Declaration of Robert Nussbaum
` 
`
`IN THE UNITED STATES PATENT AND TRADEMARK OFFICE
`
`In re Patent of: Chuu et al.
`U.S. Patent No.: 8,318,430, claims 19-30
`Issue Date: 27 November 2012
`U.S. Serial No.: 13/368,035
`Filing Date: 7 February 2012
`
`Title: METHODS OF FETAL ABNORMALITY DETECTION
`
`I, Robert Nussbaum, declare as follows:
`I. INTRODUCTION
`
`1)
`
`I am the Holly Smith Distinguished Professor of Medicine and Chief of the Division of
`
`Genomic Medicine in the Department of Medicine at the University of California, San
`
`Francisco (“UCSF”) and a member of the UCSF Institute of Human Genetics. I received my A.
`
`B. from Harvard College in 1971 and my M.D. from the Harvard-MIT Joint Program in Health
`
`Sciences and Technology. I studied Internal Medicine at Washington University & Barnes
`
`Hospital and Medical Genetics at the Baylor College of Medicine. I am familiar with DNA
`
`sequencing technology including massively parallel DNA sequencing methods used for
`
`detecting genetic disorders or for determining genetic characteristics. I have published
`
`numerous scientific papers and lectured extensively on human genetics.
`
`2)
`
`I have been retained by Ariosa Diagnostics to provide my expert opinions regarding
`
`United States Patent No. 8,318,430 (“the ‘430 patent”). More specifically, I have been asked to
`
`give my opinion about the meanings of certain terms that were introduced into the ‘430 claims,
`
`explain the differences between random and targeted DNA sequencing approaches, and
`
`compare the methods of the ‘430 claims to methods described by prior patents and publications.
`
`I submit this declaration in support of Ariosa’s Petition for Inter partes Review.
`
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`Declaration of Robert Nussbaum
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`II. EXPERT QUALIFICATIONS AND CREDENTIALS
`
`3)
`
`I have worked in the fields of medicine, pediatrics, and medical genetics and
`
`diagnosis for over 30 years. A copy of my Curriculum Vitae, including a list of my publications,
`
`is attached as Nussbaum Exhibit A.
`
`4)
`
`I am the Holly Smith Distinguished Professor of Medicine and Chief of the
`
`Division of Genomic Medicine in the Department of Medicine at the University of California,
`
`San Francisco (“UCSF”) and a member of the UCSF Institute of Human Genetics.
`
`5)
`
`I received my A. B. from Harvard College in 1971 and my M.D. from the
`
`Harvard-MIT Joint Program in Health Sciences and Technology.
`
`6)
`
`I studied Internal Medicine at Washington University & Barnes Hospital, and
`
`Medical Genetics at the Baylor College of Medicine.
`
`7)
`
`From 1981-1984, I was Assistant Professor of Medicine, Baylor College of
`
`Medicine. From 1983-1993 I was an Associate Investigator, Howard Hughes Medical Institute.
`
`From 1984 to 1989 I was also an Assistant Professor of Human Genetics & Pediatrics, Univ. of
`
`Pennsylvania and later from 1989-1993 an Associate Professor of Human Genetics &
`
`Pediatrics, Univ. of Pennsylvania. In 1993 I was elevated to Professor of Genetics, Univ. of
`
`Pennsylvania and in 1994 to Chief, Genetic Disease Research Branch, National Human Genome
`
`Research Institute within NIH where I served until 2006. Concurrently from 1996-2006 I was
`
`Acting Chief of the Inherited Disease Research Branch, NHGRI.
`
`8)
`
`From 2006 I have been the Holly Smith Chair and Professor of Medicine; Chief,
`
`Genomic Medicine; Member, Institute of Human Genetics, UCSF; Professor of Neurology, at
`
`UCSF, and from 2007 to 2012 I served on the Executive Committee of the Biomedical Graduate
`
`Studies Graduate Program and Medical Scientist Training Program Council.
