throbber
Second Declaration of Cynthia Casson Morton
`IPR2013-000276
`
`
` IN THE UNITED STATES PATENT AND TRADEMARK OFFICE
`
`In re Patent of: Chuu, et al.
`
`U.S. Patent No.: 8,318,430, claims 1-18
`
`Issue Date: 27 November 2012
`
`U.S. Serial No.: 13/368,035
`
`Filing Date: 7 February 2012
`
`
`
`Title: METHODS OF FETAL ABNORMALITY DETECTION
`
`
`
`
`
`Second Declaration of Cynthia Casson Morton
`
`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. My curriculum vitae was
`
`submitted previously in this proceeding as Exhibit 1014.
`
`
`
`2)
`
`I have reviewed and am familiar with the following documents:
`
`
`
`
`
`1
`
`Ariosa Exhibit 1042
`pg. 1
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`

`

`Second Declaration of Cynthia Casson Morton
`IPR2013-000276
`
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`
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`
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`
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`
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`U.S. Patent No. 8,318,430 to Chuu et al. [“the ‘430 patent”, Ex. 1001];
`
`U.S. Pat No. 7,332,277 to Dhallan [“Dhallan”, Ex. 1004];
`
`US Patent Publication 2008/0090239 to Shoemaker, et al., [“Shoemaker”,
`
`Ex. 1008];
`
`Binladen et al., PLoS One. 2007 Feb 14;2(2):e197 [“Binladen”, Ex.
`
`1005];
`
`
`
`Decision: Institution of Inter Partes Review [Paper 11 in IPR2013-
`
`00276];
`
`
`
`Decision: Institution of Inter Partes Review [Paper 10 in IPR2013-
`
`00277];
`
`Verinata Health, Inc.’s Patent Owner Response Pursuant to 37 C.F.R.
`
`§42.120 [Paper 19 in IPR2013-00276];
`
`Verinata Health, Inc.’s Patent Owner Response Pursuant to 37 C.F.R.
`
`§42.120 [Paper 19 in IPR2013-00277];
`
`Declaration of Dr. Atul Butte for IPR2013-00276 [Ex. 2003];
`
`Declaration of Dr. Atul Butte for IPR2013-00277 [Ex. 2003]; and
`
`Transcript of the Deposition of Dr. Atul Butte [Ex. 1041].
`
`
`
`2
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`
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`
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`
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`
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`
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`Ariosa Exhibit 1042
`pg. 2
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`

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`Second Declaration of Cynthia Casson Morton
`IPR2013-000276
`
`
`
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`I.
`
`SUMMARY OF MY OPINIONS
`
`3)
`
`As described in detail below, it is my opinion that a person of ordinary
`
`skill in the art, upon reading the Binladen, Dhallan, and Shoemaker
`
`references, would have had both the technical knowledge and the motivation
`
`to combine the methods disclosed therein. In particular, they would have
`
`been motivated to utilize these combined teachings to develop a multiplexed
`
`massively parallel sequencing (MPS) method for determining fetal
`
`aneuploidy using enriched, non-random polynucleotides from cell-free DNA
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`found in pregnant women’s plasma or serum.
`
`4)
`
`Although the individual steps recited in claims 1-18 of the ‘430 patent
`
`may not be found in a single reference, a person of ordinary skill in the art in
`
`2009 would immediately understand the benefits of using multiplexing
`
`MPS, such as the methods taught in both Binladen and Shoemaker, with the
`
`locus-based assays for detection of fetal aneuploidy, such as the assays
`
`taught in Dhallan and Shoemaker. The use of cell-free DNA for
`
`determination of fetal aneuploidy was also quite well known, and a person of
`
`ordinary skill would have understood the benefits of using cell-free DNA
`
`generally obtained through a non-invasive procedure. Because employing
`
`multiplexed MPS methods using enriched DNA assays to detect fetal
`
`
`
`3
`
`Ariosa Exhibit 1042
`pg. 3
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`

