`
`UNITED STATES PATENT AND TRADEMARK OFFICE
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`_________________________
`
`BEFORE THE PATENT TRIAL AND APPEAL BOARD
`
`_________________________
`
`
`
`MYRIAD GENETICS, INC., MYRIAD GENETIC LABORATORIES, INC.,
`BIO-RAD LABORATORIES, INC., and RAINDANCE TECHNOLOGIES, INC.
`
`Petitioners
`
`v.
`
`THE JOHNS HOPKINS UNIVERSITY
`
`Patent Owner
`
`U.S. Patent No. 6,440,706
`
`_________________________
`
`Case No. To be assigned
`
`_________________________
`
`
`
`PETITION FOR INTER PARTES REVIEW OF U.S. PATENT NO. 6,440,706
`UNDER 35 U.S.C. §§ 311-319 AND 37 C.F.R. §§ 42.1-.80, 42.100-.123
`
`
`
`

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`IPR of USPN 6,440,706
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`
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`I.
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`TABLE OF CONTENTS
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`Page
`
`STATEMENT OF THE PRECISE RELIEF REQUESTED AND THE
`REASONS THEREFOR (37 C.F.R. § 42.22(A)) ........................................... 1
`
`II. OVERVIEW .................................................................................................... 1
`
`III. THE '706 PATENT DISCLOSURE AND CLAIMS ...................................... 5
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`IV. THE '706 FILE HISTORY AND REEXAMINATION FILE
`HISTORY ........................................................................................................ 6
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`V.
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`THE PERSON OF ORDINARY SKILL IN THE ART ................................. 7
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`VI. CLAIM CONSTRUCTION ............................................................................ 8
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`VII.
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`IDENTIFICATION OF THE CHALLENGE (37 C.F.R. § 42.104(B)) ....... 11
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`VIII. THE STATE OF THE ART .......................................................................... 12
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`IX. GROUND 1: CLAIMS 1-3, 7, 15-16, 19, 24, 27, 38-39, 47-48, 51,
`56, AND 59 ARE ANTICIPATED BY SIMMONDS .................................. 13
`
`X. GROUND 2: CLAIMS 8-11 AND 40-43 WOULD HAVE BEEN
`OBVIOUS IN VIEW OF SIMMONDS AND BROWN .............................. 29
`
`XI. GROUND 3: CLAIMS 20 AND 52 WOULD HAVE BEEN
`OBVIOUS IN VIEW OF SIMMONDS AND KELLOGG .......................... 35
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`XII. GROUND 4: CLAIMS 1-3, 7, 19, 24, 27, 38, 39, 51, 56, AND 59
`ARE ANTICIPATED BY SYKES ............................................................... 39
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`XIII. GROUND 5: CLAIMS 8-11, 15-16, 40-43, AND 47-48 WOULD
`HAVE BEEN OBVIOUS IN VIEW OF SYKES AND BROWN ................ 50
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`XIV. GROUND 6: CLAIMS 20 AND 52 WOULD HAVE BEEN
`OBVIOUS IN VIEW OF SYKES AND KELLOGG ................................... 56
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`XV. OBJECTIVE INDICIA DO NOT SUPPORT PATENTABILITY .............. 59
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`XVI. CONCLUSION .............................................................................................. 64
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`XVII. MANDATORY NOTICES (37 C.F.R. § 42.8(A)(1)) ................................... 66
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`
`
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`i
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`

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`I.
`
`STATEMENT OF THE PRECISE RELIEF REQUESTED AND THE
`REASONS THEREFOR (37 C.F.R. § 42.22(A))
`
`Myriad Genetics, Inc., Myriad Genetic Laboratories, Inc. (collectively,
`
`"Myriad"), Bio-Rad Laboratories, Inc., and RainDance Technologies, Inc.
`
`(collectively, "Petitioners") respectfully petition for Inter Partes Review, and seek
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`cancellation of claims 1-3, 7-11, 15-16, 19-20, 24, 27, 38-43, 47-48, 51-52, 56, and
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`59 of USPN 6,440,706 (MYR1001) as unpatentable for anticipation and/or
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`obviousness. The '706 patent is assigned to The Johns Hopkins University
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`(hereinafter "Patent Owner").