`
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`Declaration of Robert Nussbaum
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`9)
`
`I am familiar with DNA sequencing technology including massively parallel
`
`DNA sequencing methods used for determining genetic characteristics or for detecting genetic
`
`disorders.
`
`10)
`
`I have published numerous scientific papers on chromosomal anomalies, genetic
`
`analysis and sequencing a sampling of which includes:
`
`Bale, S, Rehm, HL, Nussbaum, RL, Hegde, M, Den Dunnen, J, and Willems,
`P. MutaDATABASE: a centralized and standardized DNA variation database.
`Nat. Biotechnol 29, 117-118, 2011.
`
`Bothwell SP, Farber LW, Hoagland A, Nussbaum RL. Species-specific
`difference
`in expression and splice-site choice
`in Inpp5b, an
`inositol
`polyphosphate 5-phosphatase paralogous to the enzyme deficient in Lowe
`Syndrome. Mamm Genome: 21: 458-463, 2010.
`
`Kuo Y-M, Li Z Jiao, Gaborit N, Pan AK, Orrison BM, Bruneau BG, Giasson
`BI, Smeyne RJ, Gershon MD and Nussbaum RL. Extensive enteric nervous
`system abnormalities in mice transgenic for artificial chromosomes containing
`Parkinson disease-associated α-synuclein gene mutations precede central nervous
`system changes. Hum Molec Genet, 19(9):1633-50, 2010.
`
`
`Sotiriou S, Gibney G, Andreas D. Baxevanis AD and Nussbaum RL. A
`
`single nucleotide polymorphism in the 3'UTR of the SNCA gene encoding alpha-
`synuclein is a new potential susceptibility locus for Parkinson disease.
`Neuroscience Letters, 461(2):196-201, 2009.
`
`
`
`McFarland MA, Ellis CE, Markey SP, Nussbaum RL. Proteomic analysis
`identifies phosphorylation-dependent α-synuclein protein interactions. Mol Cell
`Proteomics, 7(11):2123-37, 2008.
`
`
`
`Entezam A, Biacs R, Orrison B, Saha T, Hoffman GE, Grabczyk E,
`Nussbaum RL and Usdin K. Large Repeat expansions in a new Fragile X
`premutation mouse model. Gene. 395:125-34, 2007.
`
`
`
`Golovko MY, Rosenberger TA, Faergeman NJ, Feddersen S, Cole NB,
`Pribill I, Berger J, Nussbaum RL and Murphy EJ Acyl-CoA synthetase activity
`links wild-type but not mutant α-synuclein to brain arachidonate metabolism.
`Biochemistry, 45:6956-6966, 2006.
`
`
`
`Ellis CE, Murphy EJ, Mitchell DC, Golovko MY, Scaglia F, Barcelo-
`Coblijn GC and Nussbaum RL. Mitochondrial Lipid Abnormality and Electron
`
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`Transport Chain Impairment in Mice Lacking α-synuclein. Mol. Cell. Biol,
`25:10190-10201, 2005.
`
`
`
`Meyer-Lindenberg A, Kohn PD, Kolachana B, Kippenham S, McInerney-
`Leo, A, Nussbaum R, Weinberger DR and Berman KF. Midbrain dopamine
`synthesis and prefrontal cortical function are related in humans in vivo and
`modulated by COMT genotype, Nature Neuroscience 8:594-6, 2005.
`
`
`
`Chiba-Falek, O, Kowalak, JA, Smulson, ME, and Nussbaum, RL.
`Regulation of α-synuclein expression by Poly (ADP ribose) polymerase-1
`(PARP-1) binding to the NACP-Rep1 polymorphic site upstream of the SNCA
`gene, Amer J Hum Genet, 76(3):478-92, 2005.
`
`
`
`Ulmer, TS, Bax, A. Cole, NB and Nussbaum, RL. Structure and dynamics
`of micelle-bound human a-synuclein, J Biol Chem, 280(10):9595-603, 2005; Cole
`NB, Murphy D, Leibowitz J, diNoto L, Levine RL and Nussbaum RL: Metal-
`catalyzed oxidation of alpha-synuclein: helping to define the relationship between
`oligomers, protofilaments and filaments, J Biol Chem, 280(10):9678-90, 2005.
