Declaration of Cynthia Casson Morton
`In re: Chuu, et al., USPN 8,318,430
` 
`
`
`
`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, Cynthia Casson Morton, declare as follows:
`Credentials
`
`1) Currently I hold the position of William Lambert Richardson Professor in the Department
`
`of Obstetrics, Gynecology and Reproductive Biology at Harvard Medical School. I am
`
`also a Professor in the Department of Pathology at Harvard Medical School and the
`
`Director of Cytogenetics at Brigham and Women's Hospital.
`
`
`
`2) I received my B.S. in Biology from The College of William and Mary and my Ph.D. in
`
`Human Genetics from the Medical College of Virginia.
`
`
`
`3) I am board-certified by the American Board of Medical Genetics in PhD Medical
`
`Genetics, Clinical Cytogenetics and Clinical Molecular Genetics. I have received
`
`numerous awards including the Warner-Lambert/Parke-Davis Award of the American
`
`Society of Investigative Pathology. I am the recent past Editor of The American Journal
`
`of Human Genetics and serve/have served on the Editorial Boards of the American
`
` 
`
`1
`
`Ariosa Exhibit 1002, pg. 1
`IPR2013-00277
`
`

`
`Declaration of Cynthia Casson Morton
`In re: Chuu, et al., USPN 8,318,430
` 
`
`Journal of Medical Genetics, Genomics, Journal of Experimental Zoology, Genes,
`
`Chromosomes and Cancer, American Journal of Human Genetics, Human Genetics,
`
`International Journal of Clinical and Experimental Pathology, The Application of
`
`Clinical Genetics, Audiology and Neurotology, International Journal of Women’s Health,
`
`and Cancer Management and Research. I also serve on various scientific/medical
`
`committees, including the Board of Directors of the American Society of Human
`
`Genetics, the Council of Scientific Trustees of the Hearing Health Foundation and the
`
`Genomic Medicine Program Advisory Committee for the Veteran's Administration.
`
`
`
`4) I have published over 260 peer-reviewed manuscripts in the field of obstetrics,
`
`reproductive biology, auditory science and human medical genetics, serve as an editor of
`
`the book Current Protocols in Human Genetics and have contributed fourteen book
`
`chapters. In addition, I am an author of over 280 abstracts presented at various scientific
`
`meetings worldwide.
`
`
`
`5) I consider myself to be well-versed in the field of medical genetics, including prenatal
`
`diagnostics. My research interests include the biology of uterine leiomyomata, the
`
`biology of hearing and deafness, gene mapping and clinical and molecular cytogenetics.
`
`I have been the Director of a clinical cytogenetics laboratory since 1987, and have had
`
`long-term involvement in prenatal diagnostics. I am knowledgeable with all major
`
`developments in the field. I am the senior author on a recent publication of the first use
`
`of whole genome sequencing in the prenatal setting for diagnosis of a genetic syndrome
`
` 
`
`2
`
`Ariosa Exhibit 1002, pg. 2
`IPR2013-00277
`
`

`
`Declaration of Cynthia Casson Morton
`In re: Chuu, et al., USPN 8,318,430
`
`(N. Engl. J. Med., 367:2226—32 (2012)). My curriculum vitae is attached hereto as
`Exhibit 1014
`Morton Exhibit A.
`
`6)
`
`I have reviewed and am familiar with the following documents: U.S. Patent No.
`
`8,318,430 to Chuu et al.
`
`(“the ‘430 patent”, Petition Exhibit 1001); U.S. Pat No.
`loo KI
`7,332,277 to Dhallan (“Dhallan”, Petition Exhibit H302’); U.S. Patent Publication
`gookr
`2007/0202525 to Quake and Fan (“Quake”, Petition Exhibit 1003); U.S. Patent
`, o 0 8
`Publication 2008/0090239 to Shoemaker, et al., (“Shoemaker”, Petition Exhibit 4-004);
`
`Craig et al. Nat Methods. 2008 October; 5(10): 887-893 (“Craig”, Petition Exhibit 1005); l 0 U 7
`
`Binladen et al., PloS ONE. 2007 Feb 14;2(2):e197 (“Binladen”, Petition Exhibit 1-006); ‘O0?
`
`Complaint filed in Verinata Health, Inc. et al. v. Ariosa Diagnostics, Inc. et al., Civil
`
`Action No. 12-05501-SI (N.D. Cal) ( ); Patent Owner’s opposition to
`
`motion to dismiss in Verinata v. Ariosa
`
`Lachnical Background: Selective Enrichment and Seguencing Technigues
`
`7) Deoxyribonucleic acid, or “DNA” is the material that transfers genetic characteristics in
`
`all higher organisms. DNA is constructed of two nucleotide strands coiled around each
`
`other in a ladder-like arrangement called a double helix, with the internal rungs of the
`
`helix being formed by pairs of nucleotides, or “bases”.’ DNA is further organized into
`
`larger structures called chromosomes, and the full set of chromosomes in an organism is
`
`referred to as that organism’s genome. The genome represents the basis for all the
`
`inheritable traits of an organism, and the order of bases in the DNA of the genome forms
`
`the blueprint for all biological functions of the organism.
`Exhibit 1015
`Nature, 422:835-47 (2003), Morton Exhibit B).
`
`(Generally, see Collins, et al.,
`
`Ariosa Exhibit 1002, pg. 3
`|PR2013—OO277
`
`Ariosa Exhibit 1002, pg. 3
`IPR2013-00277
`
`