`

`Second Declaration of Cynthia Casson Morton
`IPR2013-000276
`
`
`aneuploidy in multiple samples would provide immediate benefits such as
`
`increased efficiency, speed, and a decreased cost of sequencing per sample,
`
`a person of ordinary skill would naturally have been motivated to combine
`
`the subject matter of these references to arrive at the invention as described
`
`in claims 1-18 of the ‘430 patent.
`
`5)
`
`Moreover, in my view the technical hurdles described by Verinata’s
`
`expert, Dr. Atul Butte, would not have prevented a person of ordinary skill
`
`in the art from combining the teachings of the combined references or to
`
`doubt the expected success of utilizing multiplexing with MPS to determine
`
`fetal aneuploidy [infra at ¶¶ 44-46]. Specifically, it was known in 2009 that
`
`multiplexing could be used with MPS, and in fact it was so commonplace
`
`that kits to do so were commercially available [infra at ¶¶ 38 and 40, see
`
`also Exhibit 1010]. It was also known that MPS worked perfectly well on
`
`cell-free DNA extracted from plasma samples of pregnant women, and
`
`laboratories such as Dr. Stephen Quake’s laboratory at Stanford University
`
`and Dr. Dennis Lo’s laboratory at the Chinese University of Hong Kong had
`
`published articles using MPS to quantitate maternal and fetal cell-free DNA
`
`for detection of fetal aneuploidies [see, e.g., Ex. 1033; Ex. 1036; Ex. 1011
`
`and Ex. 1045]. Finally, the use of enriched loci from the cell-free DNA in a
`
`pregnant woman’s plasma had been used to detect fetal aneuploidies, as
`
`
`
`4
`
`Ariosa Exhibit 1042
`pg. 4
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`

`

`Second Declaration of Cynthia Casson Morton
`IPR2013-000276
`
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`demonstrated by the work of Dhallan and his collaborators [see, e.g., Ex.
`
`1004]. The other technical hurdles that Dr. Butte argues would have
`
`precluded combining the exemplary techniques of Dhallan, Binladen and
`
`Shoemaker are, in my opinion, irrelevant.
`
`6)
`
`Accordingly, based on the state of the art
`
`in 2009, the
`
`commercially-available systems for massively parallel sequencing
`
`(“MPS”), the commercially available kits available for multiplexing on
`
`such MPS systems, and various publications in the field of medical
`
`genetics and prenatal diagnosis, a person of ordinary skill could have
`
`employed multiplexed MPS as the detection method for the fetal
`
`aneuploidy assay methods taught in Dhallan and Shoemaker with a
`
`reasonable expectation that combining these methods would be
`
`successful.
`
`
`
`II. A PERSON OF ORDINARY SKILL IN THE RELEVANT FIELD
`
`7)
`
`Dr. Butte, in his declaration [Ex. 2003], sets forth his view of a person
`
`of ordinary skill in the relevant field:
`
`“In my opinion, a person of ordinary skill in the relevant
`
`field in January 2010 would include someone with a
`
`master’s or Ph.D degree in molecular biology, genetics, or
`
`a related field and, through either education or work
`
`
`
`5
`
`Ariosa Exhibit 1042
`pg. 5
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`

`

`Second Declaration of Cynthia Casson Morton
`IPR2013-000276
`
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`experience, about 2-3 years of experience with nucleic
`
`acid sequencing, sample preparation, and prenatal
`
`diagnostics.”
`
`8)
`
`Importantly, a person of ordinary skill in in the relevant fields
`
`would have knowledge about the performance of the MPS systems
`
`themselves, as opposed to just experience analyzing the data produced
`
`from such sequencing systems. Knowledge of factors that affect the
`
`performance of these sequencing systems, as well as methods for
`
`adapting or troubleshooting the biochemistry associated with these
`
`systems also is, in my view, important, as it informs the person of
`
`ordinary skill on the technical challenges that may be imposed by
`
`modification of sequencing methods.
`
`9)
`
`The conclusions I reached in my previous declaration—and the
`
`views expressed in this declaration—are consistent with the expertise
`
`and knowledge of a person of ordinary skill in the art.
`
`10)
`
`I am acquainted with Dr. Butte from his time as an Instructor in
`
`Pediatrics at Children’s Hospital in Boston. I know him to be very
`
`knowledgeable of bioinformatics, medical informatics and data analysis.
`
`However, to my knowledge, Dr. Butte has not worked in or supervised a
`
`biochemical or “wet lab”, and has never performed or directly supervised the
`
`technical aspects of nucleic acid sequencing. It is not only possible, but
`
`
`
`6
`
`Ariosa Exhibit 1042
`pg. 6
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`