`
`
`
`Valente EM, Abou-Sleiman PM, Caputo V, Muqit MM, Harvey K,
`Gispert S, Ali Z, Del Turco D, Bentivoglio AR, Healy DG, Albanese A,
`Nussbaum R, Gonzalez-Maldonado R, Deller T, Salvi S, Cortelli P, Gilks WP,
`Latchman DS, Harvey RJ, Dallapiccola B, Auburger G, Wood NW. Hereditary
`early-onset Parkinson's disease caused by mutations in PINK1. Science
`304(5674):1158-60. Epub 2004 Apr 15.
`
`
`III. BASES FOR OPINIONS AND MATERIALS REVIEWED
`
`
`
`11) The opinions set forth in my Declaration are based on my personal knowledge
`
`gained from my education, professional experience, and on the review of the documents and
`
`information described in this declaration.
`
`12)
`
`I have reviewed and am familiar with the following documents:
`
`ARIOSA Exhibit 1001: U.S. Patent No. 8,318,430 to Chuu et al. (“the 430
`patent”).
`
`ARIOSA Exhibit 1004: U.S. Pat No. 7,332,277 to Dhallan (“Dhallan ‘277”).
`
`ARIOSA Exhibit 1005: Binladen et al., PLoS One. 2007 Feb 14;2(2):e197
`(“Binladen”).
`
`ARIOSA Exhibit 1006: U.S. Patent Publication 2007/0202525 to Quake and
`Fan. (“Quake ‘525”).
`
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`ARIOSA Exhibit 1007: Craig, et al. Nat Methods. 2008 October ; 5(10): 887–
`893 (“Craig”).
`
`ARIOSA Exhibit 1008: U.S. Patent Publication 2008/0090239 to Shoemaker et
`al., (“Shoemaker”).
`
`
`
`IV. SCIENTIFIC AND TECHNOLOGICAL BACKGROUND
`
`13) Deoxyribonucleic acid or “DNA” is the hereditary material in humans and all
`
`other organisms except for certain viruses. The information in DNA is stored as a code made
`
`up of four chemical bases: adenine (A), guanine (G), cytosine (C), and thymine (T). DNA
`
`bases pair up with each other, A with T and C with G, to form units called base pairs. Each
`
`base is attached to a sugar molecule and each sugar molecule to a phosphate molecule.
`
`Together, a base, sugar, and phosphate are called a nucleotide. Nucleotides are arranged in two
`
`long strands that form a spiral called a double helix. The structure of the double helix is
`
`somewhat like a ladder with the base pairs forming the ladder’s rungs and the sugar and
`
`phosphate molecules forming the vertical sidepieces (“side rails”) of the ladder. The entirety
`
`of the DNA present in an individual human is referred to as a human genome.
`
`14) The order, or sequence, of the nucleotides in DNA forms a genetic “code”,
`
`similar to the way in which letters of the alphabet appear in a certain order to form words and
`
`sentences. The genetic code provides the informational basis for the biological functions
`
`necessary for human development and maintenance. By understanding the order of bases in
`
`DNA one can identify specific genes, protein structures, and regulatory elements involved in
`
`the biological processes of an individual. The goal of DNA sequencing techniques is to
`
`determine the order of the bases in a DNA region, thereby providing information on the genetic
`
`code of an individual or population. Determination of DNA sequences has become
`
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`indispensable in numerous applied fields in biotechnology, including diagnostics, forensic
`
`biology, and biological systematics.
`
`15)
`
`In many circumstances, analysis of DNA sequences is focused on a subset of
`
`DNA molecules. The DNA sequence of interest can be enriched from a sample containing it
`
`and other DNA sequences using various methods, including amplification methods or other
`
`DNA replication methods that increase the number of copies of the DNA sequence of interest
`
`in a sample to assist in subsequent analysis of the enriched sequence.
