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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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`
`
`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. 7,824,889
`
`_________________________
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`Case No. To be assigned
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`_________________________
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`
`
`PETITION FOR INTER PARTES REVIEW OF U.S. PATENT NO. 7,824,889
`UNDER 35 U.S.C. §§ 311-319 AND 37 C.F.R. §§ 42.1-.80, 42.100-.123
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`

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`IPR of USPN 7,824,889
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`I.
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`TABLE OF CONTENTS
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`Page
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`STATEMENT OF THE PRECISE RELIEF REQUESTED AND THE
`REASONS THEREFOR (37 C.F.R. § 42.22(A)) ........................................... 1
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`II.
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`OVERVIEW .................................................................................................... 1
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`III. THE '889 PATENT DISCLOSURE AND CLAIMS ...................................... 5
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`IV. THE '889 FILE HISTORY AND REEXAMINATION FILE
`HISTORY ........................................................................................................ 7
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`V.
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`THE PERSON OF ORDINARY SKILL IN THE ART ................................. 8
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`VI. CLAIM CONSTRUCTION ............................................................................ 9
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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, 5, 8-9, 12-15, AND 18-22 OF THE '889
`PATENT ARE ANTICIPATED BY SIMMONDS ...................................... 13
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`X. GROUND 2: CLAIMS 16-17 WOULD HAVE BEEN OBVIOUS IN
`VIEW OF SIMMONDS AND BROWN ...................................................... 27
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`XI. GROUND 3: CLAIMS 4, 6, AND 7 WOULD HAVE BEEN
`OBVIOUS IN VIEW OF SIMMONDS AND HEID .................................... 32
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`XII. GROUND 4: CLAIMS 1, 5, 8-9, 12-15, AND 18-22 ARE
`ANTICIPATED BY SYKES......................................................................... 37
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`XIII. GROUND 5: CLAIMS 16-17 WOULD HAVE BEEN OBVIOUS IN
`VIEW OF SYKES AND BROWN ............................................................... 51
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`XIV. GROUND 6 CLAIMS 4, 6, AND 7 WOULD HAVE BEEN
`OBVIOUS IN VIEW OF SYKES AND HEID ............................................. 55
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`XV. OBJECTIVE INDICIA DO NOT SUPPORT PATENTABILITY .............. 58
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`XVI. CONCLUSION .............................................................................................. 63
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`XVII. MANDATORY NOTICES (37 C.F.R. § 42.8(A)(1)) ................................... 64
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`i
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`I.
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`STATEMENT OF THE PRECISE RELIEF REQUESTED AND THE
`REASONS THEREFOR (37 C.F.R. § 42.22(A))
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`Myriad Genetics, Inc., Myriad Genetic Laboratories, Inc. (collectively,
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`"Myriad"), Bio-Rad Laboratories, Inc., and RainDance Technologies, Inc.
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`(collectively, "Petitioners") respectfully petition for Inter Partes Review, and seek
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`cancellation of claims 1, 4-9, 12-22 of USPN 7,824,889 (the "'889 patent")
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`(MYR1001) as unpatentable for anticipation and/or obviousness. The '889 patent
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`is assigned to The Johns Hopkins University (hereinafter "Patent Owner").1
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`II. OVERVIEW
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`Claims 1, 4-9, and 12-22 of the '889 patent should be canceled as anticipated
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`and/or obvious. MYR1002, ¶¶20-22. Independent claims 1 and 19 recite a method
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`that the Patent Owner calls "digital PCR." MYR1002, ¶¶10-19. The figure below
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`shows the basic steps of the method, which involve distributing a DNA sample into
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`compartments such that each compartment contains, ideally, one or zero molecules
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`of DNA from the sample, carrying out PCR in each compartment, and then
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`1 Petitioners note that the Ex Parte Reexamination Certificate for the '889
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`patent lists the NIH and DHHS as assignees, rather than JHU. Nonetheless, in
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`litigation asserting the '889 patent against Myriad, JHU stated, "The '889 patent is
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`assigned to and owned by JHU." MYR1031, ¶114.
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`analyzing the resulting amplified DNA molecules, to determine how many
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`compartments contain each different template DNA molecule
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`IPR of USPN 7,824,889
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`MYR1018, 541.
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`
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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 '889 patent.2
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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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`2 The earliest application to which the '889 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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`2
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`IPR of USPN 7,824,889
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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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`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
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`did nothing more than add a snappy name to the prior art method of LDPCR.
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`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