`

`Second Declaration of Cynthia Casson Morton
`IPR2013-000276
`
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`probable – and in fact his own deposition testimony corroborates - that he
`
`was not aware of the evolution of molecular and biochemistry techniques
`
`used in MPS in 2008-2009, including the sample multiplexing employed in
`
`the art with MPS (as evidenced by the Binladen and Shoemaker references)
`
`and the development of products for use with available MPS systems, such
`
`as the Illumina, Inc. multiplexing kit.1
`
`11) It is thus my view that a person of ordinary skill (such as a first year
`
`postdoctoral student working in a molecular genetics “wet” laboratory
`
`in 2009) would have had both the knowledge of the state of the art of
`
`MPS and an ability to understand the basic scientific premises described
`
`in Binladen and Shoemaker. A person of ordinary skill in the art would
`
`have understood the scientific basis of methods for detecting fetal
`
`aneuploidy taught
`
`in Dhallan and Shoemaker, and would have
`
`understood that combining such methods with multiplexing and MPS to
`
`process multiple samples simultaneously would require nothing more
`
`than routine and highly predictable modifications.
`
`
`1 When Dr. Butte was asked if he supervises a wet laboratory in his current
`
`position at Stanford, Dr. Butte answered “No.” [Ex. 1041 at p. 20:4] Furthermore,
`
`Dr. Butte stated that in 2009 neither he nor anyone in his laboratory was a direct
`
`customer of Illumina. [Ex. 1041 at p. 22:24 – 23:6]
`
`
`
`7
`
`Ariosa Exhibit 1042
`pg. 7
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`

`

`Second Declaration of Cynthia Casson Morton
`IPR2013-000276
`
`
`
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`III. THE COMBINED TEACHINGS OF DHALLAN, BINLADEN AND
`SHOEMAKER REFERENCES DESCRIBE ALL SUBJECT
`MATTER IN CLAIMS 1-18 OF THE ‘430 PATENT
`
`
`12)
`
`As I previously testified in my first declaration, each of Dhallan,
`
`Binladen and Shoemaker teach selective amplification of DNA [Ex. 1002 at
`
`¶¶ 39 and 103]. Both Dhallan and Shoemaker teach selective amplification
`
`of non-random polynucleotide sequences for prenatal detection of fetal
`
`aneuploidy [Ex. 1002 at ¶ 103]. Dhallan teaches use of sequencing of
`
`maternal and fetal cell-free DNA to detect selectively-targeted and amplified
`
`fetal and maternal loci indicative of individual chromosomes [Ex. 1002 at ¶¶
`
`46 and 110]. More specifically Dhallan teaches selective amplification of
`
`selected loci in cell-free DNA from a maternal plasma or serum sample [Ex.
`
`1002 at ¶¶ 39 and 103].
`
`13)
`
`Also in my previous testimony, I described the teachings of
`
`Shoemaker and Binladen on the use of nucleic acid indices (also referred to
`
`as nucleic acid tags, bar codes or locator elements) in conjunction with
`
`multiplexed sequencing systems to identify samples from different sources,
`
`whether from different individuals (Binladen) or from samples taken from
`
`the same individual at different time points (Shoemaker) [Ex. 1002 at ¶¶ 17-
`
`18, 40 and 104; and Exs. 1005 and 1008 generally]. Further, Dhallan and
`
`
`
`8
`
`Ariosa Exhibit 1042
`pg. 8
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`

`

`Second Declaration of Cynthia Casson Morton
`IPR2013-000276
`
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`Shoemaker both teach quantification of maternal and fetal alleles from a
`
`maternal sample [Ex. 1002 at ¶¶ 47-48 and 111-112], including alleles from
`
`cell-free DNA of a maternal serum or plasma sample. These alleles are from
`
`selected loci of interest known to be associated with a specific chromosome,
`
`e.g., chromosome 21. So Dhallan and Shoemaker both teach using detection
`
`and quantification of alleles associated with individual chromosomes to
`
`identify a fetal aneuploidy [Id.].
`
`14)
`
`Binladen and Shoemaker both teach the use of nucleotide sample
`
`indices (irrespective of whether they are referred to as barcodes, unique tags
`
`or locator elements) with massively parallel sequencing systems to analyze
`
`multiple samples simultaneously [Ex. 1002 at ¶¶ 17-18, 40 and 105; and
`
`Exs. 1005 and 1008 generally]. Further, both Dhallan and Shoemaker
`
`specifically use 100 different loci on each chromosome in their analysis of
`
`the loci from different chromosomes [Ex. 1002 at ¶¶ 42 and 106].
`
`15)
`
`Thus, as described in detail in my previous declaration in relation to
`
`claims 1-18 [Ex. 1002] and that of Ariosa expert, Prof. Robert Nussbaum
`
`[Ex. 1003], each and every limitation of the claims of the ‘430 patent are
`
`taught by Dhallan, Binladen and Shoemaker. Specific teachings of each
`
`reference are described in more detail below.
`
`
`
`
`
`9
`
`Ariosa Exhibit 1042
`pg. 9
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`

