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`UNITED STATES PATENT AND TRADEMARK OFFICE
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`____________
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`BEFORE THE PATENT TRIAL AND APPEAL BOARD
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`____________
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`OXFORD NANOPORE TECHNOLOGIES, INC.
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`Petitioner
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`v.
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`PACIFIC BIOSCIENCES OF CALIFORNIA, INC.
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`Patent Owner
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`____________
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`Case No. Unassigned
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`Patent 9,738,929
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`____________
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`PETITION FOR INTER PARTES REVIEW OF CLAIMS 1-17
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`OF U.S. PATENT NO. 9,738,929
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`TABLE OF CONTENTS
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`I.
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`INTRODUCTION ............................................................................................ 1
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`A. Summary of Unpatentability Grounds ............................................................... 1
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`II. MANDATORY NOTICES, STANDING, AND FEES ................................. 2
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`A. Mandatory Notices ............................................................................................. 2
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`B. Certification of Grounds for Standing ............................................................... 2
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`C. Fees .................................................................................................................... 3
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`III. OVERVIEW OF THE ’929 PATENT ........................................................... 3
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`A. Overview of the Disclosure of the ’929 Patent .................................................. 3
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`B. Overview of the Prosecution History ................................................................. 6
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`IV. LEVEL OF ORDINARY SKILL IN THE ART ........................................... 9
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`V. STATE OF THE ART ................................................................................... 10
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`A. Nanopore Sequencing Using Enzyme Chaperones ......................................... 10
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`B. Sequencing of Complementary Strands to Improve Accuracy ....................... 12
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`VI. SUMMARY OF PRIOR ART ...................................................................... 13
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`A. Nanopore Sequencing using Enzyme Chaperones was Known in the Art Prior
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`to the Earliest Priority Date Claimed by the ’929 Patent ......................................... 13
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`B. Sequencing of Both Strands of a Polynucleotide was Known in the Art Prior
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`to the Earliest Priority Date Claimed by the ’929 Patent ......................................... 17
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`C. Various Linkers for Connecting Complementary Strands of DNA were
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`Known in the Art Prior to the Earliest Priority Date Claimed by the ’929 Patent .. 23
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`VII. THERE
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`IS A REASONABLE LIKELIHOOD THAT THE
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`CHALLENGED CLAIMS ARE UNPATENTABLE ......................................... 25
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`A. Ground 1: Claims 1-8, 10-11 and 16 are obvious over Akeson and Gupte ..... 25
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`B. Ground 2: Claim 12 is obvious over Akeson, Gupte and Miner ..................... 48
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`C. Ground 3: Claim 17 is obvious over Akeson, Gupte and Akeson ’433. ......... 50
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`D. Ground 4: Claims 1-8, 10-11 and 13 are obvious over Akeson, Sanger and
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`Makrigiorgos. ........................................................................................................... 52
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`E. Ground 5: Claim 9 is obvious over Akeson, Gupte and Makrigiorgos. .......... 70
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`F. Ground 6: Claim 12 is obvious over Akeson, Sanger, Makrigiorgos and
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`Miner ........................................................................................................................ 74
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`G. Ground 7: Claims 14 and 15 are obvious over Akeson, Sanger, Makrigiorgos
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`and O’Dea. ............................................................................................................... 76
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`VIII. CONCLUSION ............................................................................................. 79
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`LIST OF EXHIBITS
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`Exhibit No.
