U.S. Patent No. 7,064,197
`Declaration of Dr. Norman Nelson
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`UNITED STATES PATENT AND TRADEMARK OFFICE
`BEFORE THE PATENT TRIAL AND APPEAL BOARD
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`BECTON DICKINSON AND COMPANY,
`Petitioner
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`v.
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`ENZO LIFE SCIENCES, INC.
`Patent Owner
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`U.S. Patent No. 7,064,197
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`SYSTEM, ARRAY AND NON-POROUS SOLID SUPPORT
`COMPRISING FIXED OR IMMOBILIZED NUCLEIC ACIDS
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`DECLARATION OF DR. NORMAN NELSON
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`Page 1 of 63
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`BD EXHIBIT 1002
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`Declaration of Dr. Norman Nelson
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`UNITED STATES PATENT AND TRADEMARK OFFICE
`BEFORE THE PATENT TRIAL AND APPEAL BOARD
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`BECTON DICKINSON AND COMPANY,
`Petitioner
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`v.
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`ENZO LIFE SCIENCES, INC.
`Patent Owner
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`U.S. Patent No. 7,064,197
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`SYSTEM, ARRAY AND NON-POROUS SOLID SUPPORT
`COMPRISING FIXED OR IMMOBILIZED NUCLEIC ACIDS
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`DECLARATION OF DR. NORMAN NELSON
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`Page 1 of 63
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`BD EXHIBIT 1002
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`U.S. Patent No. 7,064,197
`Declaration of Dr. Norman Nelson
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`I, Norman Nelson, do hereby declare:
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`1.
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`I am making this declaration at the request of Becton Dickinson and
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`Company (“BD”) in the matter of the Inter Partes Review of U.S. Patent
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`No. 7,064,197 to Rabbani et al. (“the ’197 patent”).
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`2. My qualifications are established by my resume, which I understand is
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`provided as Exhibit A to this Declaration.
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`3.
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`I am being compensated for my work on this matter, but my opinions are
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`based on my own views of the patented technology and the prior art. My
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`compensation in no way depends on the outcome of this proceeding or the
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`content of my testimony.
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`4.
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`In preparing this Declaration, I reviewed and considered the ’197 patent, the
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`prosecution history of the ’197 patent, and the documents listed at the end of
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`this declaration. Importantly, I have reviewed the related Petition, which I
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`understand BD will file at the United States Patent and Trademark Office
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`(USPTO) at the same time as this Declaration is filed at the USPTO.
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`I.
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`5.
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`QUALIFICATION AND EXPERIENCE
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`I obtained a Ph.D. in Chemistry, with a focus in Biochemistry, in 1982 from
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`University of California, San Diego. I also received a Bachelor of Science
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`in Chemistry from California Institute of Technology in 1976.
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`U.S. Patent No. 7,064,197
`Declaration of Dr. Norman Nelson
`I have nearly 31 years of experience in molecular diagnostics and nucleic
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`6.
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`acid chemistry, particularly nucleic acids analysis. I am and was very
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`knowledgeable about conventional techniques for attaching nucleic acids to
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`other moieties like solid supports or labels. I worked for Gen-Probe
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`Incorporated (now acquired by Hologic, Inc.)—a pioneer and leader in
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`molecular diagnostics—for 27 years (June 1985-August 2012). While at
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`Gen-Probe, I co-invented, reduced to practice, and played a key role in
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`commercialization of multiple core technologies involving nucleic acids
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`analysis, which are currently in FDA-approved products.
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`7.
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`I started my career at Gen-Probe as a scientist (1985-2005), where I
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`developed and implemented key nucleic acids-based technologies and
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`assays, including nucleic acids capture/immobilization and labeling
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`techniques, hybridization, amplification and detection of nucleic acids. As
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`the Director of Biochemistry at Gen-Probe (2005-2009), I led a
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`multidisciplinary team in the development of multiplexed nucleic acids-
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`based assays. And as the Senior Director of Discovery Research at Gen-
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`Probe (2009-2012), I focused on the development and commercialization of
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`various nucleic acids-based diagnostic products.
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`8.
