MYR 1043
`Myriad Genetics, Inc. et al. (Petitioners) v. The Johns Hopkins University (Patent Owner)
`IPR For USPN 7,824,889
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`Page 1 of 9
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`Myriad Genetics, Inc. et al. (Petitioners) v. The Johns Hopkins University (Patent Owner)
`IPR For USPN 7,824,889
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`Page 1 of 9
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`research since about 1989. My current research is focused on cancer genes and
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`markers of gynecological cancers. Throughout my career I have followed new
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`developmentsin the field by reading of the scientific literature, active research,
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`and interactions with colleagues. Because of my training and experience,|
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`consider myself knowledgeable in various aspects of nucleic acid amplification.
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`This includes technologies that are used to analyze DNA sequencesand variations
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`in DNA sequences.
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`.
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`In 2004 I co-authored a review article on digital PCR that appeared in Expert
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`Reviewsin Molecular Diagnostics, appended as Exhibit 3.
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`I draw from that
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`review as well, as applications of digital PCR more current than at that time with
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`which [ am familiar.
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`.
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`[have also been informed that Johns Hopkins University (JHU) ownsU.S. patents
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`7,915,015 (“015 patent”) 7,824,889 (889 patent’) and 6,440,706 (°° 706
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`patent”) and has licensed them to LabCorp (Esoterix), and Exact Sciences.
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`.
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`.
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`[have reviewed the ‘015 patent, the ‘889 patent, and the ‘706 patent, including
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`the original claims and the amendmentsfiled July 9, 2014, (attached as Exhibit 2).
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`Ihave been asked to review and summarize the state of the digital PCR field. The
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`statements that I make include my reading and interpretation of the statements as
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`represented in the exhibits. The readings andinterpretations are my own,and I
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`have no stake in the outcome of the re-examination proceedings.
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`. Lunderstandthat the “digital PCR” methods described in the claimsof the three
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`subject patents involve (1) analysis of two different analytes and (2) comparing
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`the numberof assay samples containing one of the analytes to the number of
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`assay samples containing the other analyte. While I understand that the inventors
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`coined and applied the term “digital PCR”to their methods, I understand that
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`manyin this field subsequently adopted the term “digital PCR” and use it more
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`broadly.
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`I have attempted in this declaration to refer only to examples of digital
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`PCRthat share the two features stated above, rather than the broader usage.
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`10.
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`Asanillustration of the different ways that the term is often used in thefield,
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`Dayet al., Methods 59:101-107, 2013, describes two types of digital PCR as
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`those which (1) calculate absolute abundance of a target sequence and those
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`which (2) obtain a relative abundance by comparing to an internal reference
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`sequence. Exhibit 12, paragraph spanning pages 101-102. Dayrefers to the latter
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`type as the more commonuse. Ibid.
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`Thelatter type is what I understandis
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`described in the claims of the three subject patents.
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`11.
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`The study of DNA sequencevariation is important for many areas of research. Prior
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`to digital PCR, conventional PCR did not allow the identification and quantification
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`of rare molecular genetic changes because conventional PCR amplifies a pool of
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`DNAtemplates from thestarting material. Digital PCR is useful for amplifying a
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`single DNA template from limiting dilution samples, therefore transforming the
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`exponential, analog signals from conventional PCRto linear, digital signals,
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`allowing statistical analysis of the PCR products. Digital PCR has been applied in
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`the quantification of muantalleles and detection ofallelic imbalancein clinical
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`specimens, providing a useful molecular diagnostic tool for cancer detection.
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`Exhibit 3, page 46, col. 2, text box. Digital PCR has also been appliedin the
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`-3-
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`quantification of muantalleles and detection of allelic imbalance in fetal
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`abnormalities.
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`12.
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`In 2004, in our review article, we noted twelve different examples in twelve
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`different scientific publications in the scientific literature in which digital PCR
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`had been used for molecular analysis of clinical samples. These involved
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`detection of cancer mutations, detection ofallelic imbalance, detection of loss of
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`heterozygosity, quantitative detection of tumor suppressor gene expression.
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`Exhibit 3, Table 1.
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`13.
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`The digital PCR technique is especially powerful in experiments requiring
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`quantitative investigation of individual alleles in DNA samplesisolated from a
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`mixed cell population. Exhibit 3, page 46, col. 1, last full paragraph.