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`Description
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`1001
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`1002
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`1003
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`1004
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`1005
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`1006
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`1007
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`1008
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`U.S. Patent No. 9,738,929
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`Expert Declaration of Dr. Patrick Hrdlicka
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`Curriculum Vitae of Dr. Patrick Hrdlicka
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`U.S. Patent Application Publication No. 2006/0063171
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`(“Akeson”)
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`U.S. Patent No. 6,087,099 (“Gupte”)
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`U.S. Patent Publication No. 2005/0142559 (“Makrigiorgos”)
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`Miner et al., Nucleic Acids Res., 32(17):e135 (2004) (“Miner”)
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`O’Dea and McLaughlin, Current Protocols in Nucleic Acid
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`Chemistry 5.3.1-5.3.8 (2000) (“O’Dea”)
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`1009
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`Sambrook, Fritsch and Maniatis, Molecular Cloning, A
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`1010
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`1011
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`1012
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`Laboratory Manual (1989, 2nd Ed.) (“Sambrook”)
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`U.S. Patent No. 5,795,782 to Church et al. (“Church”)
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`U.S. Patent No. 6,404,907 (“Gilchrist”)
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`Sanger, “Determination of Nucleotide Sequences in DNA,”
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`Science 214:1205-1210 (1981) (“Sanger”)
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`1013
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`Prosecution History for U.S. Patent No. 9,738,929
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`1014
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`1015
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`1016
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`U.S. Patent No. 6,936,433 (“Akeson ’433”)
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`Declaration of Dr. Sylvia Hall-Ellis
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`Struhl, “Cloning cookbook for the laboratory,” Nature 316:222
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`(July 18, 1985).
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`I. INTRODUCTION
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`Oxford Nanopore Technologies, Inc. (“Oxford” or “Petitioner”) requests
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`inter partes review (“IPR”) under 35 U.S.C. §§ 311-319 and 37 C.F.R. § 42.100 et
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`seq. of Claims 1-17 of U.S. Patent No. 9,738,929 (“the ’929 Patent”).
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`Petitioner asserts that there is a reasonable likelihood that the challenged
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`claims are unpatentable and requests review of, and cancellation of, the challenged
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`claims under 35 U.S.C. § 103.
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`A. Summary of Unpatentability Grounds
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`Ground
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`Summary
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`1
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`2
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`3
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`4
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`5
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`6
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`7
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`Claims 1-8, 10-11 and 16 are obvious over Akeson and Gupte
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`Claim 12 is obvious over Akeson, Gupte and Miner
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`Claim 17 is obvious over Akeson, Gupte and Akeson ’433
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`Claims 1-8, 10-11 and 13 are obvious over Akeson, Sanger and
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`Makrigiorgos
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`Claim 9 is obvious over Akeson, Gupte and Makrigiorgos
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`Claim 12 is obvious over Akeson, Sanger, Makrigiorgos and Miner
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`Claims 14 and 15 are obvious over Akeson, Sanger, Makrigiorgos
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`and O’Dea
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`II. MANDATORY NOTICES, STANDING, AND FEES
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`A. Mandatory Notices
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`Real Party in Interest: The real party in interest is Oxford Nanopore
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`Technologies, Inc. Out of an abundance of caution, Petitioner also identifies
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`Oxford Nanopore Technologies, Ltd., the parent company of Oxford Nanopore
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`Technologies, Inc., and Metrichor Ltd., a corporate affiliate of Oxford Nanopore
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`Technologies, Inc., as parties in interest.
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`Related Matters: The ’929 Patent is subject to a pending lawsuit entitled
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`Pacific Biosciences of California, Inc., v. Oxford Nanopore Technologies, Inc.,
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`which was consolidated into actions 1:17-cv-00275-LPS, 1:17-cv-01353-LPS (D.
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`Del.), in which Petitioner Oxford Nanopore Technologies, Inc. is a defendant.
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`Lead Counsel: Lead Counsel is Steven Lendaris (Reg. No. 53,202) and
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`Back-up Counsel is Carolyn Pirraglia (Reg. No. 75,365), each of Baker Botts
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`L.L.P.
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`Service Information: Baker Botts L.L.P., 30 Rockefeller Plaza, 45th Floor,
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`New York, NY 10112; Tel. (212) 408-2500; Fax (212) 408-2501. Petitioner
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`consents to service by electronic mail at Oxford929IPR@bakerbotts.com. Powers
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`of Attorney are filed concurrently herewith under 37 C.F.R. § 42.10(b).
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`B. Certification of Grounds for Standing
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`Petitioner certifies that the ’929 Patent is available for IPR. Petitioner is not
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`barred or estopped from requesting IPR of the ’929 Patent.
