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`UNITED STATES PATENT AND TRADEMARK OFFICE
`_______________
`
`BEFORE THE PATENT TRIAL AND APPEAL BOARD
`_______________
`
` ILLUMINA, INC.,
`Petitioner,
`
`v.
`
` THE TRUSTEES OF COLUMBIA UNIVERSITY
`IN THE CITY OF NEW YORK
`Patent Owner.
`_______________
`
`IPR2020-01177 (Patent 10,435,742)
`
`DECLARATION OF KENNETH A. JOHNSON, PH.D.
`
`Columbia Ex. 2048
`Illumina, Inc. v. The Trustees
`of Columbia University
`in the City of New York
`IPR2020-01177
`
`
`
`
`
`Table of Contents
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`I.
`
`Introduction and Qualifications ....................................................................... 1
`
`II. Materials Considered ....................................................................................... 5
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`III. The Person of Ordinary Skill in the Art .......................................................... 5
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`IV. Scope of Testimony ......................................................................................... 6
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`V.
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`Tsien ................................................................................................................. 6
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`VI. Dower ............................................................................................................... 9
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`VII. Hiatt ............................................................................................................... 12
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`A. Hiatt’s Methods Are Not SBS ............................................................. 12
`
`B.
`
`Hiatt Discloses A Very Large Number Of Capping
`Groups ................................................................................................. 14
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`VIII. Hovinen .......................................................................................................... 21
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`IX. Unpredictability ............................................................................................. 23
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`X.
`
`“
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`” and “Chemical Linker” ...................................................................... 26
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`I.
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`INTRODUCTION AND QUALIFICATIONS
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`1.
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`I have been retained on behalf of The Trustees of Columbia University
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`in the City of New York (“Columbia”) in connection with the challenge by Illumina,
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`Inc. (“Illumina”) to the claims of U.S. Patent No. 10,435,742 (the “patent-at-issue”).
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`2.
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`I am being compensated for my time consulting in this matter at the rate
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`of $700 per hour. I have no financial interest in the outcome of this proceeding and
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`my compensation is in no way contingent upon my opinions or the outcome of this
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`proceeding.
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`3.
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`I am the Roger Williams Centennial Professor of Biochemistry at the
`
`University of Texas at Austin and the President and founder of KinTek Corporation,
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`a company noted internationally for its manufacture of instruments and software that
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`I designed for advanced kinetic analysis.
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`4.
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`I earned a Bachelor of Science in Chemistry with Honors and Highest
`
`Distinction from the University of Iowa in 1971. I earned a Ph.D. in Molecular
`
`Biology from the University of Wisconsin in 1975 for work done with Professor
`
`Gary Borisy.
`
`5.
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`From 1975 to 1979 I was a postdoctoral scholar working with Dr.
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`Edwin W. Taylor at the University of Chicago Department of Biophysics and
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`Theoretical Biology. During this time I was supported by fellowships from the
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`National Institutes of Health and the Muscular Dystrophy Association.
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`6.
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`Starting as an Assistant Professor in the Department of Biochemistry
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`and Biophysics at The Pennsylvania State University in March 1979, I advanced to
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`the rank of Paul Berg Professor of Biochemistry before leaving in August 1998.
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`7.
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`Since August of 1998, I have been the Roger Williams Centennial
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`Professor of Biochemistry at The University of Texas at Austin initially in the
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`Department of Chemistry and Biochemistry, which was subsequently reorganized to
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`the Department of Molecular Biosciences.
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`8.
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`In 1987 I founded KinTek Corporation to manufacture and market
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`instruments that I designed to perform single turnover and transient-state kinetic
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`analysis. I also designed and worked closely with computer programmers to develop
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`a novel approach for modeling and fitting kinetic data.
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`9.
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`As a principal investigator, I have authored more than 175 original
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`publications and review articles in peer-reviewed journals including Science,
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`Nature, The Proceedings of the National Academy of Sciences, Journal of Biological
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`Chemistry, Biochemistry, Journal of Molecular Biology, Journal of Cell Biology,
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`Antimicrobial Agents and Chemotherapy, Nucleic Acids Research, Journal of
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`Physical Chemistry, and Journal of the American Chemical Society. My
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`publications have been cited more than 18,000 times.