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`I have been working as a consultant in the field of nucleic acids-based
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`diagnostics, DNA sequencing and Genomics since 2012.
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`U.S. Patent No. 7,064,197
`Declaration of Dr. Norman Nelson
`9. My research work has led to 37 issued U.S. patents and over 100 issued or
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`pending patent applications worldwide.
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`10.
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`I have co-authored over 20 peer-reviewed journal articles and over 35
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`technical poster presentations in field of nucleic acids-based diagnostics. I
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`have also delivered numerous technical presentations at conferences. And I
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`have also chaired or served in the program committee of many conferences
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`and symposia related to my field of work.
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`11.
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`I have extensive experience in the field of nucleic acid immobilization,
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`hybridization, and detection—the technical field of the ’197 patent. Upon
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`joining Gen-Probe, I extensively researched and studied the existing field of
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`nucleic acids analysis going back to the mid-1970s. Study and knowledge of
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`the prevailing technologies was a requirement for my success as a scientist
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`in developing novel or improved technologies. After BD retained me for
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`preparing this Declaration, I have re-familiarized myself with the pre-1983
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`scientific literature and patent publications in the field of nucleic acid
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`immobilization, hybridization, and detection (the earliest priority date listed
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`on the face of the ’197 patent).
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`II.
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`RELEVANT LEGAL STANDARDS
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`12. The opinions I express in this declaration involve the application of my
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`technical knowledge and experience to the evaluation of certain prior art
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`U.S. Patent No. 7,064,197
`Declaration of Dr. Norman Nelson
`with respect to the ’197 patent. In addition, I understand that the following
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`legal principles apply, as explained to me by BD’s legal counsel.
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`13.
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`I understand that, in proceedings like this one before the USPTO, a claim in
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`an unexpired patent shall be given its broadest reasonable interpretation in
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`light of the specification of the patent in which it appears. I also understand
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`that district courts may apply a different claim construction standard, and
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`that claims there should be given their ordinary meaning to a person having
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`ordinary skill in the art at the relevant timeframe in light of the claim
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`language, patent specification, and prosecution history. I have read Section
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`VIII of the Petition which sets out the interpretation of certain claim terms in
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`the Petition. I agree with the statements made in that section. I have been
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`informed of certain terms that have already been interpreted by a court. My
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`opinions in this declaration remain the same under either claim construction
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`standard discussed in the Petition or the claims construction standard from
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`the court.
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`14.
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`I understand that a patent claim can be unpatentable if it is anticipated in
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`view of the prior art. I understand that anticipation of a claim requires that
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`every element of a claim be disclosed expressly or inherently in a single
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`prior art reference, arranged as in the claim.
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`U.S. Patent No. 7,064,197
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`I understand that a patent claim can be unpatentable for obviousness only if
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`15.
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`the invention described in the claim would have been obvious to a person of
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`ordinary skill in the art at the time the invention was made, taking into
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`account (1) the scope and content of the prior art, (2) the differences
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`between the prior art and the claimed invention, and (3) the level of ordinary
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`skill in the art, and (4) any secondary considerations of non-obviousness,
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`including commercial success of products or processes using the invention,
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`long felt need for the invention, failure of others to make the invention,
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`industry acceptance of the invention, and copying of the invention by others.
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`16.
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`I further understand that multiple references can be combined with one
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`another, or pursuant to the knowledge of a person of ordinary skill in the art,
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`to render a claim obvious. I also understand that there must be a reason that
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`would have prompted a person of ordinary skill in the relevant field to
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`combine the features of the prior art in the way the claimed invention does.
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`17.
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`I also understand that a person skilled in the art must reasonably expect that
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`the combination will work.
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`18.
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`In determining whether a piece of prior art could have been combined with
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`other prior art or with other information within the knowledge of a person
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`having ordinary skill in the art, I understand that it is proper to conclude that
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`a claim is obvious if it is no more than a predictable use of prior art elements
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`according to their established functions and the person of ordinary skill in
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`the art would have reasonably expected success.
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`III. LEVEL OF ORDINARY SKILL IN THE ART
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`19.