`

`Second Declaration of Cynthia Casson Morton
`IPR2013-000276
`
`
`IV. THE TEACHINGS OF DHALLAN ALLOW DETECTION OF
`SELECTED LOCI USING VARIOUS TECHNIQUES
`
`16)
`
`Dr. Butte in his declaration testified that, in his opinion, one of
`
`ordinary skill in the art in January 2010 would have recognized that the
`
`teachings of Binladen or Shoemaker could not reasonably be combined with
`
`those of Dhallan because the technologies were incompatible [Ex. 2003 at ¶¶
`
`215, 218-219]. In Dr. Butte’s view, the so-called “Dhallan Process”
`
`“critically relies on restriction digestible primers for amplification” to
`
`incorporate fluorescent labels into a 5' overhang left after digestion of the
`
`nucleic acids [Id.]. These fluorescent products are detected by visualizing
`
`the products on a gel and “analyzing band intensities for each of the alleles”
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`to “determine the presence or absence of chromosomal abnormalities” [Ex.
`
`2003 at ¶ 220].
`
`17)
`
`I agree with Dr. Butte that certain embodiments in Dhallan teach the
`
`particular detection methods Dr. Butte characterizes as the “Dhallan
`
`Process”; however, Dhallan also teaches (and Dr. Butte ignores) a number of
`
`detection methods which do not require the use of restriction digestible
`
`primers. Dhallan describes multiple embodiments of the invention,
`
`including various embodiments that utilize different methods for detection of
`
`the nucleic acids. In fact, Dhallan describes a wide variety of methods for
`
`sequence determination of the selected loci:
`
`10
`
`
`
`
`
`Ariosa Exhibit 1042
`pg. 10
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`

`

`Second Declaration of Cynthia Casson Morton
`IPR2013-000276
`
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`In some embodiments, determining the sequence includes using a
`
`method that is allele specific PCR, mass spectrometry, hybridization,
`
`primer extension, fluorescence resonance energy transfer (FRET),
`
`sequencing, Sanger dideoxy
`
`sequencing, DNA microarray,
`
`GeneCHIP arrays, HuSNP arrays, CodeLink Arrays, BeadArray
`
`Technology, MassARRAY, MassEXTEND, SNP-IT, TaqMan,
`
`InvaderStrand Assay, southern blot, slot blot, dot blot, or MALDI-
`
`TOF mass spectrometry.
`
`
`
`[Ex. 1004 at col. 6: 26-34]. Moreover, Dhallan also teaches quantitation of
`
`tens to hundreds to thousands of loci from multiple chromosomes [Ex. 1004 at
`
`col. 7:9-16].
`
`18)
`
`Many of the detection mechanisms extant in 2002 and 2003 required
`
`fluorescence detection; thus, Dhallan proposes various embodiments to
`
`incorporate labels into the amplicons of selected loci including, as Dr. Butte
`
`reports, endonuclease digestion [e.g., Ex. 1004 at col. 8: 9-31; col 10:41-52;
`
`and col. 10:53 - col. 11:4]. However, Dhallan also teaches fragmentation of
`
`DNA and extension to incorporate labels [e.g., Ex. 1004 at col. 11: 61 - col.
`
`12: 17]; in vitro transcription of the loci followed by RNase A cleavage and
`
`detection using a SpectroCHIP [e.g., Ex. 1004 at col. 12: 40-47];
`
`incorporation of labelling nucleotides during amplification [e.g., Ex. 1004 at
`
`col. 13:66 - col. 13:5]; exonuclease treatment and labelling of loci [e.g., Ex.
`
`
`
`11
`
`Ariosa Exhibit 1042
`pg. 11
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`