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`10.
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`I have been invited to present lectures on 188 occasions at universities,
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`biotechnology and pharmaceutical companies and international conferences.
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`11.
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`I received the Pfizer Award in Enzyme Chemistry and the Penn State
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`Faculty Scholar Award, and I am a Fellow of The American Association for the
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`Advancement of Science and a Fellow of the Biophysical Society. From 1983-1989
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`I was an Established Investigator of the American Heart Association. I was elected
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`to organize Gordon Conferences in two fields and to organize the Enzyme
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`Mechanisms Conference.
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`12. Since 1985 I have pioneered in the development and application of
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`accurate methods
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`to quantify DNA polymerase
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`fidelity and establish
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`structure/function relationships governing nucleotide selectivity. I have published
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`70 papers and review articles on DNA polymerase mechanisms and nucleotide
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`selectivity involving studies on several enzymes: Klenow fragment of DNA
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`polymerase I, T7 DNA polymerase, HIV reverse transcriptase, Taq polymerase,
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`human mitochondrial DNA polymerase, dengue virus polymerase, hepatitis C viral
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`RNA-dependent RNA polymerase.
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`13.
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`In my work on DNA polymerases, I have provided novel insights into
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`understanding nucleotide recognition and fidelity and the structure/function
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`relationships governing discrimination of polymerases against nucleotide analogs.
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`This work has included analysis of the evolution of resistance to nucleoside analogs
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`by HIV reverse transcriptase and the toxic side effects of these nucleoside analogs
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`due to their incorporation by the human mitochondrial DNA polymerase.
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`14.
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`I have served on the Editorial Board of the Journal of Cell Biology and
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`the Journal of Biological Chemistry, and I have served as a peer-reviewer on
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`numerous papers for journals including Nature, Science, Journal of Biological
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`Chemistry, Biochemistry, Analytical Biochemistry, Biophysical Journal, Journal of
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`the American Chemical Society, Plos One, Journal of Molecular Biology, Journal of
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`Physical Chemistry, and Nucleic Acids Research.
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`15.
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`I have published a critically acclaimed textbook on modern kinetic
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`analysis of enzyme mechanisms. This book highlights the novel approaches that I
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`have developed for quantifying enzyme specificity, including examples of my work
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`on DNA polymerase fidelity in which we established a new paradigm for
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`understanding the role of conformational dynamics in enzyme specificity.
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`16. From 1994 to 2001 I was a consultant for Applied Biosystems. In this
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`capacity, I advised the research team on the application of knowledge gained from
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`DNA polymerase kinetics to optimize their four-color Sanger sequencing methods.
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`17. Based on my extensive experience in DNA polymerase kinetics,
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`nucleotide selectivity and DNA sequencing methods, and because I was active in the
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`field from 1985 through 2000, I am qualified to judge what a person of ordinary skill
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`in the art would have known and understood in October of 2000.
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`18. My curriculum vitae describes in greater detail my professional
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`experience and qualifications. Ex. 2016.
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`II. MATERIALS CONSIDERED
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`19. Appended to my Declaration is a list of documents that I have
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`considered in connection with this Declaration.
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`III. THE PERSON OF ORDINARY SKILL IN THE ART
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`20.
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`I understand that the claims of the patent-at-issue must be assessed from
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`the perspective of a hypothetical person of ordinary skill in the relevant art (“POSA”)
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`as of the date the invention described in the claims was made. For the purposes of
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`this Declaration, I have been asked to assume the invention was made by October 6,
`
`2000.
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`21.
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`I understand that a POSA has knowledge of the relevant art, as
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`described in published and issued patents and in scientific publications, and
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`possesses ordinary creativity and a basic skill set. For the purposes of this
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`proceeding, I rely upon Illumina’s criteria for defining a POSA, which I have
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`reproduced below:
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`[A] POSA would have been a member of a team of
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`scientists developing nucleotide analogues, researching
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`DNA polymerases, and/or addressing DNA sequencing
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`techniques. Such a person would have held a doctoral
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`degree in chemistry, molecular biology, or a closely
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`related discipline, and had at least five years of practical
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`academic or industrial laboratory experience.