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`It is my understanding that when considering the claims of the ’197 patent
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`and the prior art, I must do so based on the perspective of one of ordinary
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`skill in the art at the relevant effective filing date. Even though I am an
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`expert, I consider myself totally qualified as to the level of ordinary skill at
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`the relevant time(s). My understanding is that the purported effective filing
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`date of the ’197 patent is January 27, 1983, but that the ’197 patent may in
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`fact be entitled to a priority date no earlier than May 9, 1985. All of my
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`analyses below are based on the level of skill in the art at least as early as of
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`January 27, 1983. I offer no opinion here regarding the effective filing date
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`of the ’197 patent, as I understand that is a legal determination, and I am not
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`a lawyer.
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`20.
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`I understand that several factors are to be considered in determining who
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`would be a person of ordinary skill in the art. These factors include: (1) the
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`types of problems encountered in the art; (2) the prior art solutions to those
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`problems; (3) the rapidity with which innovations are made; (4) the
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`sophistication of the technology; and (5) the educational level of active
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`workers in the field.
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`21. Based on these factors as well as my experience and expertise, a person of
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`ordinary skill in the field of nucleic acid detection, immobilization and
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`hybridization as of both the 1983 and the 1985 filing dates would have (i)
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`possessed or would have been actively pursuing an advanced degree in
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`organic chemistry and/or biochemistry, (ii) attained at least two years of
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`experience in a chemistry or biochemistry laboratory and would have been
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`familiar with nucleic acid chemistry, and (iii) have been knowledgeable of
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`conventional techniques for attaching nucleic acids to other moieties like
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`solid supports or labels. This level of skill applies to all my obviousness
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`IV.
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`22.
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`analyses below.
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`SUMMARY OF MY OPINIONS
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`I have been asked to consider the ’197 patent and certain prior art references
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`and to offer my opinions on the relation of that prior art to the claims of the
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`’197 patent.
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`23. The ’197 patent in general describes methods for fixing or immobilizing
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`nucleic acids to solid supports, which can be subsequently hybridized to
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`polynucleotide probes capable of generating a soluble signal. Ex. 1001,
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`1:23-32; 5:40-46; 5:61-6:32. Broadly, the ’197 patent claims relate to non-
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`porous solid supports with fixed or immobilized nucleic acids, and systems
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`and arrays comprising such non-porous solid supports.
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`24. Two strands of nucleic acids hybridize to one another through hydrogen
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`bonding between complementary nucleotides (bases) that naturally pair with
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`one another. Under the Watson-Crick base pairing model, the nucleotide
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`“A” pairs with the nucleotide “T” on the opposite strand, and the nucleotide
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`“C” pairs with the nucleotide “G” on the opposite strand. In RNA
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`molecules, “T” is replaced by “U” to form an “A-U” base pair. To be
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`considered “hybridizable,” a nucleic acid strand does not have to hybridize
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`along its entire length to another strand—there only has to be some degree of
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`possible hybridization between complementary nucleotides of two strands of
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`nucleic acids.
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`25. Fixation or immobilization of nucleic acid sequences in hybridizable form to
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`different types of solid supports, including non-porous solid supports, was
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`known more than a year before the January 27, 1983, filing date of the first
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`application. I discuss several such publications in detail below, such as Ex.
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`1006, Ex. 1007, Ex. 1008, and Ex. 1019. Hybridizable single-stranded
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`nucleic acids bound to solid supports were routinely used for identifying
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`biological materials in samples and separating biological materials from
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`samples prior to January 27, 1983. See, e.g., Ex. 1007, p. 301, right col.,
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`first paragraph (discussing (1) analytical methods to detect nucleic acids and
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`(2) affinity chromatography and sample preparation (separating biological
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`material)).
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`26. As explained in detail below, Fish (Ex. 1006) teaches every technical detail
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`of claims 1, 6, 8, 9, 12, 13, 14, 15, 16, 27, 32, 33, 34, 38, 41, 61, 62, 63, 69,
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`70, 72, 73, 74, 78, 100, 191, 193, 194, 212, 213, 218, 222, 225, 226, 227,
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`230, and 233 of the ’197 patent.