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`absolute DNA quantitation. It is based on the use of a qualitative all-
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`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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`IPR of USPN 7,824,889
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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
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`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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`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 '889
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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
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`called "digital PCR." Notably, while much of this paper is reproduced in the
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`IPR of USPN 7,824,889
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`specification of the USPN 6,440,706 patent ("the '706 patent"), which the '889
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`patent incorporates by reference, 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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`which the '889 patent claims priority, however, 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."
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`As the 1999 PNAS paper admits, there were "several precedents" to "digital
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`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 '889 Patent for
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`which inter partes review is sought is invalid as anticipated and/or obvious over
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`these precedents. No secondary considerations or objective indicia can save the
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`challenged claims (§XV).
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` If anything,
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`the secondary consideration of
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`simultaneous invention supports cancellation of all of the challenged claims.
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`III. THE '889 PATENT DISCLOSURE AND CLAIMS
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`The '889 patent, titled "Digital Amplification," issued on 2/23/2007, from
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`App. No. 11/709,742, filed on 2/23/2007. MYR1001. The '889 patent claims
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`priority to provisional App. No. 60/146,792 filed 8/2/1999.
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`IPR of USPN 7,824,889
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`The '889 Claims. The '889 patent has 22 claims, 18 of which are challenged
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`here.2 Claims 1 and 19 are the only independent claims of the '889 patent, both of
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`which are challenged here. Claim 1 is exemplary and provided below, as amended
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`during the ex parte reexamination:
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`Claim 1. A method for determining an allelic imbalance in a
`
`biological sample, comprising the steps of:
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`distributing isolated nucleic acid template molecules to form a set
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`comprising a plurality of assay samples, wherein the nucleic acid
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`template molecules are isolated from the biological sample;
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`amplifying the template molecules within [a] the set [comprising a
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`plurality of assay samples] to form a population of amplified
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`molecules in [each of the] individual assay samples of the set[,
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`wherein the template molecules are obtained from a biological
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`sample];
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`analyzing the amplified molecules in the assay samples of the set to
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`determine a first number of assay samples which contain a selected
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`genetic sequence on a first chromosome and a second number of assay
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`samples which contain a reference genetic sequence on a second
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`chromosome, wherein between 0.1 and 0.9 of the assay samples yield
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`an amplification product of at least one of the selected and the
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`reference genetic sequences;
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`2 Petitioners do not concede the validity of any of the unchallenged claims.
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`IPR of USPN 7,824,889
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`comparing the first number of assay samples to the second number of
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`assay samples to ascertain an allelic imbalance in the biological
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`sample.
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`IV. THE '889 FILE HISTORY AND REEXAMINATION FILE HISTORY
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`The Mullis chapter and Simmonds were not before the USPTO during initial
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`prosecution of the '889 patent. MYR1005. Sykes was disclosed, but never
`
`discussed. Id. To overcome a double patenting rejection over the '706 patent, the
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`applicants submitted a terminal disclaimer. MYR1044. Therefore, the prosecution
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`history of the '706 patent is relevant to the '889 patent.
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`The '706 and '889 patents were the subject of ex parte reexamination
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`proceedings, during which multiple claims were amended to overcome rejections
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`over the prior art. MYR1002, ¶¶5-6, 43-44; MYR1008; MYR1009. Although
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`Simmonds and Sykes were nominally before the Patent Office during the ex parte
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`reexamination proceedings, the Mullis chapter was not before the Patent Office,
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`and Simmonds and Sykes were never discussed during those proceedings.
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`MYR1002, ¶¶5-6, 43-44. Instead, the proceedings focused on different prior art,
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`which involved the distribution of whole cells, rather than isolated nucleic acids,
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`into compartments. The claims were amended to specify that the method involves