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`IV. SCOPE OF TESTIMONY
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`22.
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`I have been asked to provide testimony regarding a number of prior art
`
`references, including WO 91/06678 (“Tsien”), U.S. Patent No. 5,547,839
`
`(“Dower”), U.S. Patent No. 5,763,594 (“Hiatt”), Hovinen et al., “Synthesis of 3´-O-
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`(ω-Aminoalkoxymethyl)thymidine 5'-Triphosphates, Terminators of DNA
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`Synthesis that Enable 3'-Labelling,” J. Chem. Soc. Perkin Trans. 1, 211-217 (1994)
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`(“Hovinen”), Prober et al., “A System for Rapid DNA Sequencing with Fluorescent
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`Chain-Terminating Dideoxynucleotides,” Science, 238:336-341 (1987) (“Prober”),
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`and Zhu, Z., et al., “Directly Labeled DNA Probes Using Fluorescent Nucleotides
`
`With Different Length Linkers,” Nucleic Acids Res., 22:3418-3422 (1994) (“Zhu”).
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`I have also been asked to provide testimony regarding the unpredictability of
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`modified nucleotide incorporation, and testimony regarding the claim terms “
`
`”
`
`and “chemical linker.”
`
`V. TSIEN
`
`23. Tsien (WO 91/06678) is a PCT application, titled “DNA Sequencing,”
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`which relates to “an instrument and a method to determine the nucleotide sequence
`
`in a DNA molecule without the use of a gel electrophoresis step.” Ex. 1031 at Title
`
`and Abstract.
`
`24. Tsien was filed in October 1990 and published in May 1991. Ex. 1031
`
`at Cover Page.
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`25.
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`It is my understanding that Columbia and Illumina agree that Tsien
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`describes hypothetical methods for practicing Sequencing by Synthesis (“SBS”).
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`26. Under the heading “Blocking Groups and Methods for Incorporation,”
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`Tsien describes “[t]he criteria for the successful use of 3'-blocking groups,”
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`including:
`
`(1) the ability of a polymerase enzyme to accurately and
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`efficiently incorporate the dNTPs carrying the 3'-blocking
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`groups into the cDNA chain,
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`(2) the availability of mild conditions for rapid and
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`quantitative deblocking, and
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`(3) the ability of a polymerase enzyme to reinitiate the
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`cDNA synthesis subsequent to the deblocking stage.
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`Ex. 1031 at 20-21. As seen in the above excerpt, Tsien’s first criteria requires both
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`“accurate[]” and “efficient[]” incorporation. I have been asked to explain the
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`difference between accurate incorporation and efficient incorporation.
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`27.
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`“Accuracy,” which is also known as fidelity, refers to a polymerase
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`incorporating the correct nucleotide, as opposed to an incorrect nucleotide. In DNA,
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`there are four different types of nucleotides, adenine (A), guanine (G), cytosine (C),
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`and thymine (T). Two strands of polynucleotides form a double helix structure, and
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`bonds between complementary base pairs hold the chains together, with G pairing
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`with C and A pairing with T:
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`
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`28. To duplicate (or “synthesize”) DNA, the two strands of the double helix
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`are separated. One of the strands serves as a “template” that can be used to
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`synthesize a new complementary strand, known as the “growing strand.” A
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`polymerase incorporates nucleotides into the growing strand using the template
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`strand as a guide:
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`29. As explained above, “accuracy” refers to the polymerase incorporating
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`the correct nucleotide as dictated by the template and the base pairing rules (C with
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`G, A with T). If the next nucleotide in a template strand is G (e.g., as in the image
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`above), then based on the base pairing rules, the polymerase should incorporate a C.
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`A polymerase will sometimes make an error and incorporate the incorrect nucleotide
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`(in this example, something other than C). If a polymerase incorporates the correct
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`nucleotide 90% of the time, then that polymerase has a 90% accuracy of
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`incorporation.
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`30.