`
`16) DNA replication and amplification techniques used to enrich a particular
`
`sequence can be performed using enzymes called “DNA polymerases” that catalyze these
`
`processes and a short strand of nucleotides that is complementary to and selectively hybridizes
`
`with the DNA sequence of interest called a “primer”. The hybridized primer serves as a
`
`starting point for DNA synthesis of the template DNA and is required because DNA
`
`polymerases can only add new nucleotides to an existing strand of DNA. In fact, all
`
`amplification and replication techniques require the hybridization of a complementary primer
`
`to the template DNA of interest to enable the replication of a strand of DNA. The polymerase
`
`starts replication at the 3'-end of the primer, and copies the opposite strand of single-stranded
`
`DNA template. Each replication process produces a copy of the opposite strand of a single-
`
`stranded DNA molecule.
`
`17) The most common amplification method used to selectively enrich DNA of
`
`interest from a sample is the polymerase chain reaction (“PCR”) amplification. Two primers
`
`containing sequences complementary to one or the other strand of DNA and flanking the target
`
`region are hybridized to the template DNA and a heat-stable DNA polymerase is used to
`
`extend the primers across the target region. This amplification method relies on thermal
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`cycling, consisting of cycles of repeated heating and cooling of the reaction for DNA melting
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`and enzymatic replication of the DNA. As PCR progresses, the DNA generated is itself used
`
`as a template for replication, setting in motion a chain reaction in which the DNA template is
`
`exponentially amplified.
`
`18) Once DNA molecules have been enriched, they can be analyzed using a variety
`
`of techniques. Single molecules can be identified using “sequencing” to determine the order or
`
`sequence of the bases in an enriched DNA molecule.
`
`19) The original techniques for determining the sequence of bases in DNA were
`
`developed in the 1970’s. Increased demand for low-cost sequencing has driven the
`
`development of faster and more efficient high-throughput, massively-parallel or “next-
`
`generation sequencing” techniques. Such massively parallel sequencing techniques permit the
`
`accurate counting of millions of single DNA molecules isolated or generated from a sample.
`
`The first massively parallel sequencing technique was described in 2005 with the publication
`
`of the sequencing-by-synthesis technology developed by 454 Life Sciences, Inc. (Margulies, et
`
`al., Nature, 437:376-80 (2005), Nussbaum Exhibit B) and the multiplex colony sequencing
`
`protocol by George Church’s lab (Shendure, et al., Science, 309:1728-32 (2005), Nussbaum
`
`Exhibit C). These strategies used several hundred thousand sequencing templates arrayed in
`
`picotiter plates or agarose thin layers, so that the sequences can be analyzed in parallel. Since
`
`that time, additional methods and systems have been developed that allow for massively
`
`parallel sequencing including pyrosequencing, as commercialized by 454 Life Sciences;
`
`sequencing by ligation, as commercialized in the SOLiD™ technology, Life Technology, Inc.,
`
`Carlsbad, CA;
`
`sequencing-by-synthesis methods using modified nucleotides,
`
`as
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`commercialized in TruSeq™ and HiSeq™ technology by Illumina, Inc., San Diego, CA; PacBio
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`RS™ by Pacific Biosciences of California, Inc., Menlo Park, CA; sequencing by ion detection
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`technologies, as commercialized by Ion Torrent, Inc., South San Francisco, CA; sequencing of
`
`DNA nanoballs, commercialized by Complete Genomics, Inc., Mountain View, CA; and
`
`nanopore-based sequencing technologies, as developed by Oxford Nanopore Technologies,
`
`LTD, Oxford, UK.