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`C. Fees
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`The Office is authorized to charge any fees that become due in connection
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`with this Petition to Deposit Account No. 02-0384, Ref. No. 078288.0152, as well
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`as any additional fees that might be due in connection with this Petition.
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`III. OVERVIEW OF THE ’929 PATENT
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`A. Overview of the Disclosure of the ’929 Patent
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`The disclosure of the ’929 patent is primarily directed to methods for
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`performing intermittent detection during sequencing techniques that use optically
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`detectable labeling groups. Ex. 1001, 2:46-54, 5:19-40, 8:31-35, 20:15-36.
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`According to the ’929 patent, “one drawback to the use of optically detectable
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`labeling groups is that prolonged exposure of chemical and biochemical reactants
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`to [] light sources, alone, or when in the presence of other components, e.g., the
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`fluorescent groups, can damage such reactants.” Id., 1:55-59. The ’929 patent
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`states that the disclosed methods are useful “for mitigating photo-induced damage
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`in an illuminated reaction by subjecting the illuminated reaction to intermittent
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`illumination rather than constant illumination,” “particularly [for] reactions that
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`employ fluorescent or fluorogenic reactants.” Id., 5:19-22, 20:15-22.
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`The ’929 patent further describes nucleic acid templates that are completely
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`contiguous and
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`include “a double-stranded portion comprised of
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`two
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`complementary sequences and two single-stranded linking portions.” Id., 66:22-
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`27. The ’929 patent also includes a series of paragraphs and figures incorporated
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`into the specification via a preliminary amendment from Provisional Application
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`No. 61/099,696 (“the ’696 provisional”). Id., 37:3-38:59, Figures 20 and 21.
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`These paragraphs and figures relate to nucleic acid templates that are either
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`completely contiguous or partially contiguous and include a double-stranded
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`segment. Id. The ’929 patent asserts that “the templates of the invention by virtue
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`of their inclusion of double-stranded segments, provide consensus through the
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`identification of both the sense and antisense strand of such sequences ….” Id.,
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`37:46-50.
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` More specifically, when describing the completely contiguous
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`templates, the ’929 patent contends that “[t]hese template molecules are
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`particularly useful as nucleotide sequence data generated therefrom comprises both
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`sense and antisense nucleotide sequences for the double-stranded portion … [that]
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`provides duplicative or redundant sequence information.” Id., 66:37-43.
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`Although the disclosure of the ’929 patent is primarily directed to
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`intermittent detection of sequencing reactions,1 the claims of the ’929 patent are
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`1 The working examples of the ’929 patent relate to sequencing-by-synthesis of a
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`nucleic acid template that “comprised a double-stranded region and two single-
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`stranded linker portions” and obtaining sequence data by intermittent illumination.
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`Id., 78:53-55, 79:11-29, 81:38-65.
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`directed to methods for nanopore-based sequencing of a polynucleotide comprising
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`a double-stranded portion, where an enzyme chaperone is used to control the
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`passage of the polynucleotide through the nanopore. Id., 82:35-83:32. The claims
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`also require obtaining nucleotide sequence information of the complementary
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`strands of the double-stranded portion and determining a consensus sequence
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`based on such information. Id. The ’929 patent includes 17 claims, only the first
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`of which is independent. Id.
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`In the few paragraphs of the disclosure that relate to nanopore sequencing,
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`the ’929 patent discloses that methods of fabrication and use of nanopores for
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`polynucleotide sequencing were well known in the art and cites to 9 references that
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`discuss nanopore sequencing of polynucleotides using enzyme chaperones.2 Id.,
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`77:33-41. In particular, the ’929 patent states that “the pattern of variations in the
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`2 The ’929 patent states “[f]or additional discussion of methods of fabrication and
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`use of nanopores, see, e.g., U.S. Pat. No. 5,795,782; Kasianowicz, J. J., et al.
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`(1996) Proc Natl Acad Sci USA 93(24):13770-3; Ashkenas, N., et al.
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`(2005) Angew Chem
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`Int Ed Engl 44(9):1401-4; Winters-Hilt, S., et al.
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`(2003) Biophys J 84:967-76; Astier, Y., et al. (2006) J Am Chem Soc 128(5):1705-
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`10; Fologea, D., et al. (2005) Nano Lett 5(10):1905-9; Deamer, D. W., et al.