`
`“Efficiency,” on the other hand, is a function of the extent to which a
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`polymerase incorporates nucleotides into the growing strand. In particular,
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`efficiency standards for SBS were defined by Tsien as the requirement to achieve at
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`least 98% completion of the reaction for incorporation of each nucleotide. Ex. 1031
`
`at 16. A polymerase’s accuracy does not dictate its efficiency, and vice versa. A
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`polymerase’s incorporation can be accurate and efficient, accurate and inefficient,
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`inaccurate and efficient, or inaccurate and inefficient.
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`VI. DOWER
`
`31. Dower (U.S. Patent 5,547,839) is a patent, titled “Sequencing of surface
`
`immobilized polymers utilizing microflourescence detection,” which was filed in
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`December 1990 and issued in August 1996. Ex. 1030 at Cover Page.
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`32.
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`It is my understanding that the parties agree that Dower describes
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`hypothetical methods for practicing SBS. As an SBS method, Dower requires
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`efficient incorporation. Ex. 1030 at 26:6-12.
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`33. Dower describes four nucleotides with what Dower characterizes as
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`“small” capping groups, stating: “Examples of
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`these compounds are
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`deoxynucleotide triphosphates with small blocking groups such as acetyl, tBOC,
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`NBOC and NVOC on the 3'OH.” Ex. 1030 at 25:41-64.
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`34.
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`I have been asked to consider whether Dower’s use of the term “small”
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`would convey to a POSA the same meaning as the use of the term “small” in
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`Columbia’s patent-at-issue. Based on my review of Dower, the patent-at-issue, and
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`statements made by Columbia to the Patent Office, it is clear that Dower’s meaning
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`of “small” is different than Columbia’s meaning of “small.”
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`35.
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`It is my understanding that Columbia explained to the Patent Office that
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`the term “small” in the patent-at-issue refers to capping groups that have a diameter
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`less than 3.7Å. Ex. 1136 at 3-5. It is also my understanding that Columbia explained
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`to the Patent Office that under this standard for “small,” the 2-nitrobenzyl capping
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`group is not small (5.0Å in diameter), whereas the MOM capping group is small
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`(2.1Å in diameter). Ex. 1136 at 4-5.
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`36. Dower, on the other hand, uses the term “small” to characterize capping
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`groups that are larger than 2-nitrobenzyl, e.g., NBOC, and NVOC. Below are the
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`structures for NBOC, NVOC, and 2-nitrobenzyl:
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`
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`37. As can be seen above, both NBOC and NVOC contain within them the
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`2-nitrobenzyl structure, confirming that NBOC and NVOC are larger than 2-
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`nitrobenzyl and thus do not satisfy Columbia’s meaning of “small.”
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`38. Moreover, all four of Dower’s capping groups (acetyl, tBOC, NBOC,
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`and NVOC) are excluded from Columbia’s claims because they do not meet the
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`claim requirement that an ester not be selected.
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`VII. HIATT
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`A. Hiatt’s Methods Are Not SBS
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`39. Hiatt (U.S. Patent No. 5,763,594) is a patent, titled “3' Protected
`
`Nucleotides
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`for Enzyme Catalyzed Template-Independent Creation of
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`Phosphodiester Bonds,” which was filed in June 1995 and issued in June 1998. Ex.
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`1043 at Cover Page.
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`40.
`
`It is my understanding that Illumina contends that Hiatt discloses
`
`capping groups that are “being used for the same purpose” as the capping groups
`
`used in SBS. However, Illumina does not say what that “same purpose” is. While
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`both Hiatt and SBS are, very generally speaking, directed to the synthesis of
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`polynucleotides, their purposes are quite different. Illumina does not address the
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`differences in purpose, which a POSA would have understood.
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`41. Hiatt’s methods are for making synthetic nucleic acids of a
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`predetermined sequence. Hiatt discloses “[a] method for the stepwise creation of
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`phosphodiester bonds between desired nucleosides resulting in the synthesis of
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`polynucleotides having a predetermined nucleotide sequence[.]” Ex. 1043 at
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`Abstract. Hiatt’s method is not a DNA sequencing method, nor could it be used for
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`DNA sequencing or SBS.