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`14.
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`Vogelstein and Kinzler published their original scientific paper on digital PCR in
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`Proc. Natl. Acad. Sciences USA 96: 9236-9241 (1999). Exhibit 19. I understand
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`that the paper served asthe basis for the application underlying the three subject
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`patents, as its text and figures appear to have been incorporated entirely in the
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`application. Exhibit 18.
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`Recognition in the Art
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`15.
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`According to Google Scholar™,the original digital PCR publication of inventors
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`Vogelstein and Kinzler, Proc. Natl. Acad. Sciences USA 96: 9236-9241 (1999),
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`has been cited in 532 scholarly publications in its archive from 2009-2014.
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`Exhibit 4. That is an indication of its unusually high impactin the scientific
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`community. According to the Altmetric™ score, this article was in the 88th
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`percentile of a sample of the 1888361 tracked articles of a similar age published
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`within six weeks on either side in al journals. Exhibit 4, page 2.
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`16.
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`I am aware of a numberofscientific conferences on digital PCR that have been
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`organized in the US and in Europe. One, put on by Cambridge Health Tech
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`Institute, October 6-8, 2014, the third annual such conference, describes digital
`PCR as“creating waves across the diagnostic landscape”in its conference
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`announcement. Exhibit 5, emphasis added. One of the featured presentations at
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`last year’s conference wastitled “Use of digital PCR in Oncology: Changing the
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`paradigm for systemic therapy.” Exhibit 6, emphasis added. The organizers of
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`the 2013 digital PCR conference in San Diego, CA,stated that digital PCR “has
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`already shownpotential to be a disruptive technology in manyareas of
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`diagnostics.” Exhibit 7, emphasis added. The existence of these conferences as
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`well as the descriptions they use are indications of the high importanceofdigital
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`PCRin the scientific community.
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`17.
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`Another conference, put on by an organization called Global Engage, will holdits
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`second annual event in Europe on “qPCR and digital PCR.” Exhibit 8. The first
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`such congress in 2013 reportedly had 150 attendees, and over 200 attendees are
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`expected in 2014. Global Engageindicatesthat “increasing numbersof real-time
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`PCRusers [are] purchasing digital PCR [machines] due to its reduction in cost,
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`absolute quantification, improved sensitivity, precision and greater robustness.”
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`Exhibit 8. This reflects the growing adoption of digital PCR (broadly used) in the
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`scientific and diagnostic communities.
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`18.
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`Hahnet al., Expert Rev. Mol. Diag. 9:613-621, 2009, describes non-invasive
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`detection of Down syndromeasa “long-sought goal.” It further teaches that
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`application of digital PCR or shot-gun sequencing to analysis ofcell-free fetal
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`DNA maybethe fulfillment of this goal. Exhibit 16, abstract, lines 1-3. Hahn
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`further refers to these techniques as providing a paradigm shift in prenatal
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`diagnosis. Exhibit 16, abstract lines 3-6.
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`19.
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`Tsui et al., Current Opinion in Hematology 19: 462-468, 2012, reviews analyses
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`of fetal nucleic acid in maternal plasma. Exhibit 17. Tsui indicates that digital
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`PCRhas enabled high quantitative precision for maternal plasma DNA analyses.
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`Abstract, lines 7-9. Tsui further touts the importance of digital PCR in detecting
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`fetal monogenicdiseases, stating, “To obtain an analytical precision that would
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`allow discrimination of the small concentration differences between the mutant
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`and wild-type DNA,quantification based on molecular counting, such asdigital
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`PCR, has been employed. Exhibit 17, page 463, col. 2, lines 11-16. Tsui refers to
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`this as a “technically challenging” determination to which digital PCR has
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`provided one approachto address. See “Key Points” at Exhibit 17, page 463.
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`Advantages
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`20.
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`A later review article than mine, by Vikova et al., Med. Sci. Monit. 16:RA85-91,
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`2010, describes digital PCR in Figure 2. Exhibit 9. Vikova indicates the
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`advantages of Digital PCR over real-time PCR. Vikovaasserts that “DigPCR
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`outperformsreal-time PCR in precision which is neededespecially in the
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`screening and detection of aneuploidy. Digital PCR has been provenaneffective
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`approach in noninvasive prenatal diagnostics of trisomy 21.” Exhibit 9. RA87,
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`last paragraph, citations omitted. Vikovaalso asserts that for detecting
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`monogenic diseases the “advantage lies in the digital relative mutation dosage
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`approach. Effective quantification of allele frequency by digital PCR makes
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`possible the precise evaluation of balance/imbalance between mutant and wild-
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`typealleles.” Exhibit 9, RA88, first paragraph, citation omitted.