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`(2000) Trends Biotechnol 18(4):147-51; and Church, G. M. (2006) Scientific
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`American 294(1):52.” Ex. 1001, 77:33-41.
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`current passing through the nanopore as the polynucleotide is drawn through may
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`be monitored and analyzed to determine the nucleotide sequence of the
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`polynucleotide,” and that “[a] polynucleotide may be drawn through the nanopore
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`by … enzyme chaperones to guide the polynucleotide through the nanopore.” Id.,
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`77:26-33.
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`B. Overview of the Prosecution History
`U.S. Patent Application No. 15/383,965 (“the ’965 application”), which
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`matured into the ’929 patent, was filed on December 19, 2016 as a Track One
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`application, and included 18 claims. Ex. 1013, 109-110, 172. On December 20,
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`2016, a preliminary amendment was filed to incorporate disclosure from the ’696
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`provisional. Id., 174-181. In particular, the incorporated disclosure of the ’696
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`provisional related to “template configurations and methods for using these
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`configurations in template directed sequencing processes.” Id., 175-178. Patent
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`Owner represented that the basis for this amendment was the incorporation by
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`reference of the ’696 provisional into the ’929 patent, and the claimed priority to
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`the ’696 provisional by the ’929 patent. Id., 180; see also Ex. 1001, 1:12-14,
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`36:61-37:1.
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`In a first non-final Office Action on the merits, dated March 14, 2017, then
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`pending claim 1 was rejected under 35 U.S.C. § 101. Ex. 1013, 652. Specifically,
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`it was found that the pending claims were directed to an abstract idea because the
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`recited steps of “monitoring variations,” “analyzing monitored variations,” and
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`“determining consensus sequence” could be interpreted as “reading data on a paper
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`or the gathering of data … to obtain desired sequence information.” Id., 653. It
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`was further found that “the active step of introducing polynucleotide comprising a
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`region of interest to a sequence analysis system … does not make clear how the
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`steps of ‘monitoring’, ‘analyzing’ and ‘determining’ are intended to take place
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`and/or what active processes are involved in these steps,” and thus “does not
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`transform the abstract idea … into a patent eligible application of the abstract idea
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`such that the claims amount to significantly more than the abstract idea.” Id., 653-
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`654.
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`Additionally, claims 1-8 were rejected on the ground of nonstatutory double
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`patenting as being unpatentable over the claims of U.S. Patent No. 9,057,102 (“the
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`’102 patent”). Id., 655. It was found that the specification of the ’102 patent
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`disclosed that the recited sequences could be scaffold or consensus sequences,
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`which comprised overlapping redundant sequences, and that claims 1-8 of the ’965
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`application are obvious over the teachings of the claims of the ’102 patent. Id.,
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`657-658. A telephonic interview was conducted on March 28, 2017, during which
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`Patent Owner proposed claim amendments later submitted in a response, dated
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`March 31, 2017, to overcome the § 101 rejection. The amended claim 1 specified
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`that “the nanopore is ‘in a membrane’ (previous claim 3); ‘applying a voltage
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`across the membrane’ (previous claim 6); ‘monitoring variations in ionic current
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`through the nanopore’ (previous claim 7); and ‘analyzing the monitored variations
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`in ionic current to obtain nucleotide sequence information for the polynucleotide.’”
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`Id., 697. Patent Owner argued in its response, dated March 31, 2017, that amended
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`claim 1 “clearly sets forth specific non-abstract steps for determining a nucleotide
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`sequence of a region of interest using a specific sequence analysis platform.” Id.
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`Additionally, Patent Owner submitted a Terminal Disclaimer over the ’102 patent
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`to overcome the double patenting rejection. Id., 698. The Patent Owner also
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`added new dependent claims 19-34 and independent claim 35.