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`42. That Hiatt’s methods could not be used for SBS is confirmed by the
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`fact that Hiatt’s methods utilize a template-independent polymerase, whereas SBS
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`requires a template-dependent DNA replicating polymerase. As I explained in my
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`discussion regarding Tsien, when synthesizing DNA, a single strand of DNA serves
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`as a “template,” which a polymerase uses to determine which nucleotide should next
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`be incorporated into the growing strand of DNA based on the base pairing rules. The
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`types of polymerases which can use such a template are known as template-
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`dependent DNA replicating polymerases. The purpose of SBS is to accurately
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`replicate the complementary sequence of a strand of DNA (i.e., a template), and that
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`purpose can only be achieved by using a template-dependent DNA replicating
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`polymerase. Hiatt, on the other hand, does not use a template-dependent DNA
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`replicating polymerase. Instead, Hiatt uses a template-independent polymerase,
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`specifically Terminal Transferase, which is incapable of replicating DNA with a
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`sequence specified by a template strand. Hence, Hiatt’s method of synthesizing
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`polynucleotides is incompatible with DNA sequencing. Illumina has not provided
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`any evidence to demonstrate that the MOM capping group would work with a
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`polymerase that is suitable for SBS.
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`43. As I noted above in my discussions of Tsien and Dower, there are
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`particular criteria that a capping group must meet in order to be useful for SBS, such
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`as accurate and efficient incorporation. Hiatt’s methods do not require accurate
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`incorporation because there is no template to accurately copy. Nor do they require
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`efficient incorporation because all that matters is that at least some amount of
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`nucleotide is incorporated. The accuracy of DNA synthesis using Hiatt’s methods
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`relies on an operator to add only a particular nucleotide at each step of the synthesis.
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`Accuracy is not enforced by the Terminal Transferase.
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`B. Hiatt Discloses A Very Large Number Of Capping Groups
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`44.
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`It is my understanding that Illumina contends that “Hiatt identifies a 3'-
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`O-MOM capping group as a preferred embodiment.” Petition at 16. That statement
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`does not acknowledge that Hiatt teaches that all disclosed capping groups are
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`preferred embodiments. Hiatt teaches that “[i]n preferred embodiments, the
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`nucleoside 5' phosphates of the present invention are deoxynucleoside 5'
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`triphosphate having a removable blocking moiety protecting the 3' position which
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`can be any of the blocking groups disclosed in this specification or equivalents of
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`those groups.” Ex. 1043 at 12:15-19 (emphasis added).
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`45.
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` Hiatt discloses an immense number of capping groups, each of which
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`is indicated as preferred, as noted above. For example, Hiatt discloses that
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`“[n]ucleotides having a removable blocking moiety protecting the 3' position
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`suitable for use with the present invention have a structure corresponding to Formula
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`1 …
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`… R1 can be an ester linkage, COR1', which forms the structure nucleotide-3'-O-
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`CO-R1'. R1' can be any alkyl or aryl group compatible with the utilization of the
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`molecule by the enzyme for the creation of an internucleotide phosphodiester bond.”
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`Ex. 1043 at 10:21-43. Hiatt goes on to disclose that “[r]emovable blocking moieties
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`including formate, benzoyl formate, acetate, substituted acetate, propionate,
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`isobutyrate, levulinate, crotonate, benzoate, napthoate and many other esters have
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`been described in detail (See, Greene, T. W., Protective Groups in Organic
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`Chemistry, John Wiley & Songs, New York, 1981).” Ex. 1043 at 10:46-54.
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`46. Hiatt also discloses that “[t]he present invention also contemplates a
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`nucleoside 5'-phosphate having a removable blocking moiety protecting the 3'
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`position which is an ester and which has the following formula:
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`…wherein R1 is any aliphatic or aromatic organic ester.” Ex. 1043 at 10:64-11:14.