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`Second Declaration of Cynthia Casson Morton
`IPR2013-000276
`
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`1004 at col. 6: 36-42]; and the use of a probe using a reporter dye that
`
`anneals to the locus of interest [e.g., Ex. 1004 at col. 14: 15-25]. Thus, in
`
`my view Dhallan discloses a number of labelling and detection methods,
`
`only one of which is drawn to use of restriction endonucleases.
`
`19)
`
`Dr. Butte, in my opinion, is taking a significantly more limited view
`
`of the teachings of Dhallan than one of ordinary skill in the art would have
`
`taken. It is my understanding that all embodiments described in Dhallan are
`
`relevant for the analysis of what a person of ordinary skill would have
`
`understood as being taught by Dhallan – not just the particular method
`
`identified by Dr. Butte.
`
`20)
`
`In my opinion, one of ordinary skill in the art in 2009 would view
`
`Dhallan as teaching a variety of methods for concurrent determination of
`
`sequences of multiple, specific loci of interest on multiple chromosomes for
`
`use in prenatal diagnosis.
`
`21)
`
`There were, however, a number of technological advances in
`
`detection methods between the priority date of Dhallan (circa 2002/2003)
`
`and January 2010. Specifically, massively parallel, next generation
`
`sequencing was developed and commercialized during this time [Ex. 1002 at
`
`¶15 and Ex. 1003 at ¶19], and by 2008 and 2009 massively parallel
`
`
`
`12
`
`Ariosa Exhibit 1042
`pg. 12
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`

`

`Second Declaration of Cynthia Casson Morton
`IPR2013-000276
`
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`sequencing was already being applied to prenatal detection of fetal
`
`aneuploidy [e.g., Ex. 1033; Ex. 1036; Ex. 1011; Ex. 1045].
`
`22)
`
`Molecular biology methods and systems are continually optimized,
`
`and researchers working in molecular genetics would have been aware of
`
`new and improved detection methods to enhance their research activities. In
`
`my view, one of ordinary skill in the art would not have continued to utilize
`
`the first- or second- generation detection methods disclosed in Dhallan to
`
`detect the non-random loci analyzed in Dhallan once the far-superior next
`
`generation, massively parallel sequencing techniques that were developed
`
`and commercialized in the mid-2000s became available and were widely
`
`adopted.
`
`
`
`
`
`
`
`V. THE TEACHINGS OF BINLADEN ARE USEFUL FOR
`MULTIPLEXED SEQUENCING OF MATERNAL SAMPLES
`
`23)
`
`Dr. Butte in his declaration states that the tags of Binladen are
`
`unsuitable for fetal aneuploidy detection [Ex. 2003 at ¶¶198-207].
`
`Specifically, Dr. Butte takes issue with the fact that Binladen teaches tagging
`
`of individual species of animals rather than different individuals of the same
`
`species [e.g., Ex. 2003 at ¶ 60]. Dr. Butte in his declaration states further
`
`that Binladen amplified and detected mitochondrial DNA rather than
`
`genomic DNA [e.g., Ex. 2003 at ¶ 61], and that Binladen discloses a non-
`
`13
`
`Ariosa Exhibit 1042
`pg. 13
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`

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`Second Declaration of Cynthia Casson Morton
`IPR2013-000276
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`trivial sequencing error rate using their particular dinucleotide tags [e.g., Ex.
`
`2003 at ¶¶ 62-68]. I disagree with Dr. Butte’s assessment of Binladen.
`
`24)
`
`First, I believe it is immaterial that Binladen’s sample tags are
`
`indicative of individuals from different species, as opposed to different
`
`individuals of the same species. Sample tags (also known in the art as
`
`barcodes, indexes or indices or locator elements) are used commonly to
`
`“tag” different sample sources of nucleic acids, where a different tag is used
`
`to tag each source. Once nucleic acid sequences from each source are
`
`differentially tagged, nucleic acid sequences from each source or sample can
`
`be pooled for sequencing then sorted into their original sample source using
`
`the tags [e.g., Ex. 1005 at p. 2]. It is my opinion that a scientist of ordinary
`
`skill in the art would understand that sample tags are indicative of a template
`
`source, whether the source is different individuals of the same species,
`
`individuals of different species, or many samples taken, e.g., at different
`
`time points from the same individual. The nucleotide tag is used after
`
`sequencing to “deconvolute” the pooled sequences to identify the source of
`
`each sequence.
`
`25)
`
`In addition, Dr. Butte states that because the nucleic acids used in
`
`Binladen was mitochondrial DNA rather than genomic DNA from maternal
`
`plasma or serum, one of ordinary skill would not view the teachings of
`
`
`
`14
`
`Ariosa Exhibit 1042
`pg. 14
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`