`
`Declaration of Cynthia Casson Morton
`In re: Chuu, et al., USPN 8,318,430
` 
`
`
`
`8) Over the past two decades, scientists working in the disciplines of genetics and genomics
`
`have determined the sequence of genes from the genomes of many organisms, as well as
`
`determined the entire sequence of an organism’s genome. (Collins 2003, supra, Morton
`
`Exhibit B) In doing so, for example, the sequences of genes from different individuals of
`
`the same species can be compared and the sequences of genes from different species can
`
`be compared. (Collins 2003, supra, Morton Exhibit B) Although it is of scientific
`
`interest to look at entire genomes, very often it is desirable to look at only selected
`
`regions in a genome. Because the genome of an organism is very large compared to
`
`selected regions of interest such as genes, in order to be able to analyze only selected
`
`regions it is usually necessary to increase the amount of the selected regions of interest in
`
`comparison to the rest of the sample, to isolate the selected regions of interest from the
`
`rest of the sample, or to do both (Erlich, et al., Science, 252(5013):1643-51 (1991),
`
`Morton Exhibit C).
`
`
`
`9) Enrichment of selected nucleic acids in a sample—that is, enrichment of selected regions
`
`of interest as compared to the rest of the genome—can be performed by any method that
`
`results in an increased copy number of the selected nucleic acids relative to the other
`
`nucleic acids in the sample. A common feature of all selective enrichment techniques is
`
`the hybridization of sequence-specific segments of DNA to the selected nucleic acids.
`
`Commonly used methods for selectively enriching nucleic acids are selective
`
`amplification techniques, including linear amplification or exponential amplification.
`
`Both of these selective amplification methods result in an increase in copy number of the
`
` 
`
`4
`
`Ariosa Exhibit 1002, pg. 4
`IPR2013-00277
`
`

`
`Declaration of Cynthia Casson Morton
`In re: Chuu, et al., USPN 8,318,430
` 
`
`selected nucleic acids originally found in the sample. This increase in copy number of
`
`the selected nucleic acid regions facilitates analysis by increasing the amount of material
`
`available for such analysis.
`
`
`10) A first example of selective amplification is polymerase chain reaction (“PCR”)
`
`amplification, which can increase the copy number of the selected nucleic acids into
`
`millions of copies. (Erlich 1991, supra, Morton Exhibit C) The PCR amplification
`
`method relies on thermal cycling—cycles of repeated heating and cooling of the sample
`
`nucleic acids to denature and enzymatically replicate the selected nucleic acids. In the
`
`amplification process, the sequence-specific segments of DNA used to hybridize to the
`
`selected nucleic acids are generally referred to as “primers.” (Erlich 1991, supra, Morton
`
`Exhibit C) Once the primers are allowed to hybridize to the selected nucleic acids, a
`
`reaction is carried out using a heat-stable DNA polymerase to extend the primer along the
`
`length of the selected nucleic acids. (Erlich 1991, supra, Morton Exhibit C) After a
`
`period of time—typically minutes—the polymerization or copying process is halted by
`
`cooling the reaction, then the reaction is initiated once more by heating. As this cycle of
`
`heating and cooling is repeated, copies of the selected nucleic acids that are generated in
`
`each cycle are themselves copied in subsequent cycles, setting in motion a cyclical
`
`reaction in which the selected nucleic acids are amplified exponentially. (Erlich 1991,
`
`supra, Morton Exhibit C)
`
`
`
`11) Another example of selective amplification is rolling circle amplification (“RCA”). In
`
`RCA, the sequence-specific primer hybridizes to a selected nucleic acid, and the free end
`
`of the primer anneals to a small circular DNA template. (Generally, see Lizardi, et al.,
`
` 
`
`5
`
`Ariosa Exhibit 1002, pg. 5
`IPR2013-00277
`
`