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`27. Also as explained in detail below, a person of ordinary skill in the art would
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`have understood that claims 31, 64, 68, 101, 192, and 195 would have been
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`obvious in view of Fish, and that claims 38, 78, and 218 would have been
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`obvious based on Fish in view of Gilham.
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`28. Also as explained in detail below, VPK (Ex. 1008) also teaches every
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`technical detail of claims 1, 6, 8, 9, 12, 13, 14, 15, 16, 27, 31, 32, 34, 38, 61,
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`62, 63, 68, 69, 70, 72, 74, 79, 100, 191, 192, 193, 194, 213, 219, 226, 227,
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`and 236 of the ’197 patent.
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`29. Also as explained in detail below, claims 16, 38, 64, 78, 101, 195, 218, 222,
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`and 230 would have been obvious in view of Noyes, VPK and
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`Ramachandran
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`30. Also as explained in detail below, claims 33, 41, 73, 212, 225, and 233
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`would have been obvious in view of VPK and Metzgar.
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`In my conclusions of obviousness of certain claims, I consider that the
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`31.
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`claimed solid supports and systems are no more than a predictable use of
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`prior art elements according to their established functions and that one of
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`ordinary skill in the art would have reasonably expected success in making
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`such solid supports and systems. They involve prior art technologies used in
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`suggested and predictable ways to achieve expected results.
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`V.
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`THE ’197 PATENT
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`32. The ’197 patent describes non-porous solid supports with fixed or
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`immobilized nucleic acids, and systems and arrays comprising such non-
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`porous solid supports. Ex. 1001, Title and Abstract.
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`33. The ’197 patent identifies conventional microtiter well plates, glass plates
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`having “an array of depressions or wells,” and polystyrene plates having
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`wells as examples of non-porous solid supports to which nucleic acids may
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`be fixed or immobilized. Ex. 1001, 8:65-9:5; 11:56-58; 12:7-26; and 12:54-
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`58.
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`34. The ’197 Patent also explains that polynucleotide analyte sequences fixed or
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`immobilized to the non-porous solid supports may be hybridized to
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`complementary polynucleotide or oligonucleotide probes. See e.g., id. at
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`1:23-32; 5:61-6:32; 8:65-9:30. I understand from BD’s legal counsel, as
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`well as my own extensive experience with patents that although not required
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`by any of the challenged claims, the ’197 Patent discusses that the
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`complementary probe may be provided with a chemical label capable of
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`generating a soluble signal. The ’197 Patent also discusses that hybridized
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`probe-analytes may be identified by detecting or quantifying the soluble
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`signal, which in turn may indicate the presence of the specific analyte
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`sequence in a sample of interest.
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`35. As described below, non-porous supports having fixed or immobilized
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`nucleic acids in hybridizable form, and systems comprising such non-porous
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`solid supports, were known. Below I discuss the following publications:
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`Fish (Ex. 1006), Noyes (Ex. 1007), VPK (Ex. 1008), Gilham (Ex. 1019) and
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`Metzgar (Ex. 1009) and Ramachandran (Ex. 1028).
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`VI. CLAIMS 1, 6, 8, 9, 12, 13, 14, 15, 16, 27, 31, 32, 33, 34, 41, 61, 62, 63, 68,
`69, 70, 72, 73, 74, 79, 100, 191, 192, 193, 194, 212, 213, 219, 222, 225, 226,
`227, 230, 233, AND 236 ARE ANTICIPATED BY FISH.
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`A.
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`Fish (Ex. 1008)
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`36. Fish discloses a microradioimmunoassay system for measurement of anti-
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`double-stranded DNA (dsDNA) antibodies (antibodies that bind to dsDNA).
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`Ex. 1006 [Fish], Abstract. Although the main focus was directed toward ds-
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`DNA-specific antibodies, Fish also disclose the measurement of antibodies
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`that bind ssDNA (ssDNA). It was necessary to measure this activity in
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`serum samples in order to optimize the specificity of dsDNA-binding
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`antibodies and design the best performing assay for ds-DNA activity.