`

`Second Declaration of Cynthia Casson Morton
`IPR2013-000276
`
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`Binladen as applicable to fetal aneuploidy detection [Ex. 2003 at ¶ 60].
`
`Again, I disagree with Dr. Butte’s conclusion. My view—which I believe
`
`would be shared by others of ordinary skill in the art—is that once a DNA
`
`sample is isolated, whether it be genomic DNA or mitochondrial DNA, the
`
`steps of selective amplification and isolation and the subsequent sequencing
`
`of amplification products are equally applicable.
`
`26)
`
`Dr. Butte states that mitochondrial DNA differs from a blood sample
`
`with fetal and maternal genomic DNA because mitochondrial DNA consists
`
`of a single haploid genome, rather than two diploid genomes (e.g., a mixed
`
`sample of fetal and maternal nucleic acids) [e.g., Ex. 2003 at ¶¶ 204 and
`
`206]. However, it is my opinion that one of ordinary skill in the art would
`
`view DNA as DNA. First, it should be noted that isolation of cellular
`
`nucleic acids typically results in a “mixed sample” of mitochondrial and
`
`genomic DNA and RNA. The primers used to select and amplify specific
`
`loci are added to the combination of nucleic acids from the cell, and
`
`mitochondrial and genomic DNA would both be expected to be amplified as
`
`long as appropriate primers are used. Ultimately, the amplified sequences
`
`from both the mitochondrial and genomic DNA can then be sequenced. It is
`
`my view that one of ordinary skill in the art would not be dissuaded from
`
`using the methods of Binladen with a mixed sample of fetal and maternal
`
`
`
`15
`
`Ariosa Exhibit 1042
`pg. 15
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`

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`Second Declaration of Cynthia Casson Morton
`IPR2013-000276
`
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`nucleic acids, particularly when utilizing single molecule counting methods
`
`such as next generation, massively parallel sequencing. In fact, such single
`
`molecule counting techniques would be even more effective in complex
`
`mixtures of genomes, as single molecule counting techniques are far
`
`superior to prior art techniques in identifying low level nucleic acids present
`
`in a mixed sample.
`
`27)
`
`Finally, Dr. Butte states that the tags of Binladen are unsuitable for
`
`fetal aneuploidy detection because the tags tested by Binladen resulted in
`
`variations in sequence distribution and a non-trivial error rate [e.g., Ex. 2003
`
`at ¶¶ 199-203]. However, again I disagree with Dr. Butte. Binladen
`
`discloses use of different tags and examined the observed and expected
`
`sequence distributions of these tags. Binladen found that when using
`
`dinucleotide tags, the identity of the dinucleotide tag has an important effect
`
`[Ex. 1005 at p. 7]. Binladen also found that tetranucleotide primers resulted
`
`in a lower rate of sequence mis-assignment [Id.]. However, Binladen
`
`proposes “primer design guidelines” for the tag sequences to be used with
`
`multiplexed massively parallel sequencing [Ex. 1005 at p. 8]. In particular,
`
`Binladen provides guidance as to both the optimal nucleotide content (e.g., a
`
`conserved 5' nucleotide) and length (e.g., longer and of identical length) to
`
`
`
`16
`
`Ariosa Exhibit 1042
`pg. 16
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`

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`Second Declaration of Cynthia Casson Morton
`IPR2013-000276
`
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`enhance performance [Ex. 1005, p. 7].2 Thus, it is my opinion that it would
`
`have been well within the skill of one of ordinary skill in the art, such as a
`
`first-year postdoctoral student, to design and optimize sample tags for use
`
`with massively parallel sequencing of multiple maternal DNA samples.
`
`28)
`
`Further, not only does the Binladen publication provide suggestions
`
`that might improve efficiency and accuracy of the tags, but Binladen lays a
`
`blueprint for how one of ordinary skill would test sample tags to be used in
`
`an experiment. For example, Daines, et al. (“High-Throughput Multiplex
`
`Sequencing to Discover Copy Number Variants in Drosophila”, Genetics,
`
`182:935-41 (2009) [Ex. 1046]) modified Solexa sequencing primers by
`
`adding 3-bp barcodes. The modified sequencing primers were used to
`
`prepare libraries such that the 5' ends of the sequencing products indicated
`
`the sample source. In Daines, et al., DNA of various microbes were
`
`differentially amplified and labeled with barcoded oligonucleotide adapters
`
`and the resulting libraries were mixed in equal molar amounts and
`
`sequenced simultaneously on one lane of an Illumina Genome Analyzer.
`
`Daines, et al., using essentially the same methods to assess the accuracy of
`
`
`2 I testified to this in my deposition. When asked if Binladen reports wide
`
`variations in results, I replied, “[b]ut he provides the reason why” [Ex. 2005 at
`
`101:10].
`
`
`
`17
`
`Ariosa Exhibit 1042
`pg. 17
`
`