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`In a final Office Action, dated June 5, 2017, the Examiner withdrew the §
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`101 rejection and the double patenting rejection. Id., 784. However, the Examiner
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`rejected most of the pending claims over U.S. Publication No. 2003/0044816 to
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`Denison et al. (“Denison”). Id., 785. Specifically, it was found that although
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`Denison did not “expressly teach determining the consensus sequence information
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`… [it] provides the capacity of obtaining redundant sequence information in order
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`to obtain consensus information.” Id., 786. Thus, the Examiner contended that
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`“[i]t would have been prima facie obvious to one of ordinary skill in the art at the
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`time of the claimed invention to carry out the invention with a reasonable
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`expectation of success since such steps of detecting desired consensus sequences
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`and redundant sequences using the sequencing methodology of Denison [sic] is
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`within the ordinary artisan capabilities and would not negatively alter or modify
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`the results of sequencing a desired target sequence.” Id., 787.
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`In a response filed June 5, 2018, Patent Owner amended claim 1 to
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`incorporate the subject matter of claim 26 and the intervening claim 15, and recite
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`“wherein the polynucleotide comprises a double-stranded portion comprising
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`complementary strands of the region of interest” and “wherein the redundant
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`sequence information comprises the nucleotide sequence of the complementary
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`strands.” Id., 808, 811. Patent Owner argued that “claim 26 was not rejected
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`under prior art but rather because it was dependent on a rejected base claim,” and
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`thus “[c]laim 1 is patentable over Denison [sic] et al.” Id., 811. Additionally,
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`Patent Owner canceled claims 9, 10, 12, 15, 19-26 and 35, in view of the
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`amendments to claim 1. Id.
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`Subsequently, a Notice of Allowance was mailed on June 23, 2017, and the
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`Issue Fee was paid on the same day. Id., 816-820, 833. The ’929 Patent was
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`issued on August 2, 2017. Id., 858-859.
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`IV. LEVEL OF ORDINARY SKILL IN THE ART
`The level of ordinary skill in the art is evidenced by the references. See In re
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`GPAC Inc., 57 F.3d 1573, 1579 (Fed. Cir. 1995). A person of ordinary skill in the
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`art (POSA) for this patent possesses a Ph.D. or an equivalent amount of experience
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`in molecular biology, genetics, biochemistry or a related field. A POSA in the art
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`area would have knowledge of DNA sequencing techniques including Maxam-
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`Gilbert and Sanger sequencing, as well as other techniques available on or before
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`the priority date of the ’929 Patent such as Applied Biosystems/Life Technologies,
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`Solexa/Illumina, Helicos and PacBio sequencing. Ex. 1002, ¶¶31-32.
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`V.
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`STATE OF THE ART
`A. Nanopore Sequencing Using Enzyme Chaperones
`The notion of using nanopores to sequence polynucleotides, such as DNA
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`and RNA, has been around for decades. A typical nanopore sequencing system
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`generally contains two chambers that are connected through a nanopore—a hole
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`with a diameter on the order of one nanometer—embedded in a substrate. See,
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`e.g., Ex. 1010, Figure 1; Ex. 1004, Figure 10C.
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`Ex. 1010, Figure 1.
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`During sequencing, a polynucleotide of interest, either double-stranded or single-
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`stranded, is added into one of the chambers, and an electric field is then applied
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`across the substrate, generating an electric force that pulls the negatively charged
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`polynucleotide through the nanopore. Ex. 1010, 1:40-2:58; see also id., Figure 1,
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`Ex. 1004, Figure 2. As individual nucleotides of the polynucleotide pass through
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`the nanopore, each nucleotide transiently blocks the nanopore, causing a change of
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`ionic current and producing an electric signal that correlates to the size, shape and
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`identity of the nucleotide. Ex. 1010, 6:14-22, Figure 3. Measuring such
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`nucleotide-distinct signals allows one to determine the nucleotides that make up
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`the polynucleotide in a sequential manner, in other words, the nucleotide sequence
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`of the polynucleotide. Id.
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`The use of enzyme chaperones, also referred to herein as “molecular
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`motors,” in nanopore sequencing was well recognized as of the priority date of the
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`’929 patent. Exemplary molecular motors can be used with nanopore sequencing
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`include DNA polymerases, RNA polymerases, ribosomes, exonucleases and
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`helicases. Id., 4:28-30; Ex. 1004, ¶¶[0008]-[0009], [0013], [0047], claim 3. Such
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`molecular motors bind to the polynucleotide of interest and assist its translocation
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`through the nanopore, and thus allowing for the sequencing of the polynucleotide.