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`21.
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`Lo et al., Proc. Natl. Acad. Sciences USA 104:13116-13121, 2007, explored the
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`use of digital PCR “to achieve finer degree of quantitative discrimination” than
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`possible with real-time PCR. Exhibit 10 at page 13116, col. 2, last paragraph.
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`The technique could successfully detect aneuploidy even whenthefetal fraction is
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`a minor population of a sample. Exhibit 10, page 13121, column1, lines 3-4.
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`22,
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`Lun et al., Clin. Chem 54:1664-1672, 2008, demonstrated a higher degree of
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`precision of the digital PCR to real-time PCR for detection of amounts of X and Y
`chromosomes using the ZEX/ZFYloci. Exhibit 11, page 1664, column1,
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`paragraph3.
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`23.
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`Sedlak ef al., Expert Rev. Mol. Diag. 14:501-507, 2014, teaches that digital PCR
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`is superior to qPCR (quantitative or real-time PCR) for ratiometric assays.
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`Exhibit 15, page 504,col. 2, lines 43-46. Sedlak uses the assay to detect both
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`replicating viral DNA and chromosomally integrated viral DNA. Exhibit 15,
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`page 502,col. 2, first full paragraph.
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`24.
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`Day emphasizesthe sensitivity and ability to achieve quantitation of rare variants
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`of digital PCR. Exhibit 12, page 102, first full paragraph. Daylists the positive
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`attributes of digital PCR as including rare variant detection, estimating copy
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`numbervariation, minimal template requirements, ease of analysis, and
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`integration with next generation sequencing. Section 3, spanning pages 102-103.
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`Commercial Activities
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`25.
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`It is my understanding that, a number of apparatus manufacturers have developed
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`products to carry out digital PCR. These include Fluidigm Corp. Life
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`Technologies, Bio-Rad Laboratories, and RainDance. These and other platforms
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`for PCR are compared in Table 1 of Day et al., Methods 59:101-107, 2013.
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`Exhibit 12.
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`26.
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`Global Engage in announcingits digital PCR and qPCR conference, reported that
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`“the gene amplification market [is] predicted to grow to $1.9 billion by 2015.”
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`Exhibit 8. This predictionis not limited to digital PCR,or relative digital PCR,
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`but may nonetheless suggest substantial commercial activities.
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`27.
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`Baker, Nature Methods 9:541-544, 2012 surveys the commercial digital PCR
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`offerings. Exhibit 13. The machines offered by Fluidigm and Life Technologies
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`can run either digital PCR or qPCR(real-time PCR). Exhibit 13, page 542-543,
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`spanning paragraph and page 543, second full paragraph. Digital PCR is more
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`accurate and less ambiguous but more expensive than qPCR. Exhibit 13, page
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`541, col. 3, last paragraph. The RainDance and Bio-Rad machines perform only
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`digital PCR but not qPCR. Exhibit 13, page 543, col. 2, last paragraph. Baker
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`comparesthe four instruments in Exhibit 13, Table 1.
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`28.
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`Rochealso markets an apparatus which employs digital PCR for genotyping.
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`Exhibit 14. The Light Cycler™ is used to detect JDH/ mutations. See Fig.4.
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`Although manydifferent techniques are part of the workflow, Roche describes the
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`digital PCR as the “all important second step” which “allowsrelative
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`quantification of mutant tumor cell DNA in a blood sample.” Page5,
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`“Conclusion,” col. 2, lines 3-5.
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`29.
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`Ideclare that all statements (prepared by Sarah A. Kagan) made herein of my
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`own knowledgeare true and thatall statements made on information and belief
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`are believed to be true; and further that these statements are made with the
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`knowledgethat willful false statements are punishable by fine or imprisonment, or
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`both, under §1001 of Title 18 of the United States Code and that such willfulfalse
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`statements may jeopardize the validity of the claims or the patent.
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`oa
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`le-Ming Shih
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`August 6, 2014
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`Date
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