`

`Second Declaration of Cynthia Casson Morton
`IPR2013-000276
`
`
`tags or barcodes as Binladen, concluded that the reads generated exhibited
`
`an extremely low error and cross-contamination rate [Id., at p. 938].
`
`29)
`
`Thus, it is my opinion that one of ordinary skill in the art as defined
`
`by Dr. Butte—or even a scientist with less training—would view the
`
`Binladen reference as simply teaching use of different nucleotide “tags” or
`
`“barcodes” to indicate different sources of DNA that, once tagged, can be
`
`pooled and sequenced. Once sequenced, the sequence of the tags/barcodes
`
`are used to deconvolute the sequence information to identify the source of
`
`each sequence. Moreover, it is my opinion that one of ordinary skill, upon
`
`reading the guidance provided by Binladen on optimal content and length of
`
`the tags [Ex. 1005 at pp. 7-8], would be able to easily apply the teachings of
`
`Binladen to optimize the tags to decrease the error rate and increase the
`
`accuracy of putative sample tags.
`
`
`
`18
`
`
`
`
`
`Ariosa Exhibit 1042
`pg. 18
`
`

`

`Second Declaration of Cynthia Casson Morton
`IPR2013-000276
`
`
`
`VI. SHOEMAKER
`SAMPLE
`TEACHES
`OF
`INDIVIDUAL
`IDENTIFICATION
`MULTIPLEXED SEQUENCING
`
`TAGS
`SAMPLES
`
`FOR
`IN
`
`30)
`
`Dr. Butte in his declaration takes the position that the “locator
`
`elements” of Shoemaker would not be recognized by one of ordinary skill in
`
`the art as sample tags or barcodes that indicate the sample source of a
`
`nucleic acid [e.g., Ex. 2003 at ¶¶ 70-73]. Instead, Dr. Butte states the locator
`
`elements of Shoemaker identify “bins” which correspond to individual wells
`
`[Id.]. However, because Shoemaker’s individual wells contain single cells,
`
`it is my opinion that one of ordinary skill in the relevant fields would
`
`understand that in Shoemaker the locator tags that identify an individual cell
`
`are actually being used to identify the sample source of the nucleic acids.
`
`Dr. Butte in his deposition testimony acknowledged that this is the case [Ex.
`
`1041 at p. 109:22 – 110:6]:
`
` Q: Does this [particular passage in Shoemaker] describe the
`
`use of a unique tag sequence for an individual cell?
`
`A: My understanding is that the unique tag sequence identifies
`
`a well. And if they believe one cell is in one well, it would
`
`correspond to one cell.
`
`Q: But Shoemaker does say that the methods comprise
`
`labeling one or more regions of genomic DNA in each cell,
`
`correct?
`
`19
`
`
`
`
`
`Ariosa Exhibit 1042
`pg. 19
`
`

`

`Second Declaration of Cynthia Casson Morton
`IPR2013-000276
`
`
`A: It does say, "The methods comprised labeling one or more
`
`regions of genomic DNA in each cell," yes.
`
`
`
`31)
`
`It is my opinion that one of ordinary skill in the art would understand
`
`that the locator elements used in Shoemaker are nucleotide tags used to
`
`identify a source of nucleic acids used in sequencing, whether that source is
`
`different cells (e.g., Shoemaker), samples taken from the same human at
`
`different time points (e.g., Shoemaker), samples taken from different
`
`individuals (e.g., samples from many different individual species such as
`
`those disclosed in Binladen), or samples from individual human patients
`
`(e.g., the ‘430 patent).
`
`
`
`
`
`
`
`
`VII. BINLADEN AND DHALLAN CAN BE REASONABLY
`COMBINED
`
`32)
`
`Dr. Butte in his declaration states that in his opinion, one of ordinary
`
`skill in the art would recognize that the teachings of Binladen cannot
`
`reasonably be combined with those of Dhallan, due to Dhallan’s reliance on
`
`restriction endonuclease digestible primers. Dr. Butte states that the tags
`
`taught by Binladen would be cleaved off by the restriction enzyme that
`
`Dhallan uses to generate the 5' overhang [e.g., Ex. 2003 at ¶¶ 214-225].
`
`However, as discussed above at ¶ 18, the teachings of Dhallan are not
`
`20
`
`Ariosa Exhibit 1042
`pg. 20
`
`