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`II. OVERVIEW
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`Claims 1-3, 7-11, 15-16, 19-20, 24, 27, 38-43, 47-48, 51-52, 56, and 59 of
`
`the '706 patent should be canceled as anticipated and/or obvious. MYR1002, ¶¶20-
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`22. Independent claims 1 and 38 recite a method that Patent Owner calls "digital
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`PCR." MYR1002, ¶¶10-19. The figure below shows the basic steps of the
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`method, which involve distributing a DNA sample into compartments such that
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`each compartment contains, ideally, one or zero molecules of DNA from the
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`sample, carrying out PCR in each compartment, and then analyzing the resulting
`
`amplified DNA molecules to determine how many compartments contain each
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`different template DNA molecule:
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`IPR of USPN 6,440,706
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`MYR1018, 541.
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`The steps comprising what the Patent Owner calls "digital PCR" were well
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`known in the art before the earliest possible priority date for the '706 patent.1
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`MYR1002, ¶11. In the prior art, this method was often called "limiting dilution
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`analysis" or "limiting dilution PCR" ("LDPCR") because the sample is diluted
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`down to the point at which some compartments will be "positive," i.e., contain a
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`PCR-amplified product, and some will be "negative," i.e., contain no PCR-
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`amplified product. Id. For LDPCR, terms such as "assay samples," "replicates,"
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`"compartments," "sample chambers," "wells," or "microreactors" all represent the
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`1 The earliest application to which the '706 patent claims priority is
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`provisional application 60/146,792, filed 8/2/1999. MYR1011. Given that,
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`Petitioners rely almost exclusively on prior art under 35 U.SC. §102(b), they are
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`not aware of any claim to an earlier priority date that would affect any of the
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`arguments set forth herein. Petitioners reserve the right to respond should Patent
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`Owner allege an earlier priority date.
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`
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`2
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`IPR of USPN 6,440,706
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`same functional element – a separate space where a diluted single template
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`molecule can undergo PCR without cross-contamination, and produce pure or
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`homogeneous amplified product. Id. As discussed in detail below, Patent Owner
`
`did nothing more than add a snappy name to the prior art method of LDPCR.
`
`By 1994, Kary Mullis, the Nobel Prize winning inventor of PCR, had edited
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`a book on PCR (MYR1014) that included a chapter on quantitative PCR, the use of
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`PCR to quantitate amounts of nucleic acids in a sample. The Mullis chapter
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`discloses and discusses the work of multiple groups of scientists at the time who
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`were carrying out and publishing work involving LDPCR. MYR1002, ¶15. A
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`common feature of this work is that it involved diluting and distributing nucleic
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`acids down to the single molecule level in assay samples or compartments,
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`amplifying the single molecule templates using PCR, and counting or otherwise
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`analyzing the amplified templates in the assay samples or compartments. As the
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`Mullis chapter disclosed in 1994:
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`The principle of limiting dilution can also be called on to achieve
`
`absolute DNA quantitation. It is based on the use of a qualitative all-
`
`or-none endpoint and on the premise that one or more targets in the
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`reaction mixture give rise to a positive endpoint. . . . Accurate
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`quantitation can be achieved by performing multiple replicates at
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`serial dilutions of the material to be assayed (Simmonds, 1990; Lee
`
`et al. 1990; Sykes et al. 1992). At the limit of dilution, where some
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`end points are positive and some are negative, the number of targets
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`3
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`IPR of USPN 6,440,706
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`present can be calculated from the proportion of negative endpoints by
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`using Poisson statistics. . . . This method quantitates the total number
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`of initial DNA targets present in a sample. In this type of quantitative
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`format, it is mandatory that PCR be optimized so that reliable
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`detection of one or a few DNA targets occurs. Therefore, as long as
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`the one copy level still gives a positive signal, the quantitation is not
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`dependent on the amplification efficiency. This represents a major
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`advantage of this PCR format.
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`MYR1014, 78 (emphases added); MYR1002, ¶15.
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`As the Mullis chapter discloses, multiple groups of scientists, including
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`Simmonds (MYR1012) and Sykes (MYR1013) – authors of two prior art
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`references discussed in detail below – were carrying out LDPCR and publishing
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`the results of their work prior to the earliest possible priority date for the '706
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`patent. MYR1002, ¶16.
