmentalized self-replication, " attributed to Ghadessy et al., and published in the journal of Proceedings of the National Academy of Sciences ("PNAS") on April 10, 2001 ("Ghadessy"). (ATX 1099; Sarkis Tr. at 187:23-188:13; DTX33) Also on December 19, 2002, Dr. Sarkis sequenced test fragments that 454 used as controls and obtained good sequencing results. (Sarkis Tr. at 192:19-193:2; ATX 1105 at 94)

         42. Drs. Sarkis, Leamon, and Berka referenced the seminar given at Yale by Dr. Ong in their notebooks. (Sarkis Tr. at 186:18-187:22; ATX 1105 at 93; Berka Tr. at 303:1-4; ATX 1095 at 69; ATX 1113 ¶ 23; Leamon Tr. at 309:18-310:8; ATX 1114 ¶¶ 27-28) Dr. Leamon included an excerpt of the Ghadessy paper in his lab notebook and noted his belief that the emulsion information discussed by Dr. Ong could be used for emulsion PCR using a sepharose bead. (Sarkis Tr. at 188:3-190:1; Leamon Tr. at309:18-310:8; ATX 1097 at2-3; ATX 1114 ¶¶ 27-28) Dr. Leamon also noted that "Andrew Griffith's group has used emulsion based bead PCR bead capture for translation studies which suggests that the beads maintain their discrete micelle identity, " and included an excerpt from an article by Armin Sepp, Dan Tawfik, and Andrew Griffiths ("Sepp"), regarding formation of emulsions, on the next page of his notebook on December 19, 2002. (ATX 1097 at 3-4) The Sepp article is entitled "Microbead display by in vitro compartmentalization selection for binding using flow cytometry, " and was published in Federation of European Biochemical Societies Letters ("FEB S") in November 2002. (ATX 1100)

         43. On December 20, 2002, the day after attending the lecture at Yale and reviewing the Ghadessy and Sepp references, Dr. Leamon performed an experiment and successfully prepared beads in a water-in-oil emulsion. (Sarkis Tr. at 190:2-16; ATX 1097 at 5-6; Leamon Tr. at 310:9-21; ATX 1114¶29; Levy Tr. at 446:24-447:17) Dr. Leamon included pictures in his notebook that demonstrate that some of the micelles were suitably-sized for the sepharose beads. (ATX1114¶29)

         44. Also on December 20, 2002, again the day after attending the lecture at Yale, Dr. Sarkis performed an emulsion PCR experiment with PCR-generated test fragments TF1, TF2, TF.3, TF4, TF5, TF6, F6, and N7, and successfully amplified the test fragments. (Sarkis Tr. at 193:22-195:7; ATX 1105 at 95-96; Levy Tr. at 446:24-447:17) Dr. Sarkis's December 20, 2002 experiment evidenced conception of the invention of the Count, as further explained below.

         45. Dr. Sarkis testified that the test fragments used by 454 originally came from Curagen Corporation, a sister company to 454. (Sarkis Tr. at 193:3-21) The fragments were labeled by the well number that they came from on the microliter plates, and 454 knew the sequence of all of these test fragments. (Id.) The sequence of the F6fest fragment is recorded in Dr. Sarkis's lab notebook many times and was used for comparison each time the test fragment was sequenced in a reaction. (Id.)

         46. The F6 test fragment was PCR amplified by Mr. Airman, a 454 research assistant, who testified by declaration about making the F6 test fragments. (Airman Tr. at 271:25-273:20; ATX 1120¶¶ 6-10; ATX 1108; ATX 1109) As recorded in his notebook, Mr. Airman successfully sequenced the F6 test fragments on July 22, 2002. (Id.; ATX 1109 at 13)

         47. Ms. Lanza (now Ms. Thompson) testified that she recorded the sequence of the F6 test fragment, which was a PCR-generated fragment commonly used at 454, in her notebook on January 23, 2003. (Lanza, Tr. at 280:1-20; ATX 1132 at 134; ATX 1126 ¶ 11) The sequence of F6 is identified on page 134 of her notebook, i.e., ATX 1132 at 134.