`
`20) Once improvements in sequencing techniques allowed the quantitative analysis
`
`of individual DNA molecules, complementary biochemical analysis techniques and new
`
`efficiencies of sample processing were introduced to enhance the use of such sequencing
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`systems. The simultaneous analysis of multiple samples was achieved by uniquely identifying
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`DNA from a particular source or individual by “tagging” or “indexing” DNA molecules from
`
`that source to construct a tagged or indexed library of that source’s or individual’s DNA
`
`fragments. Each DNA molecule in the indexed library is thus associated with a unique index
`
`designating the molecule as being from a particular source or individual. Since different
`
`libraries are identified by different DNA tags, they can be pooled and more economically and
`
`efficiently sequenced in a single reaction. After sequencing of the pooled DNA from different
`
`libraries, the sequences from the different libraries can easily be de-convoluted using the
`
`unique tags that identify an individual’s DNA. This use of multiplexed indexed sequencing
`
`greatly improved the efficiency and scalability of analysis of multiple samples simultaneously,
`
`and improved productivity by reducing research time, effort, and reagent use.
`
`21)
`
`In fact, as of 2008, indexed multiplexing was so widespread as a technique that
`
`the company Illumina, Inc. offered a commercially available kit for production and analysis of
`
`indexed libraries from different samples of origin. This kit enabled the sequencing of 96
`
`different samples on a single flow cell and was designed to be used with Illumina’s
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`multiplexed sequencing platform, the Illumina Genome Analyzer™. According to the Illumina
`
`documentation (Nussbaum, Exhibit D), “To make multiplexed sequencing on the Genome
`
`Analyzer available to any laboratory, Illumina offers the Multiplexing Sample Preparation
`
`Oligonucleotide Kit and the Multiplexing Sequencing Primers and PhiX Control Kit.” The
`
`Illumina documentation (Exhibit D) graphically illustrates the multiplexing process. Various
`
`possible uses of this kit with the sequencing platform are discussed, including “resequencing of
`
`targeted regions in many individuals” for association with human disease.
`
`22)
`
`Following sequencing on the Genome Analyzer, Illumina’s commercially-
`
`available Pipeline Analysis software provided de-convolution of the combined sequencing
`
`technology: “Using Illumina’s Pipeline Analysis software, each index is associated with a
`
`particular read-pair, identifying samples for downstream analysis.”
`
`23)
`
`So, as a molecular geneticist in 2008, I would have had the ability to order a
`
`commercially available kit for production of enriched and indexed libraries, which I could have
`
`analyzed on a commercially-available massively parallel sequencing platform sold by the same
`
`vendor. Moreover, the software for sample identification and de-convolution of the
`
`multiplexed sequencing data would have been provided with the multiplexing kit and
`
`sequencing platform.
`
`24) On the diagnostic front, DNA sequencing had been used to develop tests to
`
`identify overabundance or under representation of certain fetal chromosomes (aneuploidy)
`
`using a peripheral blood sample from a pregnant woman. In the 1990¹s it was discovered that
`
`cell-free fetal DNA was present in maternal blood plasma and serum in concentrations high
`
`enough to be readily detectable (Kazakov, et al., Cytology, 37(3):232-236 (1995), Nussbaum
`
`Exhibit F; Lo, et al., The Lancet, 350:485-87 (1997), Nussbaum Exhibit E). As early as 2002,
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`scientists were using the then-available sequencing techniques to analyze targeted cell free
`
`DNA fragments in maternal plasma to identify fetal abnormalities including aneuploidies. In
`
`particular, regions containing single nucleotide polymorphisms on putative aneuploidy
`
`chromosomes were targeted and compared to targeted regions on control chromosomes.
`
`Aneuploidies that were tested include trisomies, where an individual has three copies of a
`
`particular chromosome instead the normal (euploid) two copies. Some of the more common
`
`trisomies include those of chromosomes 13 (Patau syndrome), 18 (Edwards syndrome) and 21
`
`(Down syndrome). These initial techniques using cell-free DNA did not use massively parallel
`
`sequencing, because it was not yet available.
`
`25) As reviewed below, these massively parallel sequencing techniques became
`
`available and I am aware of four different, commercially-available noninvasive prenatal
`
`diagnostic tests that detect abnormal distribution of a fetal chromosome. All four of these tests
`
`utilize the same massively parallel sequencing platform, the IlluminaHiSeq™, to perform
`
`single molecule counting of maternal and fetal nucleic acid sequences.
`
`26) A person of skill in the art of medical genetics would be familiar with the
`
`information described above.