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`Some five years after publication of the Mullis chapter, two professors and
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`named co-inventors working for Patent Owner, Vogelstein and Kinzler, published
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`a paper in PNAS (MYR1017) in which they described the steps of what they called
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`"digital PCR." Notably, while much of this paper is reproduced in the
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`specification of the '706 patent, there is one important difference. The PNAS
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`paper stated that "there are several precedents for the approach described here."
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`MYR1017, 9239 (emphasis added). In the applications filed with the USPTO to
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`4
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`IPR of USPN 6,440,706
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`which the '706 patent claims priority, however, the Patent Owner abandoned the
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`candor of the PNAS paper and did not include the statement regarding the
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`existence of "several precedents." MYR1002, ¶17.
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`As the 1999 PNAS paper admits, there were "several precedents" to "digital
`
`PCR." The 1994 Mullis chapter and cited references confirm the existence of such
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`precedents beyond any reasonable dispute. Every claim of the '706 patent for
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`which inter partes review is sought is invalid as anticipated and/or obvious over
`
`these precedents. No secondary considerations or objective indicia can save the
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`challenged claims (§XV).
`
` If anything,
`
`the secondary consideration of
`
`simultaneous invention supports cancellation of all of the challenged claims.
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`III. THE '706 PATENT DISCLOSURE AND CLAIMS
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`The '706 patent, titled "Digital Amplification," issued on 8/27/2002, from
`
`App. No. 09/613,826, filed on 7/11/2000. MYR1001. The '706 patent claims
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`priority to provisional App. No 60/146,792 filed 8/2/1999. Id.
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`The '706 Claims. The '706 patent has 64 claims, 26 of which are challenged
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`here.2 Claims 1 and 38 are the only independent claims challenged here. Claim 1
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`is exemplary and provided below, as amended during the ex parte reexamination:
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`2 Petitioners do not concede the validity of any of the unchallenged claims.
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`Those claims, however, require the use of molecular beacon probes or other steps
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`5
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`IPR of USPN 6,440,706
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`Claim 1. A method for determining the ratio of a selected genetic
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`sequence in a population of genetic sequences, comprising the steps
`
`of:
`
`(a)
`
`diluting isolated nucleic acid template molecules [in] isolated
`
`from a biological sample to form a set comprising a plurality of assay
`
`samples;
`
`(b)
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`amplifying the template molecules within the assay samples to
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`form a population of amplified molecules in the assay samples of the
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`set;
`
`(c)
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`analyzing the amplified molecules in the assay samples of the
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`set to determine a first number of assay samples which contain the
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`selected genetic sequence and a second number of assay samples
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`which contain a reference genetic sequence;
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`(d)
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`comparing the first number to the second number to ascertain a
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`ratio which reflects the composition of the biological sample.
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`MYR1001.
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`IV. THE '706 FILE HISTORY AND REEXAMINATION FILE HISTORY
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`The Mullis chapter, Simmonds, and Sykes were not before the USPTO
`
`during initial prosecution of the '706 patent. MYR1004.
`
`
`that make them less relevant to modern DNA analysis and, therefore, less
`
`deserving of the PTAB's attention.
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`
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`6
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`IPR of USPN 6,440,706
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`The '706 patent was the subject of an ex parte reexamination proceeding,
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`during which multiple claims were amended to overcome rejections over the prior
`
`art. MYR1002, ¶¶4-6, 43-44; MYR1008. Although Simmonds and Sykes were
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`nominally before the Patent Office during the ex parte reexamination proceeding,
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`the Mullis chapter was not before the Patent Office and Simmonds and Sykes were
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`never discussed during that proceeding. MYR1002, ¶¶4-6, 43-44. Instead, the
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`proceeding focused on different prior art, which involved the distribution of whole
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`cells, rather than isolated nucleic acids, into compartments. Id. The claims were
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`amended to specify that the method involves "isolated" nucleic acids rather than
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`whole cells, in light of this art. MYR1002, ¶¶6, 44; MYR1035. While these
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`amendments addressed the prior art discussed during the ex parte reexamination,
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`they did nothing to address the Mullis chapter, Simmonds, Sykes, or other prior art
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`references discussed in this petition for inter partes review. Id.