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`Ex. 1010, 4:11-30; see also id., Figure 2. Moreover, the molecular motor has the
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`advantage of controlling the rate of a polynucleotide passing through a nanopore,
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`resulting in “a higher degree of resolution with regard to both the composition and
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`spatial relationship between nucleotide units within a polynucleotide.” Ex. 1004,
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`¶¶[0007], [0019], [0036]. For example, it was well known that the rate of
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`movement may be altered by changing reaction conditions, e.g., “a change in
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`voltage, pH, temperature, viscosity, or concentration of a chemical species (e.g.,
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`ions, cofactors, energy sources, or inhibitors).” Id., ¶[0009].
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`B.
`Sequencing of Complementary Strands to Improve Accuracy
`Before the priority dates of the ’929 patent, it was well recognized that an
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`increased rate of accuracy in polynucleotide sequencing can be obtained by
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`determining the sequences of both complementary strands of a double-stranded
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`polynucleotide. See Ex. 1005, Ex. 1009, Ex. 1011, Ex. 1012. As disclosed in
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`Sambrook, a widely-known manual on DNA sequencing and colloquially referred
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`to as “The Bible,”3 an increased rate of accuracy can be obtained by sequencing
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`both strands of the target DNA, comparing sequences of both strands and resolving
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`“all ambiguities and discrepancies.” Ex. 1009 (“Sambrook”), 13.20; see also Ex.
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`1011 (“Gilchrist”), 2:61-3:7 (disclosing a method of improving sequencing
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`accuracy by obtaining forward and reverse data sets and comparing these
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`sequences); and Ex. 1005 (“Gupte”), 1:13-15 (disclosing that “it is common to
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`3 See, e.g., Struhl, Nature 316:222 (1985) (Ex. 1016).
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`sequence both strands of DNA to minimize any errors which may occur in the
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`sequencing”).
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`Even as early as 1980, sequencing of both strands of a double-stranded
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`nucleic acid was known to improve the accuracy of the sequencing. This is
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`evidenced by the Nobel Laureate speech given by Frederick Sanger in 1980, in
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`which he states “it [is] necessary to determine the sequence of each region on both
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`strands of the DNA” to minimize errors during DNA sequencing. Ex. 1012, 3-4.
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`VI. SUMMARY OF PRIOR ART
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`A. Nanopore Sequencing using Enzyme Chaperones was Known in
`the Art Prior to the Earliest Priority Date Claimed by the ’929
`Patent
`1.
`U.S. Patent Publication No. 2006/0063171 (“Akeson”)
`U.S. Patent Publication No. 2006/0063171, entitled “Methods and apparatus
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`for characterizing polynucleotides,” was filed as U.S. Application No. 11/088,140
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`on March 23, 2005 and published on March 23, 2006 (hereinafter “Akeson”) and is
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`§ 102(b) prior art (Ex. 1004). Akeson was not submitted to, or considered by, the
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`Examiner, or otherwise made of record, during the prosecution of the ’929 Patent.
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`Akeson discloses methods for sequencing a polynucleotide using a nanopore
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`and an enzyme chaperone (i.e., molecular motor) “that physically interacts with a
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`[] polynucleotide.” Ex. 1004, ¶[0016]; see also id., ¶[0008]-[0009], [0017],
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`[0019]. Akeson teaches that the target polynucleotide can be “single-stranded or
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`double-stranded.” Id, ¶[0047]. Akeson further discloses that the molecular motor
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`can be a polymerase, a helicase or an exonuclease, and that the molecular motor
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`can be used to modify the rate of translocation of a nucleic acid through a
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`nanopore. Id., ¶¶[0019], [0047]-[0051], [0083].
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`As shown in Figure 10B, Akeson teaches that when “the molecular motor 26
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`is a helicase, and the polynucleotide is double-stranded DNA,” “[t]he addition of
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`an energy source [] to the cis solution activates the helicase to separate the DNA
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`and may push one of the strands [86] of the DNA through the pore structure 24.”