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`V. COMMENTS ON THE ‘430 PATENT
`
`27) The claims of the ‘430 patent describe a method that indexes or tags DNA
`
`samples obtained from different individuals, pools these indexed DNA samples for sequencing,
`
`and then de-convolutes the sequence information using the index or tag to identify DNA from a
`
`particular individual. The ‘430 claims also describe a method of determining aneuploidy by
`
`comparing the relative number of DNA fragments from a putative aneuploid chromosome to
`
`the number of fragments from a control, or reference chromosome. An abnormal ratio of
`
`fragments indicates an aneuploidy, which is characterized by missing or extra chromosomal
`
`DNA compared to a normal, euploid individual.
`
`28)
`
`I have been apprised that a patent applicant is entitled to be his or her own
`
`lexicographer and can set forth a definition of a term that is different from its ordinary and
`
`customary meaning(s). Where an explicit definition is provided by the applicant, then that
`
`definition of the term will control interpretation of the term as it is used in a claim. I have also
`
`been instructed that a claim should be read in the context of the specification and drawings, and
`
`that any special meaning assigned to a term must be sufficiently clear so that any departure
`
`from common usage would be clearly understood by a person of experience in the relevant
`
`scientific field. I therefore looked at both the definitions and other description in the
`
`specification for the meaning of the terms used in the claims.
`
`29) To construe the meanings of these terms, I have considered them based on how
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`one within the field of medical genetics or prenatal diagnosis would have understood them
`
`when read in light of the ‘430 disclosure and within the context of how these terms were used
`
`in the ‘430 claims.
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`“Selectively enrich”
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`30) The term “selectively enrich” is not explicitly defined in the specification. I
`
`have therefore based my interpretation on the common usage of the term and the context of this
`
`phrase in the ‘430 specification and claims. Examples of the meaning of this phrase are found
`
`in the specification at Col. 2, lines 40-43:
`
`In another embodiment, said selectively enriching comprises performing PCR. In
`another embodiment, said selectively enriching comprises linear amplification.
`
`In both of these stated embodiments, the phrase “selective enriching” is exemplified by a
`
`method that selectively creates copies of a particular DNA sequence. The experimental section
`
`of the ‘430 patent only describes the use of PCR for enrichment to create multiple copies of
`
`sequences. No other methods are mentioned that could be used for selective enrichment. The
`
`‘430 specification describes enrichment of DNA from “hot spots” which are selected regions in
`
`the genome as described at Col 14, lines 63-64:
`
`FIG. 1 illustrates a strategy for selecting sequences from chromosome 21 for
`enrichment.
`
`Thus, upon reading the ‘430 patent, a person in the field of medical genetics would have
`
`interpreted the term “selective enrichment” in the ‘430 patent to mean “amplification of a
`
`selected genomic region.”
`
`“generate a library derived from each fetal and maternal cell-free genomic
`DNA sample of enriched and indexed fetal and maternal non-random
`polynucleotide sequence”
`
`31) As described above, I interpret the term “selectively enriched” to mean “amplified
`
`from a selected genomic region.” So it follows that the generation of a library derived from
`
`each DNA sample would be created by amplification of selected DNA regions, where the
`
`selective amplification process is used to add an index that identifies the source (such as a
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`particular patient) of the enriched DNA sample. Since the enriched and indexed products are
`
`created by a selective amplification process,
`
`the products would be non-random
`
`polynucleotides.
`
`“Chromosome control region”
`
`32) In the specification, the term “chromosome control region” is used to describe
`
`segments from chromosomes that can actually serve as comparisons to a segment of a
`
`chromosome being tested for aneuploidy (Ex. 1001, 13:6-8, Fig. 8B).
`
`33) In order for such a comparison to be meaningful, the chromosome control region must
`
`be a segment from a chromosome not being tested for aneuploidy. Therefore, I interpret this
`
`term as including a segment of a chromosome not being tested for aneuploidy and which is
`
`presumed prior to testing to be diploid.