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`47. Hiatt also discloses that “[t]he present invention also contemplates a
`
`nucleoside 5'-phosphate having a removable blocking moiety protecting the 3'
`
`position which is an ester and which has the following formula:
`
`
`
`wherein R is selected from the group consisting of: formate, benzoylformate,
`
`chloroacetate, dicholoroacetate, trichloroacetate, trifluoroacetate, methoxyacetate,
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`triphenylmethoxyacetate, phenoxyacetate, p-chlorophenoxyacetate, 2,6-dichloro-4-
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`methylphenoxyacetate,
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`2,6-dichloro-4-(1,1,3,3-tetramethylbutyl)phenoxyacetate,
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`2,4-bis(1,1-dimethylpropyl)phenoxyacetate, chlorodiphenylacetate, phenylacetate,
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`16
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`3-phenylpropionate, 3-benzoylpropionate,
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`isobutyrate, monosuccinoate, 4-
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`oxopentanoate, pivaloate, adamanioate, crotonate, 4-methoxycrotonate, (E)-2-
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`methyl-2-butenoate, o-(dibromomethyl)benzoate, o-(methoxycarbonyl)benzoate, p-
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`phenylbenzoate, 2,4,6-trimethylbenzoate and α-naphthoate.” Ex. 1043 at 11:15-44.
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`48. Hiatt also discloses that “[t]he present invention also contemplates a
`
`nucleoside 5'-phosphate having a removable blocking moiety protecting the 3'
`
`position which is an ester and which has the following formula:
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`
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`… wherein R is selected from the group consisting of: H, CH3, CH3(CH2)N where N
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`is an integer from 1 to 12, (CH3)x+1(CH)x where x is an integer from 1 to 12,
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`(CH3)x+1(CH)x(CH2)n where x and n are independent integers from 1 to 12,
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`Cx(CH3)3x-(x-1)(CH2)n where x and n are independent integers from 1 to 12,
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`, and
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`, where R1, R3, R4, R5, and R6 is CH3, H or NO2.” Ex. 1043
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`at 11:45-12:14.
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`49.
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`
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`Hiatt also discloses that “[a]n alternative type of removable
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`blocking moiety utilizes an ether linkage which forms the structure nucleotide-3'-O-
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`R'1. In this instance R'1 can be methyl, substituted meythyl, ethyl, substituted ethyl,
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`butyl, allyl, cinnamyl, benzyl, substituted benzyl, anthryl or silyl.” Ex. 1043 at
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`12:20-24.
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`50.
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`
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`Hiatt also discloses that “[i]n other embodiments, a nucleoside 5'
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`phosphate of the present invention has a removable blocking moiety protecting the
`
`3' position which is an ether and which has the following formula:
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`… wherein R1 is an ether selected from the group consisting of a substituted or
`
`unsubstituted: aliphatic group, aromatic group or silyl group.” Ex. 1043 at 12:28-
`
`
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`45.
`
`51. Hiatt also discloses that “[i]n other embodiments, a nucleoside 5'
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`phosphate of the present invention has a removable blocking moiety protecting the
`
`3' position which is an ether and which has the following formula:
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`… wherein R1 is an ether selected from the group consisting of a methyl,
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`methoxymethyl, methylthiomethyl,
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`benzyloxymethyl,
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`t-butoxymethyl,
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`2-
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`methoxyethoxymethyl, 2,2,2-trichloroethoxymethyl, bis(2-chloroethoxy)methyl, 2-
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`(trimethylsilyl)ethoxymethyl,
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`tetrahydropyarnyl,
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`3-bromotetrahydropyranyl,
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`tetrahydrothiopyranyl,
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`4-methoxytetrahydropranyl,
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`4-
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`methoxytetrahydrothiopyranyl,
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`4-methoxytetrahydrothiopyranyl
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`S,S-dioxido,
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`tetrahydrofuranyl, tetrahydrothiofuranyl, 1-ethoxyethyl, 1-methyl-1-methoxyethyl,
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`1-(isopropoxy)ethyl, 2,2,2-trichloroethyl, 2-(phenylselenyl)ethyl, butyl, allyl,
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`cinnamyl, p-chlorophenyl, benzyl, p-methoxybenzyl, o-nitrobenzyl, p-nitrobenzyl,
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`p-halobenzyl, p-cyanobenzyl, 3-methyl-2-picolyl N-oxido, diphenylmethyl, 5-
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`dibenzosuberyl,
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`triphenylmethyl,
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`α-naphthyldiphenylmethyl,
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`p-
`
`methoxyphenyldiphenylmethyl, p-(p'-bromophenacyloxy)phenyldiphenylmethyl,
`
`9-anthryl, 9-(9-phenyl-10-oxo)anthryl, benzisothiazolyl S,S-dioxido, trimethylsilyl,
`
`triethylsilyl,
`
`isopropyldimethylsilyl,
`
`t-butyldimethylsilyl,
`
`(triphenylmethyl)dimethylsilyl, methyldiisopropylsilyl, methyldi-t-butylsilyl,
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`tribenzylsilyl, tri-p-xylylsilyl, triisopropylsilyl and triphenylsilyl.” Ex. 1043 at
`
`12:46-13:16.