`
`Declaration of Cynthia Casson Morton
`In re: Chuu, et al., USPN 8,318,430
` 
`
`Nature Genetics, 19 July 1998:225-32, Morton Exhibit D) A DNA polymerase is added
`
`to extend the primer to copy the selected nucleic acid. The DNA polymerase extends the
`
`primer continuously around the circular DNA template generating a long DNA product
`
`that consists of many repeated copies of the selected nucleic acid. By the end of the
`
`reaction, the polymerase generates many thousands of copies of the selected nucleic acid,
`
`with the chain of copies tethered to the original target DNA. (Lizardi 1998, supra,
`
`Morton Exhibit D) RCA allows for spatial resolution of the selected nucleic acid and
`
`rapid amplification of signal. RCA can result in a linear amplification process, or the use
`
`of forward and reverse primers can change a linear RCA reaction into an exponential
`
`process. (Lizardi 1998, supra, Morton Exhibit D)
`
`
`
`12) The selective amplification methods described in ¶¶ 10 and 11, above, are but two
`
`examples of selective enrichment of nucleic acids through amplification. Since the
`
`advent of PCR in the late 1980s, many variations of selective amplification have been
`
`developed, most of which rely on sequence-specific primers and on thermal cycling.
`
`Such additional selective amplification methods
`
`include strand displacement
`
`amplification (SDA) (see U.S. Pat. Nos. 5,270,184; and 5,422,252, Morton Exhibits E
`
`and F), transcription-mediated amplification (TMA) (see U.S. Pat. No. 5,399,491, Morton
`
`Exhibit G), linked linear amplification (LLA) (see U.S. Pat. No. 6,027,923, Morton
`
`Exhibit H), self-sustained sequence replication (see Guatelli et al., Proc. Nat. Acad. Sci.
`
`USA, 87, 1874 (1990), Morton Exhibit I), consensus sequence primed polymerase chain
`
`reaction (CP-PCR) (see U.S. Pat. No. 5,437,975, Morton Exhibit J), and isothermal
`
` 
`
`6
`
`Ariosa Exhibit 1002, pg. 6
`IPR2013-00277
`
`

`
`Declaration of Cynthia Casson Morton
`In re: Chuu, et al., USPN 8,318,430
` 
`
`amplification methods such as SDA (see Walker et al., Nucleic Acids Res. 20(7):1691-6
`
`(1992), Morton Exhibit K).
`
`
`
`13) When performing selective amplification methods, it is generally desirable to be able to
`
`selectively amplify more than one selected nucleic acid from a sample at a time, a
`
`technique referred to as multiplexing. (For a review, see Fan, et al., Nature Reviews
`
`Genetics, 7:632-44) (2006), Morton Exhibit L) In multiplexed amplification such as
`
`multiplex PCR, multiple sequence-specific primer sets are used in a single reaction to
`
`amplify multiple selected nucleic acids from a sample. Multiplex PCR was first
`
`described in 1988 as a method to detect deletions in the DMD gene (Chamberlain, et al.,
`
`Nuc. Acids Res., 16(23):11141-56 (1988), Morton Exhibit M), and has since been used
`
`ubiquitously in genetic research for such applications as pathogen identification, forensic
`
`studies, mutation analysis, linkage analysis, single nucleotide polymorphism genotyping,
`
`and paternity analysis (Edwards and Gibbs, PCR Methods and Applications, 3:S65-S75
`
`(1994), Morton Exhibit N and Fan 2006, supra, Morton Exhibit L). Commercial
`
`multiplexing kits for PCR are widely available. Multiplexing can also refer to selective
`
`amplification and analysis of nucleic acids from multiple samples, in which the samples
`
`are combined prior to sequencing and analysis.
`
`14) A second method for selective enrichment of nucleic acids from a sample is hybridization
`
`followed by separation of the hybridized nucleic acids from non-hybridized nucleic. In
`
`such selective hybridization techniques, the sequence-specific segments of DNA used to
`
`hybridize to the selected nucleic acids are generally referred to as “probes”, and in many
`
`methods the probes are immobilized on a planar surface (often referred to as an array) or
`
` 
`
`7
`
`Ariosa Exhibit 1002, pg. 7
`IPR2013-00277
`
`