`
`
`
`“sequence reads corresponding to enriched and indexed fetal and maternal
`non-random polynucleotide sequences”
`
`34) The phrase “corresponding to” is not used anywhere in the specification in the context
`
`of describing a sequence read and in fact this language only occurs in the claims. The only
`
`sequence reads described are those that reflect the actual sequence of the amplified
`
`polynucleotide sequences, which are the genomic sequences of the cell-free DNA used as an
`
`amplification template. The central description of the claimed methods supports this reading of
`
`the claim because the Summary of the Invention clearly describes the sequence reads as being
`
`produced by sequencing the enriched polynucleotide sequences. The ‘430 states at Col. 1, lines
`
`40-48:
`
` 
`
`In one aspect, a method for determining the presence or absence of fetal
`aneuploidy
`is provided comprising a) selectively enriching non-random
`polynucleotide sequences of genomic DNA from a cell-free DNA sample; b)
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`sequencing said enriched polynucleotide sequences; c) enumerating sequence
`reads from said sequencing step; and d) determining the presence or absence of
`fetal aneuploidy based on said enumerating.
`
`
`Therefore, I interpret the term “sequence read” as the determined sequence of the enriched
`
`polynucleotide.
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`VI. COMPARISON OF THE ‘430 CLAIMS TO THE PRIOR ART
`
`A. ALL OF THE ELEMENTS OF CLAIMS 19-30 OF THE ‘430 PATENT ARE
`DISCLOSED BY DHALLAN US PAT NO. 7,332,277
`(“DHALLAN”)
`IN
`COMBINATION WITH BINLADEN ET AL., PLOS ONE. 2007 FEB 14;2(2):E197
`(“BINLADEN”).
`
`
`35) It is my opinion that the combination of Dhallan and Binladen teaches all the elements
`
`of these claims and believe that one skilled in the art would have had impetus to combine these
`
`techniques because the combination would have clearly provided enhanced productivity and
`
`increased throughput of sample analysis. The sequencing and multiplexing technology of
`
`Binladen would have made the sequencing procedure of Dhallan less expensive, faster and more
`
`efficient because one could sequence indexed samples from many different patients in a single
`
`sequencing run instead of laboriously performing a single sequencing run for the DNA samples
`
`from each patient. Each section of claim 19 or dependent claims 20-30 is presented in bold text
`
`followed by my analysis of that part of the claim.
`
`Claim 19: A method for determining a presence or absence of a fetal
`aneuploidy in a fetus for each of a plurality of maternal blood samples
`obtained from a plurality of different pregnant women, said maternal blood
`samples comprising fetal and maternal cell-free genomic DNA, said method
`comprising:
`
`36) The preamble of Claim 19 of the ‘430 patent is disclosed in Dhallan which states at
`
`Col. 25 line 63 through Col. 26, line 6:
`
`The present invention provides a method for detecting genetic disorders,
`including but not limited to mutations, insertions, deletions, and chromosomal
`abnormalities, and is especially useful for the detection of genetic disorders of a
`fetus. The method is especially useful for detection of a translocation, addition,
`amplification,
`transversion,
`inversion, aneuploidy, polyploidy, monosomy,
`trisomy, trisomy 21, trisomy 13, trisomy 14, trisomy 15, trisomy 16, trisomy 18,
`trisomy 22, triploidy, tetraploidy, and sex chromosome abnormalities including
`but not limited to XO, XXY, XYY, and XXX.
`
` 
`
`15
`
`Ariosa Exhibit 1003, p. 15
`IPR2013-00277
`
`

`
`Declaration of Robert Nussbaum
` 
`
`
`Clearly, Dhallan discloses determining the presence or absence of fetal aneuploidies.