`

`Second Declaration of Cynthia Casson Morton
`IPR2013-000276
`
`
`limited to embodiments requiring use of a restriction endonuclease, and it is
`
`my opinion that one of ordinary skill in the art would substitute the robust
`
`next generation, massively parallel sequencing techniques developed and
`
`commercialized in the mid-2000s for the first- and second-generation
`
`detection techniques disclosed in Dhallan. It is also my opinion that in
`
`doing so, one of ordinary skill would use basic common sense and not
`
`utilize the embodiments of Dhallan where restriction sites are added to the
`
`primers, or deliberately add barcodes or sample tags to loci only to cleave
`
`them off as Dr. Butte suggests. It is my view that one of ordinary skill
`
`would recognize that in substituting next generation sequencing techniques
`
`for the detection techniques taught in Dhallan, it would be completely
`
`nonsensical to use a restriction endonuclease to remove the sample tags.
`
`
`
`VIII. SHOEMAKER AND DHALLAN CAN BE REASONABLY
`COMBINED
`
`
`33)
`
`Dr. Butte has opined that Shoemaker could not be combined with and
`
`is not compatible with Dhallan for the same reasons that Binladen cannot
`
`reasonably be combined with Dhallan; that is, because “Dhallan critically
`
`relies on restriction digestible primers followed by cleaving the primer
`
`sequence by enzyme digestion” [Ex. 2003 at ¶¶ 226-230]. However, I
`
`disagree with Dr. Butte in this instance as I do with his opinion regarding
`
`
`
`21
`
`Ariosa Exhibit 1042
`pg. 21
`
`

`

`Second Declaration of Cynthia Casson Morton
`IPR2013-000276
`
`
`combining Binladen and Dhallan [supra at ¶ 35]. The teachings of Dhallan
`
`are not limited to embodiments requiring use of a restriction endonuclease
`
`[supra at ¶ 18]. For example, Dhallan also teaches fragmentation of DNA
`
`and extension to incorporate labels [e.g., Ex. 1004 at col. 11: 61- col. 12:
`
`17]; in vitro transcription of the loci followed by RNase A cleavage and
`
`detection using a SpectroCHIP [e.g., Ex. 1004 at col. 12: 40-47];
`
`incorporation of labelling nucleotides during amplification [e.g., Ex. 1004 at
`
`col. 13:66- col. 13: 5]; exonuclease treatment and labelling of the loci [Ex.
`
`1004 at col. 6: 36-42]; and using a probe using a reporter dye that anneals to
`
`the locus of interest [e.g., Ex. 1004 at col. 14: 15-25].
`
`34)
`
`It is my opinion that one of ordinary skill in the art would have been
`
`familiar with the next generation, massively parallel sequencing techniques
`
`developed and commercialized in the mid-2000s at least by January 2010,
`
`and would have been motivated to substitute these next generation
`
`sequencing techniques, such as those described in Binladen and Shoemaker,
`
`for the first- and second-generation detection techniques disclosed in
`
`Dhallan. Essentially, it is my opinion that in January 2010, one of ordinary
`
`skill in the art would want to use the most accurate and efficient detection
`
`techniques available.
`
`
`
`22
`
`Ariosa Exhibit 1042
`pg. 22
`
`

`

`Second Declaration of Cynthia Casson Morton
`IPR2013-000276
`
`
`35)
`
`The use of improved sequencing techniques would not employ the
`
`addition of restriction sites into amplification primers and a person of
`
`ordinary skill would not be led away from using next generation, massively
`
`parallel sequencing techniques based on the embodiments of Dhallan that
`
`utilize restriction enzymes. A person of ordinary skill in the art would
`
`understand that it does not make sense to deliberately engineer amplification
`
`primers to include barcodes or sample tags to be incorporated into resulting
`
`products only to subsequently cleave them off using a restriction enzyme, as
`
`Dr. Butte suggests. One of ordinary skill would recognize that cleaving the
`
`sample tag would negate its entire purpose, and would render the detection
`
`inoperable – and thus would not use the restriction enzyme. Instead, a
`
`person of ordinary skill in the art would use one of the other techniques
`
`described by Dhallan in conjunction with next generation sequencing.
`
`
`IX. BINLADEN AND SHOEMAKER CAN BE REASONABLY
`COMBINED
`
`
`36)
`
`The teachings of Binladen and Shoemaker can also be reas

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