`
`Declaration of Cynthia Casson Morton
`In re: Chuu, et al., USPN 8,318,430
` 
`
`to a bead or other solid support. (See, e.g., Chetverin and Kramer, BioTechnology,
`
`12:1093-99 (1994), Morton Exhibit O.) The sequences of the probes are chosen to
`
`hybridize specifically with selected nucleic acids from the sample, while other nucleic
`
`acids in the sample do not hybridize to the probes. (Chetverin and Kramer 1994, supra,
`
`Morton Exhibit O) Because the probes are immobilized, the selected nucleic acids that
`
`hybridize to them will be immobilized as well, and can be separated from the
`
`unhybridized nucleic acids that are in solution. (Chetverin and Kramer 1994, supra,
`
`Morton Exhibit O)
`
`15) An additional method for selective enrichment combines the techniques of selective
`
`amplification and selective hybridization, in which selected nucleic acids from a sample
`
`are first amplified using sequence-specific primers, and where the sequence-specific
`
`primers also serve as probes to immobilize the amplification products for separation from
`
`the remaining nucleic acids in a sample. (Fan 2006, supra, Morton Exhibit L) The
`
`combination of selective amplification and hybridization allows isolation of highly-
`
`enriched selected nucleic acids from a nucleic acid sample containing, e.g., the DNA
`
`from an entire genome. The combination of selective amplification and hybridization are
`
`described, for example, in Fan 2006, supra (Morton Exhibit L).
`
`
`16) Once selected nucleic acids have been enriched, they typically are analyzed by, e.g.,
`
`DNA sequencing. The goal of sequencing DNA is to determine the order of bases of the
`
`selected nucleic acids. Within the past 6-7 years, “massively parallel sequencing” (also
`
`known as “next generation sequencing” or “NGS”), has been developed and become
`
`commercially available (see reviews of next generation sequencing technologies in
`
`Metzker, Nature Reviews, 11:31-46 (2010), Morton Exhibit P, and Kircher and Kelso,
`
` 
`
`8
`
`Ariosa Exhibit 1002, pg. 8
`IPR2013-00277
`
`

`
`Declaration of Cynthia Casson Morton
`In re: Chuu, et al., USPN 8,318,430
`
`Exhibit 1030
`Bioessays, 322524-36 (2010), Morton Exhibit Q). Massively parallel sequencing enables
`
`extremely accurate single molecule counting of literally millions of molecules in parallel,
`
`which allows one to both determine the sequence of selected nucleic acids and quantify
`
`the number of copies of the selected nucleic acids in the amplified mixture. (Metzker
`Exhibits 1029 and 1030
`
`2010, and Kircher and Kelso 2010, supra, Morton Exhibits P and Q) Massively parallel
`
`sequencing methods and systems include pyrosequencing methods, as commercialized by
`
`454 Life Sciences; sequencing-by-ligation methods, as commercialized in the SOLiDTM
`
`technology from Life Technologies, Inc. and by Complete Genomics, Inc., Mountain
`
`View, CA;
`
`sequencing—by-synthesis methods,
`
`as commercialized in the HiSeqTM
`
`technologies by Illumina,
`
`Inc., San Diego, CA;
`
`the PacBio RS system by Pacific
`
`Biosciences of California,
`
`Inc., Menlo Park, CA; and sequencing by ion detection
`
`technologies, as commercialized by Life Technologies, Inc., Carlsbad, CA (Metzker
`Exhibits 1029 and 1030
`
`2010, and Kircher and Kelso 2010, supra, Morton Exhibits P and Q).
`
`17) With the techniques described above, therefore,
`
`it
`
`is possible to perform multiplexed
`
`amplification to amplify multiple selected nucleic acids from a sample,
`
`isolate the
`
`amplified nucleic acids, sequence millions of the amplified nucleic acids in parallel, and
`
`quantify the copies of the amplified nucleic acids.
`
`In fact, the power of massively
`
`parallel sequencing in theory allows for many samples to be pooled together and
`
`analyzed simultaneously. However, if one were to pool amplification reactions from
`
`different samples into one sequencing reaction, it would be difficult to determine which
`
`selected nucleic acids came from which sample (see Binladen, et al., PLoS ONE,
`H305
`2(2):e197.doi:10.1371/journal.pone.0000l97 (2007), Petition Exhibit l-006)). To solve
`
`Ariosa Exhibit 1002, pg. 9
`|PR2013—OO277
`
`Ariosa Exhibit 1002, pg. 9
`IPR2013-00277
`
`