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`"isolated" or "cell-free" nucleic acids rather than whole cells in light of this art.
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`MYR1008, 7/9/2014 Amendment, 2; MYR1036; MYR1009, 7/9/2014 Responsive
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`7
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`IPR of USPN 7,824,889
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`Amendment to Final Office Action. While these amendments addressed the prior
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`art discussed during the ex parte reexamination, they did nothing to address the
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`Mullis chapter, Simmonds, Sykes or other prior art references discussed in this
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`petition for inter partes review.
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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
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`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 '889 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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`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
`
`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). MYR1002, ¶27.
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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
`
`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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`IPR of USPN 7,824,889
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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
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`quantitative amplification techniques, detection, and analysis. MYR1002, ¶¶27-28.
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`VI. CLAIM CONSTRUCTION
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`In accordance with 37 C.F.R. § 42.100(b), the challenged claims must be
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`given their broadest reasonable interpretations (BRI) in light of the specification
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`and prosecution history of the '889 patent. MYR1002, ¶48. Since the '706 and
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`'889 Reexamination Certificates were issued, Patent Owner has asserted the
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`challenged '889 (and '706) patent claims against Myriad's myRisk diagnostic test
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`for hereditary cancer risk. Esoterix Genetic Laboratories, LLC and The Johns
`
`Hopkins University v. Myriad Genetics, Inc. et al., Civil Action No. 1:16-cv-
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`01112-WO-JEF (M.D.N.C) (MYR1032). In the Myriad litigation, Patent Owner
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`has accused of infringement the same dilution and distribution steps found in the
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`Mullis chapter and the Simmonds and Sykes prior art references, and is effectively
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`seeking to re-patent the prior art. Given that Patent Owner has adopted a claim
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`construction in the Myriad litigation that requires only dilution and/or distribution,
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`PCR amplification, and any analysis of the PCR products (which construction was
`
`not before the USPTO during the ex parte reexamination proceedings), it is beyond
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`9
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`reasonable dispute that the challenged claims are invalid over the prior art and
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`IPR of USPN 7,824,889
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`should be cancelled by the PTAB. MYR1002, ¶¶45-47.
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`A. The Preambles Are Not Limiting
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`Under the BRI of Claims 1 and 19 of the '889 Patent, the preambles "[a]
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`method for determining an allelic imbalance in a biological sample" should be
`
`construed as non-limiting. These preambles do not recite any structure or step
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`needed to give meaning and life to the claims, or to any dependent claims. See,
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`e.g., Summit 6, LLC v. Samsung Electronics Co., Ltd., 802 F.3d 1283, 1292 (Fed.
`
`Cir. 2015) ("[g]enerally, a preamble is not limiting"); TomTom, Inc. v. Adolph, 790
`
`F.3d 1315, 1323 (Fed. Cir. 2015). No term in Claims 1 or 19, or in any claim that
`
`depends from them, refers back to these preambles, which therefore do not provide
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`any antecedent basis for the body of the claims. Instead, the steps of the claimed
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`method stand independent of these preambles, and the structure they recite is in no
`
`way dependent on any preamble language.
`
`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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`MYR1002, ¶¶49; see Summit, 802 F.3d at 1292; TomTom, 790 F.3d at 1323.
`
`B.
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`"allelic imbalance" and "allele"
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`Under the BRI, a POSA would understand that the term "allelic imbalance"
`
`should be construed to require a "loss of or an increase in copy number of one
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`10
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`IPR of USPN 7,824,889
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`allele relative to the other allele or different allele," wherein "allele" refers to "one
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`of various alternative forms of a gene or genomic sequence." MYR1002, ¶¶50.
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`These proposed constructions are consistent with
`
`the
`
`intrinsic evidence,
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`MYR1001, 1:49-51; 2:64-67; 5:24-29, and were previously agreed to by Patent
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`Owner in the litigation Esoterix Genetic Laboratories, LLC and The Johns
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`Hopkins University v. Life Technologies Corp., et al., Civil Action No. 1:12-cv-
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`01173-CCE-JEP (M.D.N.C.), where Patent Owner asserted the '889 Patent against
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`various defendants (not including Myriad). MYR1032, 2.
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`VII. IDENTIFICATION OF THE CHALLENGE (37 C.F.R. § 42.104(B))
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`Petitioners respectfully petition for inter partes review of claims 1, 4-9, and
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`12-22 of the '889 patent based on the unpatentability grounds summarized in the
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`index below. Per 37 C.F.R. § 42.6(c), copies of the cited references accompany
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`this Petition.