`
`52.
`
`
`
`Hiatt also discloses that “[i]n more preferred embodiments, a
`
`nucleoside 5' phosphate of the present invention has a removable blocking moiety
`
`protecting the 3' position which is an ether and which has the following formula:
`
`
`
`… wherein R1 is CH3, CH3(CH2)N where N is an integer from 1-10, methyl,
`
`methoxymethyl, methoxyethoxymethyl, trimethlsilyl, and triethylsilyl. In a more
`
`preferred embodiment, the nucleoside 5'-phosphate of the present invention has an
`
`R1 group which is CH(OC2H5)CH3 and R2 is triphosphate and said nucleoside 5'-
`
`phosphate is a deoxynucleoside.” Ex. 1043 at 13:35-56.
`
`53. Hiatt also discloses that “[a]dditional well known removable blocking
`
`moieties useful for protecting for hydroxyls include carbonitriles, phosphates,
`
`carbonates, carbamates, borates, nitrates, phosphoramidates, and phenylsulfenates.”
`
`Ex. 1043 at 13:57-65.
`
`
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`54. The above citations, which are non-exhaustive, demonstrate the
`
`enormous number of capping groups that Hiatt discloses for use in Hiatt’s template-
`
`independent, non-SBS methods.
`
`55.
`
`I also note that while Hiatt discloses, without explanation, various
`
`embodiments as “more preferred,” including wherein R1 could be MOM, that
`
`collection includes many combinations of R2 and R1 substituents, with no emphasis
`
`on any one of those possibilities being more preferred than any other. See Ex. 1043
`
`at 13:35-56, 14:16-30.
`
`VIII. HOVINEN
`
`56. Hovinen is a 1994 Journal of Chemical Society, Perkin Transactions 1
`
`article,
`
`titled
`
`“Synthesis of 3´-O-(ω-Aminoalkoxymethyl)thymidine 5´-
`
`Triphosphates, Terminators of DNA Synthesis that Enable 3'- Labelling.” Ex. 1060
`
`at 211.
`
`57.
`
`I note that Illumina’s Petition is silent on Hovinen’s purpose. Notably,
`
`Hovinen does not disclose methods for SBS. Instead, Hovinen describes the
`
`synthesis of modified nucleotides for use in Sanger sequencing reactions. See Ex.
`
`1060 at 211-216. Hovinen reports that “Fig. 1 shows the results obtained with Tet/z
`
`DNA-polymerase,
`
`using
`
`2',3'-dideoxyribonucleoside
`
`5'-triphosphates
`
`and
`
`compounds 9a, 10a-c and 11 as terminators.” Ex. 1060 at 213. Hovinen’s Figure 1
`
`is reproduced below:
`
`
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`21
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`
`
`58. The “[g]el pattern” shown in Figure 1 is the hallmark of Sanger
`
`sequencing, which requires the use of gel electrophoresis in order to sequence DNA.
`
`SBS, on the other hand, is a DNA sequencing technology that does not utilize gel
`
`electrophoresis.
`
`
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`22
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`59.
`
`I also note that many of the termination ladder bands are missing or
`
`extremely light in the lanes where Hovinen uses his 3’-O-capped nucleotides,
`
`indicating poor (i.e., inefficient) incorporation of those nucleotides, even for Sanger
`
`sequencing. In my experience as a consultant for Applied Biosystems, these analogs
`
`would have been rejected by those skilled in the art at the time searching for analogs
`
`to use in an SBS method that required accurate and efficient incorporation.
`
`IX. UNPREDICTABILITY
`
`60.
`
`I have been asked to provide testimony on the unpredictability of
`
`modified nucleotide incorporation as of October 6, 2000. As explained below, the
`
`prior art taught unpredictability as to whether any given polymerase could
`
`incorporate any given 3’-O-capped nucleotide.