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`Id., ¶[0090]. In addition, Akeson discloses that when “the molecular motor 26 is a
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`DNA polymerase, and the double-stranded DNA contains a nick, … the strand [8]
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`that is dislodged as the polymerase acts may be analyzed, e.g., as it traverses
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`through the nanopore.”
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` Id.
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` Akeson further discloses that the “target
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`polynucleotide 82 may have a nick in the strand near one termini of the target
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`polynucleotide 82.” Id., ¶[0086]. For example, the “break, or nick, in the
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`phosphodiester backbone towards one termini of the DNA duplex … creates a free
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`3’-terminal hydroxyl to serve as an initiation site for [a] DNA polymerase.” Id.,
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`¶[0063].
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`Id., Figures 10A-10D.
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`Akeson further discloses that “the intrinsic rate of movement of a particular
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`molecular motor may be modified, e.g., by chemical modification of the motor, by
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`changes in temperature, pH, ionic strength, the presence of necessary cofactors,
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`substrates, inhibitors, agonists, or antagonists, by the nature of the medium (e.g.,
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`the presence of nonaqueous solvents or the viscosity), by external fields (e.g.,
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`electric or magnetic fields), and hydrodynamic pressure … to start, stop, increase,
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`decrease, or stabilize the rate of movement.” Id., ¶[0083]. In particular, Akeson
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`discloses that the rate of movement of the polynucleotide can be altered “before,
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`during, or after the monitoring step.” Id., ¶[0009].
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`U.S. Patent No. 6,936,433 (“Akeson ’433”)
`2.
`U.S. Patent 6,936,433 entitled “Method and devices for characterizing
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`duplex nucleic acid molecules,” was filed on November 21, 2001, and issued on
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`August 30, 2005 (hereinafter “Akeson ’433”) and is § 102(b) prior art (Ex. 1014).
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`Akeson ’433 was not submitted to, or considered by, the Examiner, or otherwise
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`made of record, during the prosecution of the ’929 Patent.
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`Akeson ’433 discloses methods for analyzing the nucleotide sequence of
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`double-stranded polynucleotides using nanopore systems. Ex. 1014, Abstract,
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`1:43-63, claim 13. In particular, Akeson ’433 discloses methods where “a fluid
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`conducting medium that includes a duplex nucleic acid molecule is contacted with
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`a nanopore under the influence of an applied electric field and the resulting
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`changes in current through the nanopore caused by the duplex nucleic acid
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`molecule are monitored.” Id., 1:45-49. Akeson further discloses that the “during
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`application of the applied electric field, the ion current through the nanopore is
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`measured or monitored over a period of time. Measurements are typically made at
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`least every 1 s, usually at least every 0.1 s and more usually at least every 0.01 s
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`using a single nanopore. This step results in the production of a set of measured
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`current derived data files, where the set typically consists of at least about 5,
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`usually at least about 10 and more usually at least about 50 individual measured
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`data points, where the set generally includes many more data points, usually at
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`least about 100, 1000, 5000 or more.” Id., 10:48-58.
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`B.
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`Sequencing of Both Strands of a Polynucleotide was Known in
`the Art Prior to the Earliest Priority Date Claimed by the ’929
`Patent
`1.
`Sanger
`Sanger, Determination of Nucleotide Sequences
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`in DNA,” Science
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`214:1205-1210 (1981) (hereinafter “Sanger”) is a publication of Sanger’s Nobel
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`Laureate speech that was given in 1980 and published in 1981 and is § 102(b) prior
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`art (Ex. 1012). See also Ex. 1015. Sanger was not submitted to, or considered by,
`
`the Examiner, or otherwise made of record, during the prosecution of the ’929
`
`Patent.
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`Sanger discloses that “[i]n most studies on DNA one is concerned with
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`identifying the right reading frames for protein genes, and to do this the sequence
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`must be correct. Errors can readily occur in such extensive sequences and
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`confirmation is always necessary. We usually consider it necessary to determine
`
`the sequence of each region on both strands of the DNA.” Ex. 1012, 3-4.