`
`Declaration of Cynthia Casson Morton
`In re: Chuu, et al., USPN 8,318,430
`
`this problem, researchers use coded bits of DNA called “sample tags” or “sample
`
`indices” to identify the origin of the sample. That is, each copy of each selected DNA
`
`fragment in a sample is tagged so that it is possible to identify from which sample a
`
`selected nucleic acid originated.
`
`“Sample tags” or “sample indexing” of multiplexed
`
`DNA samples is described, e. g., in the following references: Binladen, eta1., PLoS ONE,
`,
`_ C,
`2(2):e197.doi:i0.1371/joumal.pone.0000197 (2007), Petition Exhibit ]4§0%;()C)raig, et al.,
`Nat. Methods, 5(10):887-93 (2008), Petition Exhibit V1-‘0g’5‘k;}_I,sizardi, et al., U.S. Pat. No.
`6,329,150,
`R; Strathmann, U.S. Pub. No. 2007/0224613,
`
`S.
`
`18) For example, Binladen, et al., PLoS ONE, 2(2):e197.doi:10.1371/joumal.pone.0000197
`(2007)
`(“Binladen”)
`(Petition Exhibit
`i!6L0bJ)Sgenerally describes performing PCR
`
`amplification of selected nucleic acids from over a dozen different species of animals
`
`using primers that in addition to being complementary to selected nucleic acids, also
`
`contain indices indicative of the sample used as a template for the amplification.
`
`including the sample indices on the primers assured that copies of selected nucleic acids
`
`could be identified as products from a particular sample (i.e., a single species), as each
`
`amplification product from the sample contained the same sample index. Copies of
`
`selected nucleic acids from different samples (i.e., from different species) contained
`
`different, uniquely identifiable sample indices. Once the copies of the selected nucleic
`
`acids were sequenced, the sequences of selected nucleic acids from each sample were
`
`grouped by virtue of their sample index for analysis.
`
`10
`
`Ariosa Exhibit 1002, pg. 10
`|PR2013—OO277
`
`Ariosa Exhibit 1002, pg. 10
`IPR2013-00277
`
`

`
`Declaration of Cynthia Casson Morton
`In re: Chuu, et al., USPN 8,318,430
`
`19) In another example, Craig, et al., Nat. Methods, 5(10):887-93 (2008) (“Craig”) (Petition
`Exhibit I082) iiescribes multiplexed sequencing of selected nucleic acids from different
`
`human individuals using “barcodes” (aka “sample tags” and “sample indices”). Where
`
`Binladen included the sample indices in the primers used to amplify the selected nucleic
`
`acids, Craig attached the sample indices to the copies of the selected nucleic acids by a
`
`process called ligation. Thus, the amplified selected nucleic acids of Craig also contained
`
`sample indices, where copies of selected nucleic acids from the same sample contained
`
`the same sample index, and copies of selected nucleic acids from different samples
`
`contained different, uniquely identifiable sample indices. As in Binladen, once the copies
`
`of the selected nucleic acids were sequenced, the sequences of selected nucleic acids
`
`from each sample could be grouped by virtue of the sample index for further analysis.
`
`Lachnical Background: Prenatal Diagnosis
`20)Prenatal diagnosisiis an established and widely-used clinical practice in modem
`
`obstetrics. Until
`
`recently, however,
`
`the direct analysis of fetal nucleic acids or
`
`chromosomes required invasive sampling of fetal
`
`tissues, e.g., via amniocentesis or
`
`chorionic villus sampling, both of which run the risk of damage to or termination of a
`
`genetically normal fetus.
`
`(See, e.g., Chiu, et al., PNAS, 105(51):20458-63 (2008),
`
`Exhibit 1033
`attached as Morton Exh1b1tT.)
`
`21) In the mid-1990s, it was discovered that cell—free fetal nucleic acids are naturally present
`
`in relatively high concentrations in maternal plasma and serum in normal pregnancies.
`Exhibit 1034
`(Kazakov, et al., Tsitologiia, 37(3):232-36 (1995), attached as Morton Exhibit U; and Lo,
`Exhibit 1035
`et al., Lancet, 3501485-87 (1997), attached as Morton Exhibit V). The relatively high
`
`11
`
`Ariosa Exhibit 1002, pg. 11
`|PR2013—OO277
`
`Ariosa Exhibit 1002, pg. 11
`IPR2013-00277
`
`