`
`61. Unpredictability is demonstrated by Metzker et al., “Termination of
`
`DNA synthesis by novel 3'-modified-deoxyribonucleoside 5'-triphosphates,”
`
`Nucleic Acids Research, 22:4259-67 (1994) (“Metzker”). Ex. 1039. Metzker
`
`disclosed a hypothetical SBS method, which he and his colleagues called “Base
`
`Addition Sequencing Scheme (BASS).” Ex. 1039 at 4259. According to Metzker,
`
`“BASS involves repetitive cycles of incorporation of each successive nucleotide, in
`
`situ monitoring to identify the incorporated base, and deprotection to allow the next
`
`cycle of DNA synthesis.” Ex. 1039 at 4259.
`
`
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`62. Metzker synthesized eight 3'-O-capped nucleotides and tested their
`
`usefulness for Sanger sequencing and/or BASS. Ex. 1039 at 4259. Metzker
`
`examined whether eight polymerases (AMV-RT, M-MuLV-RT, Klenow fragment,
`
`Sequenase, Bst DNA polymerase, AmpliTaq DNA polymerase, rTth DNA
`
`polymerase, Vent(exo-) DNA polymerase) could incorporate any of eight different
`
`3'-O-capped nucleotides (3'-O-methyl-dATP, 3'-O-acyl-dATP, 3'-O-allyl-dATP, 3'-
`
`O-tetrahydropyran-dATP, 3'-O-(4-nitrobenzoyl)-dATP, 3'-O-(2-aminobenzoyl)-
`
`dATP, 3'-O-(2-nitrobenzyl)-dATP, and 3'-O-methyl-dTTP). Ex. 1039 at 4263
`
`(Table 2).
`
`63. Metzker reported the results of his assays in Table 2. Metzker
`
`characterized the results of each assay as “-,” meaning that “no activity was
`
`detected,” as “Termination,” meaning that “the reaction containing the test
`
`compound mimicked the migration pattern of the ddNTP control,” or as “Inhibition,”
`
`meaning that “the presence of the test compound prevented the polymerase from
`
`incorporating the natural nucleotides.” Ex. 1039 at 4263. A POSA would
`
`understand that “Termination”
`
`indicated
`
`incorporation of
`
`the 3'-O-capped
`
`nucleotide.
`
`64. Metzker further characterized the results of his assays with an asterisk
`
`(“*”) to indicate “where activity was incomplete at a final concentration of 250 uM.”
`
`Ex. 1039 at 4263.
`
`
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`24
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`65. Out of the sixty-four polymerase/nucleotide combinations tested,
`
`Metzker reported some level of incorporation (reported as Termination or
`
`Termination*), only in ten instances, and only in six of those ten instances was the
`
`incorporation complete (reported as Termination):
`
`That less than 10% (6 of 64) of the polymerase/nucleotide combinations achieved
`
`complete incorporation would have demonstrated to a POSA the difficulty in
`
`achieving incorporation with a modified nucleotide.
`
`66. Additionally, Metzker’s assays would have demonstrated to a POSA
`
`the unpredictability as to which polymerase/nucleotide combinations could achieve
`
`incorporation. For example, whereas AMV-RT polymerase achieved complete
`
`incorporation with a 3'-O-methyl adenine nucleotide, it was incapable of
`
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`25
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`incorporating the 3'-O-methyl thymine nucleotide. And the opposite was true for
`
`the Bst and AmpliTaq polymerases, which completely incorporated the 3'-O-methyl
`
`thymine nucleotide but were incapable of incorporating the 3'-O-methyl adenine
`
`nucleotide.
`
`67.
`
`I understand that Illumina contends that incorporation of a larger
`
`capping group would provide an expectation of incorporation of a smaller capping
`
`group. Petition at 19. Illumina’s contention is incompatible with Metzker’s results,
`
`which demonstrated that a polymerase’s ability to incorporate a larger capping group
`
`provided no expectation of incorporation with a smaller capping group. For
`
`example, in Metzker’s assays, Bst DNA polymerase incorporated the large 2-
`
`nitrobenzyl c
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