`
`
`
`(a) obtaining a fetal and maternal cell-free genomic DNA sample from each
`of the plurality of maternal blood samples;
`
`37) The first step of Claim 19 of the ‘430 patent is disclosed by Dhallan in combination
`
`with Binladen. Claim 19 recites, “for each of a plurality of maternal blood samples obtained
`
`from a plurality of different pregnant women…”. Dhallan at Col. 5, lines 39-41 states: “Fetal
`
`DNA has been detected and quantitated in maternal plasma and serum (Lo et al., Lancet
`
`350:485-487 (1997); Lo et al., Am. J. hum. Genet. 62:768-775 (1998))”. Dhallan discloses
`
`maternal serum and plasma, which is a blood sample from a pregnant woman. Further, Dhallan
`
`at Col. 219, lines 57-63 states:
`
`Blood samples were received from 27 different clinical sites operating in 16
`different states located throughout the U.S. Blood samples were collected from
`both women carrying male and female fetuses, however, here, we report results
`obtained from woman carrying male fetuses, as the Y chromosome is the accepted
`marker when quantitating percentages of fetal DNA.
`
`As evident, Dhallan obtained blood samples from a plurality of different pregnant women.
`
`38) Binladen also discloses gathering a plurality of samples. At page 2, Binladen recites,
`
`“Currently, the method provides a means for the simultaneous sequencing, generation of single
`
`molecule sequences, and assignment of short (120 bp) from homologous PCR products obtained
`
`from multiple individuals.”
`
`
`39) Dhallan discloses the various elements found in the first two phrases of step (b):
`
`(b) selectively enriching a plurality of non-random polynucleotide sequences
`of each fetal and maternal cell-free genomic DNA sample of (a) to generate a
`library derived from each fetal and maternal cell-free genomic DNA sample
`of enriched and indexed fetal and maternal non-random polynucleotide
`sequences, wherein each library of enriched and indexed fetal and maternal
`
` 
`
`16
`
`Ariosa Exhibit 1003, p. 16
`IPR2013-00277
`
`

`
`Declaration of Robert Nussbaum
` 
`
`non-random polynucleotide sequences includes an indexing nucleotide
`sequence which identifies a maternal blood sample of the plurality of
`maternal blood samples,
`
`Dhallan at Col. 32, lines 22-25 states:
`
`
`
` In another embodiment, the template DNA is obtained from the plasma or serum
`of the blood of the pregnant female. The percentage of fetal DNA in maternal
`plasma is between 0.39-11.9% (Pertl and Bianchi, Obstetrics and Gynecology 98:
`483-490 (2001)).
`
`Further, Dhallan at Col. 219, lines 57-63 states:
`
`Blood samples were received from 27 different clinical sites operating in 16
`different states located throughout the U.S. Blood samples were collected from
`both women carrying male and female fetuses, however, here, we report results
`obtained from woman carrying male fetuses, as the Y chromosome is the accepted
`marker when quantitating percentages of fetal DNA.
`
`Thus, Dhallan discloses use of cell-free DNA and obtaining blood samples from a plurality of
`
`different pregnant women.
`
`40) Dhallan discloses at Col. 7, lines 54-63:
`
`In another embodiment, the sequence of one to tens to hundreds to thousands of
`loci of interest on the template DNA obtained from a sample of a pregnant female
`is determined… In some embodiments, determining the sequence of the alleles
`comprises amplifying alleles of a locus of interest on a template DNA using a first
`and a second primer….
`
`Dhallan also discloses at Col. 47, lines 38-40: “The template DNA [maternal and fetal DNA]
`
`can be amplified using any suitable method known in the art including but not limited to PCR
`
`(polymerase chain reaction)…”. Thus, Dhallan teaches enrichment (amplification) of a plurality
`
`of non-random polynucleotide sequences (specific loci of interest).
`
`41) Dhallan recites at Col. 48, line 64 through Col. 49, line 4:
`
`The multiple primer sets will amplify the loci of interest, such that a minimal
`amount of template DNA is not limiting for the number of loci that can be
`detected... low concentrations of each primer set can be used in a first
`amplification reaction to amplify the loci of interest.
`
`
` 
`
`17
`
`Ariosa Exhibit 1003, p. 17
`IPR2013-00277
`
`

`
`Declaration of Robert Nussbaum
` 
`
`Dhallan thus teaches using multiple primer sets to amplify loci of interest to create a library of