`
`U.S. Patent No. 6,087,099 (“Gupte”)
`2.
`U.S. Patent 6,087,099 entitled “Method for sequencing both strands of a
`
`double stranded DNA in a single sequencing reaction,” was filed on September 8,
`
`1997, and issued on July 11, 2000 (hereinafter “Gupte”) and is § 102(b) prior art
`
`(Ex. 1005). Gupte was not submitted to, or considered by, the Examiner, or
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`otherwise made of record, during the prosecution of the ’929 Patent.
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`Gupte states that “it is common to sequence both strands of DNA to
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`minimize any errors which may occur in the sequencing” and discloses methods
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`“for sequencing both strands of a double-stranded DNA molecule.” Ex. 1005,
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`1:13-15, 1:55-60, claim 1. In particular, Gupte discloses a process for generating a
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`double-stranded DNA product having a hairpin structure when sequenced “yields
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`the sequence of both strands of the original DNA.” Id., 2:10-11, 2:14-18.
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`As shown in Figure 2B, Gupte discloses the amplification of a double-
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`stranded polynucleotide (shown in Figure 2A), e.g., genomic DNA, using a pair of
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`amplification primers to generate a double-stranded polynucleotide product, where
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`each strand includes a first region (denoted C in Figure 2B) and a second region
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`(denoted C’ in Figure 2B), which are reverse complements of each other. Id.,
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`Figure 2A-B, 2:64-3:1, 6:22-28, claim 1. Gupte discloses that the amplification
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`primers can be complementary to a nucleotide sequence at the 3’ end or the 5’ end
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`of the double-stranded polynucleotide. Id., 2:42-60, 5:22-37. Gupte further
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`discloses that “when [the double-stranded polynucleotide product of Figure 2B] is
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`denatured one end of the resulting single stranded DNA loops around to form an
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`intrastrand stem-loop structure” (shown in Figure 2C)… which is then elongated to
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`produce “a double-stranded DNA [] wherein the two strands are joined by a loop”
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`(shown in Figure 2D). Id., 1:49-55, 3:10-20, 4:31-46, Figure 2C-2D. Denaturing
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`the double-stranded polynucleotide product of Figure 2D followed by PCR
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`amplification using the amplification primers results in the double-stranded
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`polynucleotide products of Figure 2E and Figure 2F. Id., 3:21-38, Figure 2E-2F.
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`Gupte discloses that the polynucleotide products disclosed in Figure 2D, Figure 2E
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`and Figure 2F can be substrates for sequencing. Id., 7:1-4; see also id., 2:61-63
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`(stating that “all the strands labeled ꞏSEQ [in Figure 2] are substrates for dye
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`primer sequencing.”).
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`Id., Figures 2A-2F.
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`U.S. Patent Publication No. 2005/0142559 (“Makrigiorgos”)
`3.
`U.S. Patent Publication No. 2005/0142559 entitled “Amplification of DNA
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`in a hairpin structure, and applications” was filed as U.S. Application No.
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`10/758,401 on January 15, 2004 and published on June 30, 2005 (hereinafter
`
`“Makrigiorgos”) and is § 102(b) prior art (Ex. 1006). Makrigiorgos was not
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`submitted to, or considered by, the Examiner, or otherwise made of record, during
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`the prosecution of the ’929 Patent.
`
`Makrigiorgos discloses methods for generating nucleic acids having hairpin
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`and dumbbell-like structures, and teaches that such structures can be sequenced.
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`Ex. 1006, ¶¶[0071], [0072], Figure 3A. As shown in Figure 3A, Makrigiorgos
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`describes the generation of a dumbbell polynucleotide structure by the addition of
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`two oligonucleotide “caps” (i.e., linkers) to a double-stranded DNA fragment. Id.,
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`Figure 3A, ¶[0028]. Makrigiorgos discloses that the double-stranded DNA
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`fragment can be a large genomic fragment as “polymerases can displace much
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`longer (>1 kb) DNA stretches during synthesis.” Id., ¶[0073].
`
`Makrigiorgo
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