`
`Declaration of Cynthia Casson Morton
`In re: Chuu, et al., USPN 8,318,430
` 
`
`concentrations made it commercially practical to conduct prenatal diagnosis in a
`
`noninvasive manner. Id. However, the percentage of cell-free fetal nucleic acids in
`
`maternal plasma or serum can be relatively low in comparison to maternal cell-free fetal
`
`nucleic acids —typically 10% or less—therefore, such fetal nucleic acid sequences are
`
`usually evaluated within a large background of maternal nucleic acids. (See, e.g., Chiu
`
`2008, supra, Morton Exhibit T).
`
`
`
`22) The easiest types of fetal nucleic acid sequences to detect in the background of maternal
`
`DNA are those where the sequences are known a priori to be physically absent in the
`
`mother. (Chiu, et al., Trends in Genetics, 25(7):324-31 (2009), attached as Morton
`
`Exhibit W) Examples include Y chromosome sequences such as SRY for fetal male sex
`
`determination and the RHD gene for blood group genotyping when the mother is known
`
`to have an RhD-negative phenotype. (Lo 1997, supra, Morton Exhibit V and Lo, et al.,
`
`N. Engl. J. Med., 339(4):1734-38 (1998), Morton Exhibit X) The mother is known not to
`
`possess a Y chromosome with SRY, and in mothers who are RhD-negative the RHD gene
`
`is generally absent or grossly deleted. (Lo 1998, supra, Morton Exhibit X) Such fetal-
`
`specific sequences are therefore easily detectable, e.g., with standard PCR methods that
`
`utilize primers specific for those sequences. (Lo 1997, supra, Morton Exhibit V and Lo
`
`1998, supra, Morton Exhibit X) In these cases, if a Y chromosome sequence is present in
`
`the maternal serum or plasma sample, the fetus is typically male; and if the RHD gene is
`
`detected in a sample from an RhD-negative mother with a deletion of RHD, the fetus is
`
`RhD-positive.
`
`
`
` 
`
`12
`
`Ariosa Exhibit 1002, pg. 12
`IPR2013-00277
`
`

`
`Declaration of Cynthia Casson Morton
`In re: Chuu, et al., USPN 8,318,430
`
`23) Detection of cell-free fetal nucleic acid sequences that are present in both the fetus and
`
`the pregnant mother presents different technical challenges, as does the detection of fetal
`Exh1b1t 1036
`
`aneuploidies or other copy number variants.
`
`(See Chiu 2009, supra, Morton Exhibit W)
`
`Detection of fetal sequences that are present in both the fetus and the pregnant mother is
`
`currently possible only through the use of highly precise methods for quantification by,
`
`e.g., amplification of the fetal and maternal nucleic acids in the sample followed by
`
`single molecule sequencing (e. g., NGS) as described in W10-16 herein.
`
`24)I am aware of four different, commercially-available noninvasive prenatal diagnostic
`
`tests that detect fetal aneuploidies.
`
`25)A person of skill in the art of prenatal diagnostics would be familiar with the information
`
`provided in M 7-24, above.
`
`final Aneugloidy Detection Prior Art
`
`26)A first reference which describes elements of the claimed invention in the ‘430 patent is
`
`Dhallan, et al., U.S. Pat. No. 7,332,277, issued 19 February 2008, and published in 2004
`‘~ L
`
`(“Dhallan”), Petition Exhibit l-02O.—UD‘hallan describes methods for quantitating ratios of
`
`alleles at
`
`loci of interest and further
`
`that a difference in ratios may indicate a
`
`chromosomal abnormality. Dhallan teaches that
`
`in some embodiments,
`
`the ratio of
`
`alleles at a locus of interest on one chromosome can be compared to the ratio of alleles at
`
`a locus of interest on another chromosome. Dhallan further teaches determining the
`
`sequence of alleles of a locus of interest in a sample using various methods. Dhallan
`
`13
`
`Ariosa Exhibit 1002, pg. 13
`|PR2013—OO277
`
`Ariosa Exhibit 1002, pg. 13
`IPR2013-00277
`
`