`
`Ground
`
`35 U.S.C. § (pre-
`AIA)
`
`§ 102
`
`§ 103
`
`§ 103
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`§ 102
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`§ 103
`§ 103
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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
`6
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`
`
`
`
`Claims
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`Reference(s)
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`1, 5, 8-9, 12-15, 18-
`22
`
`Simmonds
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`16-17
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`4, 6-7
`1, 5, 8-9, 12-15, 18-
`22
`16-17
`4, 6-7
`
`Simmonds and
`Brown
`Simmonds and Heid
`
`Sykes
`
`Sykes and Brown
`Sykes and Heid
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`11
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`IPR of USPN 7,824,889
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`The "Simmonds" reference (MYR1012) was published in February 1990 and
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`is prior art to the '889 patent under at least 35 U.S.C. §102(b). The "Sykes"
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`reference (MYR1013) was published in 1992 and is prior art to the '889 patent
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`under at least 35 U.S.C. § 102(b). The "Brown" reference (MYR1015) was filed
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`on 4/17/1997 and issued on 11/7/2000, and is prior art to the '889 patent under at
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`least 35 U.S.C. § 102(e). The Heid reference (MYR1024) was published in 1996,
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`and is prior art to the '889 patent under at least §102(b).
`
`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, ¶56. 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
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`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
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`POSAs knew long before August 2, 1999 that PCR was a powerful tool for
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`quantitation of DNA. MYR1002, ¶¶30-35. For example, by the late 1980s and
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`
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`12
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`IPR of USPN 7,824,889
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`early 1990s, POSAs knew that some of the potential pitfalls of PCR related to its
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`exponential nature – potentially biased amplification of certain sequences over
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`others and artifacts that could arise from using multiple different pairs of primers
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`in a single reaction – could be avoided
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`through
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`the use of parallel,
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`compartmentalized PCR reactions, carried out at limiting dilution. See MYR1014,
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`68; MYR1002, ¶¶32-35. Indeed, as the 1994 Mullis chapter demonstrates,
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`multiple research groups were carrying out and publishing LDPCR methods by the
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`early 1990s. Id.
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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
`
`example, Brown disclosed a platform to perform a multitude of LDPCR assays in
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`parallel in an efficient manner, and Heid disclosed the use of non-polymorphic
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`markers in the context of quantitative PCR analysis. MYR1002, ¶¶33; 129.
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`IX. GROUND 1: CLAIMS 1, 5, 8-9, 12-15, AND 18-22 OF THE '889
`PATENT 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, 5, 8-9, 12-15, and 18-22. MYR1002, ¶¶20, 57-108.
`
`A.
`
`Independent Claim 1
`
`Claim
`1. A method of
`determining an
`
`Disclosure in Simmonds (MYR1012)
`This non-limiting preamble is nonetheless disclosed:
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`
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`13
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`
`
`Claim
`allelic imbalance
`in a biological
`sample,
`comprising the
`steps of:
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`IPR of USPN 7,824,889
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`Disclosure in Simmonds (MYR1012)
`"Here we describe a modified PCR method with a sensitivity
`sufficient to detect a single molecule of target DNA. . . . By
`combining this double PCR method with a limit dilution
`approach, both the proportion of infected cells and the number
`of molecules of HIV provirus per cell can be accurately
`estimated. . . .The approach described here also provides a
`way to analyze sequence heterogeneity within populations of
`HIV provirus which avoids the complications that arise
`because of the lack of fidelity of the Taq polymerase (33) and
`other artifacts associated with amplification." (865)
`
`"Separate amplification of individual molecules from a
`mixture after dilution and distribution. If the double PCR
`can detect single DNA molecules, then 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
`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)
`
`"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. Each sample contained
`(nominally) 6.5 ag of pBH10.R3 (0.5 molecules) and 60 ag of
`lambda HAT 3 (1.2 molecules)." (867, Figure 3 legend)
`
`"Limiting-dilution PCR products from patients 75, 76, and 79,
`amplified with gag primers, were sequenced directly by using
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`
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`14
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`IPR of USPN 7,824,889
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`Disclosure in Simmonds (MYR1012)
`primer 883. . . . These dilutions gave a low frequency of
`positive reactions (Table 1) and . . . the probability was high
`that single molecules would be amplified. . . . 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); 76 (4/16), and 79 (4/14),
`representing a total of 38 assay samples.
`See preamble element above.
`
`"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)
`
`"FIG. 3. Separate amplification of single molecules from a
`mixture. . . . The first PCR contained the env and gag outer
`primers. A single sample of each PCR product was then
`amplified with radiolabeled inner env and gag primers." (867,
`Fig. 3 legend)
`
`See also preamble element above.
`
`
`
`Claim
`
`(a) distributing
`isolated nucleic
`acid template
`molecules to form
`a set comprising a
`plurality of assay
`samples, wherein
`the nucleic acid
`template
`molecules are
`isolated from the
`biological sample;
`(b) amplifying the
`template
`molecules within
`[a] the set
`[comprising a
`plurality of assay
`samples] to form a
`population of
`amplified
`molecules [each of
`the] individual
`assay samples of
`the set [, wherein
`the template
`molecules are
`obtained from a
`biological
`
`
`
`15
`
`