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`V. THE PERSON OF ORDINARY SKILL IN THE ART
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`A person of ordinary skill in the art ("POSA") is a hypothetical person who
`
`is presumed to be aware of all pertinent art, thinks along the lines of the
`
`conventional wisdom in the art, and is a person of ordinary creativity. As of
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`August 2, 1999, a POSA in the technical field of the '706 patent – molecular
`
`biology – would have had knowledge of the scientific literature concerning
`
`methods of DNA manipulation and analysis, including amplification (e.g., PCR),
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`7
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`IPR of USPN 6,440,706
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`dilution and distribution, including down to the single molecule level and using
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`techniques such as LDPCR, and methods of nucleic acid analysis (e.g., gel
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`electrophoresis, detecting certain
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`sequences using hybridization probes,
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`quantitating specific sequences in a mixture of different nucleic acids, using
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`Poisson statistics for DNA quantitation, or sequencing).
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`As of August 2, 1999, a POSA would typically have had: (1) a M.D. degree
`
`or a Ph.D. degree in molecular biology, molecular genetics, biology, or equivalent
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`discipline, plus at least two years experience in a laboratory working in the field of
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`molecular biology techniques, including in quantitative amplification techniques,
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`detection, and analysis; (2) a Master's degree in molecular biology, molecular
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`genetics, biology, or equivalent discipline, plus at least five years experience in the
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`laboratory working in the field of molecular biology techniques, including in
`
`quantitative amplification techniques, detection, and analysis. MYR1002, ¶¶27-28.
`
`VI. CLAIM CONSTRUCTION
`
`In accordance with 37 C.F.R. § 42.100(b), the challenged claims must be
`
`given their broadest reasonable interpretations (BRI) in light of the specification
`
`and prosecution history of the '706 patent. Since the '706 patent Reexamination
`
`Certificate was issued, Patent Owner has asserted the challenged '706 patent claims
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`against Myriad's myRisk diagnostic test for hereditary cancer risk. MYR1031,
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`¶31. In the Myriad litigation, Patent Owner has accused of infringement the same
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`8
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`IPR of USPN 6,440,706
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`dilution and distribution steps found in the Mullis chapter, Simmonds, and Sykes,
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`and is effectively seeking to re-patent the prior art. Given that Patent Owner has
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`adopted a claim construction in the Myriad litigation that requires only dilution
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`and/or distribution, PCR amplification, and any analysis of the PCR products
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`(which construction was not before the USPTO during the ex parte reexamination
`
`proceedings), it is beyond reasonable dispute that the challenged claims are invalid
`
`over the prior art and should be cancelled by the PTAB. MYR1002, ¶¶45-52.
`
`A. The Preambles Are Not Limiting
`
`Under the BRI of Claims 1 and 38, their preambles should be non-limiting.
`
`These preambles do not recite any structure or step needed to give meaning and
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`life to the claims, or to any dependent claims. See, e.g., Summit 6, LLC v. Samsung
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`Electronics Co., Ltd., 802 F.3d 1283, 1292 (Fed. Cir. 2015) ("[g]enerally, a
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`preamble is not limiting"); TomTom, Inc. v. Adolph, 790 F.3d 1315, 1323 (Fed.
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`Cir. 2015). No term in Claim 1 or Claim 38, or in any claim that depends from
`
`them, refers back to these preambles, which therefore do not provide any
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`antecedent basis for the body of the claims.
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`A POSA would have understood that these preambles merely recite intended
`
`uses of the claimed methods, and therefore do not limit the claims in any way.
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`Summit, 802 F.3d at 1292; TomTom, 790 F.3d at 1323; MYR1002, ¶¶49-50.
`
`B.
`
`"allele"
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`
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`9
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`IPR of USPN 6,440,706
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`
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`Claims 27 and 59 include the term "allele." MYR1001, 19:6-7; 21:9-10.
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`Table 1 of the '706 patent refers to "mutant or WT alleles," and "normal or
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`translocated alleles." Id., Table 1. A POSA would have understood the term
`
`“allele” to mean, under the BRI: “one of various alternative forms of a gene or
`
`genomic sequence.” MYR1002, ¶51. This construction for the term "allele" was
`
`agreed to by Patent Owner in a previous litigation. MYR1032, 2.
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`C.
`
`"wild–type"
`
`Claims 27 and 59 include the term "wild-type." MYR1001, 19:6-7; 21:9-10.