`
`Declaration of Cynthia Casson Morton
`In re: Chuu, et al., USPN 8,318,430
`
`teaches that, in certain embodiments, determining the sequence of alleles of a locus of
`
`interest comprises amplifying alleles of a locus of interest on a template DNA using
`
`primers. Dhallan further teaches that the methods may be used for determination of the
`
`presence or absence of chromosomal abnormalities in a fetus. The correlation between
`
`Dhallan and each element of claims 19-30 of the ‘430 patent are detailed below.
`
`27) Quake, U.S. Pub. No. 2007/0202525, published 30 August 2007 (“Quake ‘525”), Petition
`Exhibit l'0U(?3,0z1Kl’so describes elements of the claimed invention in the ‘430 patent. Quake
`
`‘525 provides non-invasive methods for determination of fetal aneuploidies by digital
`
`analysis. Quake ‘525 demonstrates that when a maternal mixed sample is diluted to a
`
`nominal value of 0.5 genome equivalents per reaction sample, massively parallel
`
`amplification via digital PCR may be performed and the relative number of fetal
`
`chromosomal sequences may be determined, which allows for the determination of the
`
`presence or absence of a fetal aneuploidy. Quake ‘525, in addition to describing digital
`
`PCR, describes the use of next generation sequencing to enumerate fetal sequences in a
`
`maternal sample. The correlation between Quake ‘525 and each element of claims 19-30
`
`of the ‘430 patent are detailed below.
`
`28) Another reference which describes elements of the claimed invention in the ‘430 patent is
`
`Shoemaker, et al., U.S. Pub. No. 2008/0090239, published 17 April 2008 (“Shoemaker”),
`5008
`Petition Exhibit 1004: with a priority date of 14 June 2006.
`
`Shoemaker describes
`
`methods for labeling selected nucleic acid regions—including regions comprising one or
`
`more polymorphisms—of genomic DNA in individual cells with different unique tags
`
`14
`
`Ariosa Exhibit 1002, pg. 14
`|PR2013—OO277
`
`Ariosa Exhibit 1002, pg. 14
`IPR2013-00277
`
`

`
`Declaration of Cynthia Casson Morton
`In re: Chuu, et al., USPN 8,318,430
` 
`
`(i.e., “labels” where each label is specific to a cell). Shoemaker describes methods for
`
`quantifying the labeled selected regions of genomic DNA from each cell. Shoemaker
`
`teaches that the methods may be used for determining the presence or absence of fetal
`
`abnormalities. Shoemaker teaches that, in some embodiments, the selected genomic
`
`regions are amplified prior to being quantified, and that “ultra deep” sequencing can be
`
`used to provide an accurate and quantitative measurement of allele abundance.
`
`Quantitative genotyping using the sequence information obtained can be used to declare
`
`the presence of fetal cells and to determine copy numbers of fetal chromosomes. The
`
`correlations between Shoemaker and each element of claims 19-30 of the ‘430 patent are
`
`detailed below.
`
`
`
`The ‘430 Patent
`
`29) Application No. 13/368,035 (“the ‘035 application”) that issued as the ‘430 patent was
`
`filed on February 7, 2012, as a continuation of and claiming priority to, Application No.
`
`13/012,222, filed on January 24, 2011. Both the ‘035 application and Application No.
`
`13/012,222 claim priority to U.S. provisional application No. 61/297,755, filed on
`
`January 23, 2010.
`
`
`
`30) The independent claims of the ‘430 patent recite the following claim elements: use of a
`
`plurality of maternal and fetal cell free DNA samples for detecting aneuploidies;
`
`enriching and indexing at least 100 non-random genomic DNA sequences of 10-1000
`
`nucleotides in length from each of two different chromosomes from each of the samples;
`
`pooling the enriched and indexed non-random genomic DNA sequences from the
`
` 
`
`15
`
`Ariosa Exhibit 1002, pg. 15
`IPR2013-00277
`
`