`

`
`
`Claim
`sample];
`(c) analyzing the
`amplified
`molecules in the
`assay samples of
`the set to
`determine a first
`number of assay
`samples which
`contain a selected
`genetic sequence
`on a first
`chromosome and a
`second number of
`assay samples
`which contain a
`reference genetic
`sequence on a
`second
`chromosome,
`wherein between
`0.1 and 0.9 of the
`assay samples
`yield an
`amplification
`product of at least
`one of the selected
`and the reference
`genetic sequences;
`
`IPR of USPN 7,824,889
`
`Disclosure in Simmonds (MYR1012)
`
`"Separate amplification of individual molecules from a
`mixture after dilution and distribution….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)…." (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." (p.
`867, Fig. 3 legend)
`
`"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.
`
`
`
`16
`
`

`

`IPR of USPN 7,824,889
`
`Disclosure in Simmonds (MYR1012)
`See step (c) above.
`
`are
`contain provirus
`lymphocytes which
`"If most
`transcriptionally silent, it might be because the provirus that
`they carry is defective. …Using the dilution, distribution, and
`double PCR method to amplify single sequences, we have
`determined a 180-bp sequence from the gag region of 19
`single provirus molecules from six patients who were
`probably infected with the same virus (19) (7 of these are
`illustrated in Table 4). Among the sequences, there are 16
`variable sites, i.e., sites at which one or more of the sequences
`differs from the consensus of all of them….Similarly, analysis
`of a 170-bp sequence from the env region of 24 single
`molecules from four patients revealed 49 variable sites and a
`minimum of eight independent deletions or insertions…."
`(870)
`
`
`
`Claim
`(d) comparing the
`first number of
`assay samples to
`the second number
`of assay samples
`to ascertain an
`allelic imbalance
`in the biological
`sample.
`
`
`
`Claim 1, preamble. To the extent the preamble is found to be limiting,
`
`Simmonds discloses it. MYR1002, ¶¶60-61. Simmonds discloses a method "to
`
`analyze sequence heterogeneity within populations of HIV provirus" by comparing
`
`two variants of the same viral gene from a biological sample. See MYR1012, 865.
`
`This method includes "separate amplification of individual molecules from a
`
`mixture after dilution and distribution." MYR1012, 867. Simmonds discloses how
`
`to determine the ratio of a given HIV provirus variant 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 discloses
`
`how to determine that out of 28 assay samples, 9 contained one provirus variant
`
`
`
`17
`
`

`

`IPR of USPN 7,824,889
`
`
`(i.e. genetic sequence) and 13 contained the other provirus variant (i.e. different
`
`genetic sequence). Id. Simmonds showed that these ratios reflected the ratios of
`
`the two variants (i.e. genetic sequences) in the original sample, namely that one
`
`variant was present at about twice the amount of the other, which was in good
`
`agreement with how the mixtures were originally created. MYR1012, Fig. 3
`
`legend. Simmonds discloses the use of sequence analysis to determine a ratio of a
`
`selected genetic sequence in a population of genetic sequences in a biological
`
`sample. 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 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.
`
`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 discloses amplifying the provirus template molecules in the assay
`
`samples of the set using PCR to form a population of amplified provirus molecules
`
`
`
`18
`
`

`

`
`in 38 assay samples of the set and 56 assay samples of the set. MYR1012, 868-
`
`IPR of USPN 7,824,889
`
`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.
`
`MYR1012, 867. Gel electrophoresis analysis showed that one provirus variant had
`
`been amplified in 9/28 (0.3) of the assay samples, while the other provirus variant
`
`had been amplified in 13/28 (0.5) of the assay samples. MYR1012, 867.
`
`Therefore, between 0.1 and 0.9 of the assay samples yielded an amplification
`
`product.
`
`Simmonds 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 to determine that 2/8 (0.3)
`
`for patient 75 contained one variant, 4/16 (0.3) for patient 76 contained more than
`
`one variant, and 4/14 (0.3) for patient 79 contained more than one variant.
`
`MYR1012, 868-869. Therefore, between 0.1 and 0.9 of the assay samples yielded
`
`an amplification product.
`
`Simmonds discloses that "[i]n order to quantify the amount of provirus, 12
`
`