         48. Dr. de Winter testified that all of the test fragments that were used as controls by the 454 scientists were PCR-generated fragments, (de Winter Tr. at 331:16-18) JHU's expert, Dr. Tyagi, testified that the test fragments, e.g., F6, would satisfy step (a) of the Count if they were generated by PCR. (Tyagi Tr, at 90:5-19)

         49. Dr. de Winter testified that he prepared adenovirus DNA libraries for use in the emulsion PCR proj ect and testified about the process or standard operating procedure that lie used to prepare the libraries, (de Winter Tr. at 324:6-9, 326:2-327:23, 330:17-331:4; ATX 1122 at 51-52, 65, 69, 71-72, 75-76, 78; ATX 1123 ¶¶ 10-11) The typical process for preparing the library would be to fragment the adenovirus, polish the ends of the fragments with polymerases or dNTPs, ligate adaptors to the ends of the polished fragments, purify the ligation products, capture on streptavidin-coated beads, and then elute the single-stranded DNA fragments. (ATX 1123¶10)

         50. Dr. de Winter included in his notebook a detailed sample preparation protocol that he used for preparing adenovirus template libraries for emulsion PCR. (de Winter Tr. at 327:1-23, 330:1-11; ATX 1122 at 60) For this particular protocol, he usually used DNase I to fragment the adenovirus, (de Winter Tr. at 329:2-15, 330:5-16; ATX 1122 at 59)

         51. Dr. de Winter testified that there are a number of ways to fragment DNA, including using restriction enzymes that recognize particular 4-base sequences, (de Winter Tr. at 331:24-332:6)

         52. Restriction enzyme digestion can be used to generate overlapping genomic sequences for sequencing because they will each cut at a different site. (Tyagi Tr. at 125:15-19; Levy Tr. at 388:9-25) As a result, if a genome is cut with one enzyme it will generate a particular set of fragments, and if it is then cut with a different enzyme, it will generate a different set of overlapping fragments. (Tyagi Tr. at 125:20-127:3; Levy Tr. at 388:15-25)

         53. Both Dr. de Winter and Dr. Sarkis testified that, depending on the experiment, they would sometimes perform emulsion PCR of the test fragments alone, and other times they would mix test fragments together with adenovirus fragments. (Sarkis Tr. at 195:19-196:1; de Winter Tr. at 332:7-333:7)

         54. Dr. Sarkis testified at trial that the 454 inventors wanted clonal amplification, so they designed their experiments with the goal of having a single effective copy per bead. (Sarkis Tr. at 182:24-183:21) Clonal amplification means amplifying a single DNA fragment in isolation from all others. (Sarkis Tr. at 217:5-23)

         55. After December 2002, 454 scientists continued to perform experiments to optimize the conditions and develop a product that could be easily used by their customers. (Sarkis Tr. at 195:8-18)

         H. The Poisson Distribution

         56. The "Poisson distribution" is a statistical tool that can be used to predict the number of times that a given event occurs within a certain interval or physical space and is well-known and widely accepted. (Levy Tr. at 358:2-15) 454's expert, Dr. Tyagi, testified that the "Poisson distribution is [a] statistical distribution, basically, that scientists use when they want to deliver single cells into a well or single molecules in a reactor and so on." (Tyagi Tr. at 98:17-24) Using the Poisson distribution, it is possible to estimate the number of beads in a population that will be attached to more than one DNA fragment, to a single DNA fragment, and to no DNA fragments. (See Levy Tr. at 3 58:11 -15)

         57. The Poisson distribution has been repeatedly validated through empirical testing and experimental verification. (Id. at 361:4-15; Tyagi Tr. at 133:9-23) Dr. Tyagi explained: "The Poisson distribution stands on its own. It's always true, " and "Poisson statistics are always true, nobody is denying that." (Tyagi Tr. at 137:2-3, 138:25-139:1) The Poisson distribution is reliable evidence of what actually transpired during the experiments performed by 454 's scientists. (Levy Tr. at 361:16-22, 498:13-24) The number of compartments present in a 454 emulsion PCR reaction can be calculated by knowing the diameter of the compartments and the volume of the aqueous solution. (Id. at 365:11-22) Knowing the number of compartments allows one to know if enough compartments have been generated to encapsulate all of the beads and all of the DNA fragments in the experimental setup. (Id.)

         58. The Poisson distribution can be used to predict whether the emulsion PCR experiments performed by 454 contained some population of beads that had only a single DNA fragment attached. (Id. at 358:9-15) The December20, 2002 experiment and each subsequent emulsion PCR experiment discussed hereinafter would be expected to conform to the Poisson distribution. (Id. at 359:23-360:11) Therefore, based on 454's experimental setups and the Poisson distribution, one can reliably conclude that ...


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