`
`Declaration of Cynthia Casson Morton
`In re: Chuu, et al., USPN 8,318,430
` 
`
`samples; sequencing the pooled, enriched and indexed non-random genomic DNA
`
`sequences from the samples; and comparing the number of non-random genomic DNA
`
`sequences from a chromosome region tested for aneuploidy with a chromosome control
`
`region from each sample to determine the presence or absence of an aneuploidy.
`
`
`31) I have been instructed that the claim elements, unless a special and particular definition is
`
`provided, should be afforded their ordinary and accustomed meaning. I have also been
`
`instructed that the plain and ordinary meaning must be supported by the description of the
`
`term as found in the patent and discussion of the terms by the Inventors during
`
`prosecution of the patent. I have thus interpreted the elements of the ‘430 patent claims
`
`following these guidelines. For example, the only forms of selective enrichment taught
`
`or described in the ‘430 patent are selective amplification techniques that increase the
`
`concentration of a selected molecule relative to other molecules; thus, the analysis I have
`
`performed has focused on selective amplification as exemplary of selective enrichment.
`
`
`In another example, the phrase “sequence reads corresponding to enriched and indexed
`
`fetal and maternal non-random polynucleotide sequences” is used in step d of claim 19.
`
`“Sequence reads” as taught in the ‘430 refers to the order of nucleotides determined from
`
`sequencing selectively enriched and indexed polynucleotides; that is, “sequence reads”
`
`are informational. However, I have interpreted the term “enriched and indexed non-
`
`random polynucleotide sequences” to mean a nucleic acid molecule that has been
`
`selectively enriched and indexed, such as a nucleic acid molecule that has been amplified
`
`using tagged primers that amplify a specific region in a genome. In contrast to the word
`
`“sequence” in “sequence reads”, the word “sequence” in the term “non-random
`
` 
`
`16
`
`Ariosa Exhibit 1002, pg. 16
`IPR2013-00277
`
`

`
`Declaration of Cynthia Casson Morton
`In re: Chuu, et al., USPN 8,318,430
` 
`
`polynucleotide sequences” refers to a physical molecule; a non-random enriched and
`
`indexed fetal or maternal nucleic acid.
`
`
`32) As described below, it is my opinion that all three of the following combinations of art
`
`teach each and every element of claims 19-30 of the ‘430 patent: Dhallan and Binladen,
`
`Quake and Craig, and Shoemaker, Dhallan and Binladen. Binladen and Craig are
`
`described briefly in relation to multiplexing and indexing at ¶¶18-19, above. Dhallan,
`
`Quake and Shoemaker are described briefly in relation to prenatal diagnostics at ¶¶26-28,
`
`above.
`
`
`
`The techniques of claims 19-30 of the ‘430 patent would have been obvious to any skilled
`
`artisan reading Dhallan U.S. Pat No. 7,332,277 in view of the general knowledge concerning
`
`indexed PCR techniques, as reflected in Binladen et al., PloS ONE. 2007 Feb 14;2(2):e197
`
`33) I have reviewed U.S. Pat. No. 7,332,277 to Dhallan (“Dhallan”) and Binladen et al., PloS
`
`ONE. 2007 Feb 14;2(2):e197 (“Binladen”). It is my view that the combination of
`
`Dhallan and Binladen discloses each element of Claims 19-30 of the ‘430 patent when
`
`the teachings of Dhallan are considered in view of the general knowledge concerning
`
`indexed PCR techniques at the time of filing, as reflected in the Binladen reference. I
`
`will discuss each equivalent element of the claims in more detail below.
`
`
`
`34) The first element of Claim 19 of the ‘430 patent is disclosed in Dhallan. Claim 19
`
`recites, “A method for determining a presence or absence of a fetal aneuploidy in a
`
`fetus…” Dhallan states at 25:63-26:6, “The present invention provides a method for
`
` 
`
`17
`
`Ariosa Exhibit 1002, pg. 17
`IPR2013-00277
`
`

`
`Declaration of Cynthia Casson Morton
`In re: Chuu, et al., USPN 8,318,430
` 
`
`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, triso