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`The specification uses the term "wild-type" to mean a variant of a particular
`
`nucleic acids sequence: "[a]t its limit, single template molecules can be amplified
`
`so that the products are completely mutant or completely wild-type." MYR1001 at
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`4:3-5; MYR1002, ¶52. The claimed method is not carried out any differently
`
`whether the products are completely mutant or completely wild type. Id. And
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`during prosecution of a related application in the same family with the same
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`specification, USPN 8,859,206, the applicants did not challenge the Patent Office's
`
`interpretation that the terms "wild type" and "mutant" could encompass two
`
`different single nucleotide polymorphisms
`
`("SNPs") during prosecution.
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`MYR1007; MYR1037, 8. Under the BRI of Claims 27 and 59, the term "wild-
`
`type" would therefore be interpreted by a POSA to mean "a variant of a particular
`
`nucleic acids sequence." MYR1002, ¶52.
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`10
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`IPR of USPN 6,440,706
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`VII. IDENTIFICATION OF THE CHALLENGE (37 C.F.R. § 42.104(B))
`
`Petitioners respectfully petition for inter partes review of claims 1-3, 7-11,
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`15, 16, 19, 20, 24, 27, 38-43, 47, 48, 51, 52, 56, and 59 of the '706 patent based on
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`the unpatentability grounds summarized in the index below. Per 37 C.F.R. §
`
`42.6(c), copies of the cited references accompany this Petition.
`
`Ground
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`35 U.S.C. § (pre-
`AIA)
`
`1
`
`2
`
`3
`
`4
`
`5
`
`6
`
`
`
`§ 102
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`§ 103
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`§ 103
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`§ 102
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`§ 103
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`§ 103
`
`Claims
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`Reference(s)
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`1-3, 7, 15, 16, 19,
`24, 27, 38, 39, 47,
`48, 51, 56, 59
`
`8-11, 40-43
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`20, 52
`
`1-3, 7, 19, 24, 27,
`38, 39, 51, 56, 59
`8-11, 15, 16, 40-43,
`47, 48
`20, 52
`
`Simmonds
`
`Simmonds and
`Brown
`Simmonds and
`Kellogg
`
`Sykes
`
`Sykes and Brown
`
`Sykes and Kellogg
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`"Simmonds" (MYR1012) was published in February 1990 and is prior art to
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`the '706 patent under at least 35 U.S.C. §102(b). "Sykes" (MYR1013) was
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`published in 1992 and is prior art to the '706 patent under at least 35 U.S.C.
`
`§ 102(b). "Brown" (MYR1015) was filed on 4/17/1997 and issued on 11/7/2000,
`
`and is prior art to the '706 patent under at least 35 U.S.C. § 102(e). "Kellogg"
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`(MYR1016) was published in 1994 and is prior art to the '706 patent under at least
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`35 U.S.C. § 102(b).
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`11
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`IPR of USPN 6,440,706
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`Grounds 1 and 4 are not redundant because Simmonds and Sykes disclose
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`experiments carried out by independent groups applying the LDPCR technique for
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`different purposes. MYR1002, ¶58. For the same reason, Grounds 2 and 5, and 3
`
`and 6 are not redundant. Id. Petitioner includes all of these grounds to avoid a
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`repeat of the Patent Owner's conduct in the ex parte reexamination, during which it
`
`focused on an ancillary point – whole cell distribution versus isolated DNA
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`distribution – to avoid discussing the real issue: the fact that the steps comprising
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`what Patent Owner calls "digital PCR" were well known in the prior art.
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`This Petition is accompanied by a supporting declaration of Petitioners'
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`technical expert, Dr. Michael L. Metzker. MYR1002.
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`VIII. THE STATE OF THE ART
`
`POSAs knew long before August 2, 1999 that PCR was a powerful tool for
`
`quantitation of DNA. MYR1002, ¶¶29-35. For example, by the late 1980s and
`
`early 1990s, POSAs knew that some of the potential pitfalls of PCR related to its
`
`exponential nature – potentially biased amplification of certain sequences over
`
`others and artifacts that could arise from using multiple different pairs of primers
`
`in a single reaction – could be avoided
`
`through
`
`the use of parallel,
`
`compartmentalized PCR reactions, carried out at limiting dilution. MYR1014, 68.
`
`Indeed, as the 1994 Mullis chapter demonstrates, multiple research groups were
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`12
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`carrying out and publishing LDPCR methods by the early 1990s. MYR1002,
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`IPR of USPN 6,440,706
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`¶¶29-35.
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`By August 1999, researchers had developed numerous high-throughput
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`technologies to improve limiting dilution analysis, including LDPCR. For
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`example, Brown disclosed a platform to perform a multitude of LDPCR assays in
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`parallel in an efficient manner, and Kellogg disclosed a commercially-available,
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`"hot start" Taq polymerase product that used heat activation to enhance specificity
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`and sensitivity in PCR reactions. Id.
`
`IX. GROUND 1: CLAIMS 1-3, 7, 15-16, 19, 24, 27, 38-39, 47-48, 51, 56,
`AND 59 ARE ANTICIPATED BY SIMMONDS
`
`As illustrated in the claim charts and discussion below, a POSA would have
`
`understood that Simmonds discloses each element of, and therefore anticipates,
`
`Claims 1-3, 7, 15-16, 19, 24, 27, 38-39, 47-48, 51, 56, and 59. MYR1002, ¶¶59-
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`108.
`
`A.
`
`Independent Claim 1
`
`Claim
`1. A method for
`determining the
`ratio of a selected
`genetic sequence
`in a population of
`genetic sequences,
`comprising the
`steps of:
`
`Disclosure in Simmonds
`This non-limiting preamble is nonetheless disclosed:
`
`"Separate amplification of individual molecules from a
`mixture after dilution and distribution. . . . it should be
`possible to separate single molecules of two types from a
`mixture of the two by dilution and distribution and to amplify
`them separately. In order to test this proposition, a mixture
`was made of two clones derived from different HIV isolates,
`pBH10.R3 (HIV-HTLV-IIIB) and lambda HAT 3 (HIV-RF),
`and the mixture was diluted, distributed, and amplified as
`
`
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`13
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`
`
`Claim
`
`(a) diluting
`isolated nucleic
`acid template
`molecules [in]
`isolated from a
`biological sample
`to form a set
`comprising a
`plurality of assay
`samples;
`(b) amplifying the
`template
`molecules within
`the assay samples
`to form a
`population of
`amplified
`molecules in the
`assay samples of
`the set;
`(c) analyzing the
`amplified
`molecules in the
`assay samples of
`the set to
`determine a first
`number of assay
`samples which
`contain the
`selected genetic
`
`IPR of USPN 6,440,706
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`Disclosure in Simmonds
`before by double PCR. . . . Figure 3, lanes 2 and 32 show the
`results of amplifying lambda HAT 3 alone, and lanes 3 and 33
`show the results of amplifying pBH10.R3 alone. . . . Of 28
`reactions, 9 showed amplification of the pBH10.R3 env
`sequence (corrected mean, 0.39 molecules per reaction),
`while 13 of 28 reactions showed amplification of the lambda
`HAT 3 env sequence (corrected mean, 0.62 molecules per
`reaction)." (867)
`See preamble.
`
`"FIG. 3. Separate amplification of single molecules from a
`mixture. A mixture of pBH10.R3 and lambda HAT 3 was
`diluted in herring sperm DNA, and the appropriate dilutions
`were distributed to 28 tubes." (867, Figure 3 legend)
`
`See preamble and step (a) above.
`
`"After dilution of the mixture of the two sequences (lanes 4
`through 31), a clear separation of pBH10.R3 and lambda
`HAT 3 env sequences was seen. Of 28 reactions, 9 showed
`amplification of the pBH10.R3 env sequence (corrected
`mean, 0.39 molecules per reaction), while 13 of 28 reactions
`showed amplification of the lambda HAT 3 env sequence
`(corrected mean, 0.62 molecules per reaction). The number
`of reactions in which the amplification of both env sequences
`was expected to occur, calculated from these numbers with
`the assumption of independent distribution, was four. The
`
`
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`14
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`
`
`Claim
`sequence and a
`second number of
`assay samples
`which contain a
`reference genetic
`sequence;
`
`IPR of USPN 6,440,706
`
`Disclosure in Simmonds
`observed number of three is in good agreement with
`expectation." (867)
`
`"FIG. 3. . . . The products were run on an acrylamide gel,
`which was exposed to X-ray film. The amplified env
`sequences of lambda HAT 3 (a; 359 bp) and pBH10.R3 (b;
`317 bp) are readily distinguishable; c (248 bp) is the
`amplified gag sequence. Lanes: 1, negative control (carrier
`DNA); 2 and 32, lambda HAT 3 alone; 3 and 33, pBH10.R3
`alone; 4 to 31, the 28 samples distributed from the diluted
`mixture." (867, Fig. 3 legend)
`
`"In order to quantify the amount of provirus, 12 of the DNA
`samples were assayed by amplification after dilution and
`distribution (Table 1). . . . In each case, some of the selected
`dilutions gave both positive and negative reactions." (868).
`
`"Limiting-dilution PCR products from patients 75, 76, and 79,
`amplified with gag primers, were sequenced directly by using
`primer 883. In the case of patient 75, 13,000 cell equivalents
`of DNA were amplified, in the case of patient 76, 25,000 cell
`equivalents were amplified, and in the case of patient 79,
`1,000 and 2,000 cell equivalents were amplified. These
`dilutions gave a low frequency of positive reactions (Table 1)
`and hence, if our conclusions are correct, the probability was
`high that single molecules would be amplified. Each
`amplification product gave an unambiguous readable
`sequence of at least 175 bases. Five of the seven sequences
`are unique, with variation both between samples derived from
`different patients and between parallel amplification reactions
`originating from the same DNA preparation (Table 4)." (869)
`
`Table 1 shows the number of assay samples for Patient 75
`(2/8) (13,000 cell equivalents); 76 (4/16) (25,000 cell
`equivalents), and 79 (4/14) (1,000 and 2,000 cell equivalents),
`representing a total of 38 assay samples. MYR1002, ¶60.
`
`"In order to quantify the amount of provirus, 12 of the
`
`
`
`15
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`

`

`IPR of USPN 6,440,706
`
`Disclosure in Simmonds
`[patient's] DNA samples were assayed by amplification after
`dilution and distribution (Table 1)." (868).
`
`Data from Table 1 are summarized for the 25,000 cell
`equivalent column as such: Patient 75 (4/6); 76 (4/16), 77
`(2/2), 78 (6/11), 80 (5/13), 82 (2/2), 83 (2/3), and 84 (3/3). In
`sum, the positive selected genetic sequences would total 28 of
`fifty-six assay samples: the addition of the numerators is 4 +4
`+ 2 + 6 + 5 + 2 + 3 + 3 = 28; the addition of the denominators
`is 6 +16 + 2 + 11 + 13 + 2 + 3 + 3 = 56. MYR1002, ¶60.
`See step (c) above.
`
`
`
`Claim
`
`(d) comparing the
`first number to the
`second number to
`ascertain a ratio
`which reflects the
`composition of the
`biological sample.
`
`
`Claim 1, preamble. To the extent the preamble is found to be limiting,
`
`Simmonds nonetheless discloses it. Simmonds discloses a method of "separate
`
`amplification of individual molecules from a mixture after dilution and
`
`distribution." MYR1012, 867. Simmonds discloses that this method can be used
`
`". . . to analyze sequence heterogeneity within populations of HIV provirus . . . ."
`
`MYR1012, 865. Simmonds also discloses how to determine the ratio of a given
`
`HIV provirus in a mixed population of proviruses using the method of separate
`
`amplification of individual molecules from a mixture after dilution and
`
`distribution. MYR1012, 867. Simmonds further discloses how to determine that
`
`out of 28 assay samples, 9 contained one provirus variant and 13 contained the
`
`
`
`16
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`

`IPR of USPN 6,440,706
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`
`other provirus variant. Id. Simmonds showed that these ratios reflected the ratios
`
`of the two variants in the original sample, namely that one was present at about
`
`twice the amount of the other, which was in good agreement with how the mixture
`
`was originally created. MYR1012, Fig. 3 legend. Simmonds further discloses the
`
`use of sequence analysis to determine the ratio of a selected genetic sequence in a
`
`population of genetic sequences. MYR1012, 868-869.
`
`Claim 1, step (a). Simmonds discloses diluting and distributing a mixture
`
`of isolated provirus template DNA molecules that were isolated from blood
`
`samples to form a set of 28 assay samples. MYR1012, 867. Simmonds further
`
`discloses diluting and distributing a mixture of isolated provirus template DNA
`
`molecules that were isolated from three patient blood samples to form a set of 38
`
`assay samples. MYR1012, 868-869, Table 1.
`
`Claim 1, step (b). Simmonds discloses amplifying the provirus template
`
`molecules in the assay samples of the set using PCR to form a population of
`
`amplified provirus molecules in the 28 assay samples of the set. MYR1012, 867.
`
`Simmonds further discloses amplifying the provirus template molecules in the
`
`assay samples of the set using PCR to form a population of amplified provirus
`
`molecules in 38 assay samples of the set. MYR1012, 868-869.
`
`Claim 1, step (c). Simmonds discloses analyzing the PCR-amplified
`
`molecules in each of the 28 assay samples of the set by gel electrophoresis.
`
`
`
`17
`
`

`

`
`MYR1012, 867. Gel electrophoresis analysis showed that one provirus variant had
`
`been amplified in 9/28 assay samples, while the other provirus variant had been
`
`IPR of USPN 6,440,706
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`amplified in 13/28 assay samples. MYR1012, 867.
`
`Simmonds also discloses analyzing the PCR-amplified molecules from three
`
`patients of the 38 assay samples of the set using a sequencing technique.
`
`MYR1012, 869-870. Sequence analysis was performed from 2/8 assay samples
`
`(patient 75), 4/16 assay samples (patient 76), and 4/14 assay samples (patient 79).
`
`MYR1012, 868. Sequence analysis demonstrated the numbers and ratios of
`
`different provirus sequence variants in other patient samples using the same
`
`method. MYR1012, 868-869.
`
`Claim 1, step (d). Simmonds compares the number of assay samples
`
`containing each different provirus variant out of the 28 assay samples. MYR1012,
`
`867. Simmonds discloses that 9 samples contain one of the provirus variants,
`
`while 13 samples contain the other provirus variant. Id. Simmonds also compares
`
`the number of assay samples containing each different provirus variant out of the
`
`38 assay samples. MYR1012, 868-869. In particular Simmonds states that "[f]ive
`
`of the seven sequences are unique, with variation both between samples derived
`
`from different patients and between parallel amplification reactions originating
`
`from the same DNA preparation (Table 4)." MYR1012, 869.
`
`
`
`18
`
`

`

`IPR of USPN 6,440,706
`
`Disclosure in Simmonds
`See Claim 1, preamble.
`
`See Claim 1, step (a).
`
`See Claim 1, step (b).
`
`See Claim 1, step (c).
`
`See Claim 1, step (d).
`
`
`
`B.
`
`Independent Claim 38
`
`Claim
`38. A method for determining the
`ratio of a selected genetic sequence in
`a population of genetic sequences,
`comprising the steps of:
`(a) distributing cell-free nucleic acid
`template molecules from a biological
`sample to form a set comprising a
`plurality of assay samples;
`(b) amplifying template molecules
`[within a set comprising a plurality of
`assay samples] to form a population
`of amplified molecules in [each of
`the] individual assay samples of the
`set;
`(c) analyzing the amplified molecules
`in the assay samples of the set to
`determine a first number of assay
`samples which contain the selected
`genetic sequence and a second
`number of assay samples which
`contain a reference genetic sequence,
`wherein at least one-fiftieth of the
`assay samples in the set comprise a
`number (N) of molecules such that
`1/N is larger than the ratio of selected
`genetic sequences to total genetic
`sequences required to determine the
`presence of the selected genetic
`sequence;
`(d) comparing the first number to the
`second number to ascertain a ratio
`which reflects the composition of the
`biological sample.
`
`
`
`
`19
`
`

`

`
`
`Claim 38, preamble, steps (a). (b). (c). Simmonds discloses these
`
`limitations, including any limitations found to be present in the preamble. See
`
`IPR of USPN 6,440,706
`
`Claim 1.
`
`Claim 38, step (c). Simmonds discloses analyzing the PCR-amplified
`
`molecules in each of the 28 assay samples of the set by gel electrophoresis.
`
`MYR1012, 867. Gel electrophoresis analysis showed that one provirus variant had
`
`been amplified in 9/28 assay samples, while the other provirus variant had been
`
`amplified in 13/28 assay samples. Id.; MYR1002, ¶91.
`
`Simmonds also discloses analyzing the PCR-amplified molecules from three
`
`patients of the 38 assay samples of the set using a sequencing technique.
`
`MYR1012, 869-870. Sequence analysis was performed from 2/8 assay samples
`
`(patient 75), 4/16 assay samples (patient 76), and 4/14 assay samples (patient 79).
`
`MYR1012, 868. Sequence analysis demonstrated the numbers and ratios of
`
`different provirus sequence variants in other patient samples using the same
`
`method. MYR1002, ¶92; MY