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`Furthermore, binding to ssDNA was a necessary control in the S1 nuclease
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`experiments (see below). Specifically, Fish discloses immobilizing of
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`double-stranded and ssDNA to plastic microtitration wells, incubating
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`patient sera in the DNA-coated wells, and detecting antibodies in patient
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`sera that bind to DNA; those antibodies are detected using second,
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`radiolabeled anti-Ig antibodies (the second antibodies bind to antibodies).
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`Id. at Abstract; Figure 1 (showing measurements of anti-DNA antibodies
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`bound to the DNA-coated wells).
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`B.
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`Fish discloses surface treatment of plastic microtitration trays
`with poly-L-lysine to bind nucleic acids in hybridizable form to
`the surface of the microtitration wells.
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`37. All of independent claims 1, 6, 8, 9, 12, 13, 14, and 15 refer to a “non-
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`porous solid support.” Claim 27 recites a “non-porous glass or a non-porous
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`plastic support.” I understand that all of the rest of the claims listed in the
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`VI header above depend from one or more of claims 1, 6, 8, 9, 12, 13, 14,
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`15, and 27, and thus also include the “non-porous solid support” limitation.
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`38. Fish discloses the binding of ssDNA to the wells of a microtitration tray
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`(also known as microtiter plate). It is well known that wells of
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`microtitration trays are non-porous. In particular, the microtitration trays
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`used in Fish are made of non-porous polyvinyl. The ’197 patent is
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`consistent with that understanding, as it states that “the polynucleotide
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`analyte sequence can be fixed “directly to a non-porous solid support, such
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`as a conventional microtiter well . . . .” Ex. 1001:12:54-57.
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`39. Moreover, wells of a microtitration tray must hold liquid in order to
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`successfully carry out the processes conducted in the wells—incubation,
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`washing, detection, etc.—and therefore, the microtitration trays must be non-
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`porous. The Patent Owner agreed with that correct technical understanding
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`when it explained that supports or systems that contain solutions must be
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`non-porous. For example, the Patent Owner stated during prosecution of
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`European Patent No. 0107440 (Application No. 84100836.0-2106) that “[a]
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`support or system...to which DNA is bound, being a depression or a well and
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`allowing the determination of the DNA whereby washing steps and substrate
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`reactions...are performed in the support/system must be non-porous.” Ex.
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`1016 at pp. 6-7. Thus, the Patent Owner itself correctly agrees that surface
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`of wells or depressions, including those of a microtitration tray, are in fact
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`non-porous.
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`40. The polyvinyl microtitration well disclosed by Fish is a non-porous solid
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`support as set forth in all of claims 1, 6, 8, 9, 12, 13, 14, 15, 16, 27, 31, 32,
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`33, 34, 41, 61, 62, 63, 68, 69, 70, 72, 73, 74, 79, 100, 191, 192, 193, 194,
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`212, 213, 219, 222, 225, 226, 227, 230, 233, and 236 (“the first set of
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`challenged claims”).
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`41.
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`I understand that each of the first set of challenged claims require the solid
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`support to have at least one or more amine(s), hydroxyl(s), or epoxide(s),
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`and a single-stranded nucleic acid must be fixed or immobilized in
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`hybridizable form to the solid support via one of those groups. Fish
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`describes coating the wells of a microtitration tray with poly-L-lysine (PLL,
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`also referred to as PPL) prior to binding of nucleic acid. Ex. 1006, p. 536,
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`left col., first two full ¶¶. As described in the section titled, “DNA coated
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`microtitration trays,” a 50 (cid:541)g/mL solution of PLL was added to each well of
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`a microtitration tray, incubated for 45 minutes at room temperature, and then
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`washed three times with normal saline. Id. This process resulted in a
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`microtitration tray coated with PLL.
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`42. The PLL treatment provides amine groups on the surface of the wells of the
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`microtitration tray. See, e.g., claims 42, 182, and 215 of the ’197 Patent
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`(reciting that surface treatment with polylysine provides amine groups) (Ex.
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`1001, 17 (claim 42), col. 23 (claim 182), col. 25 (claim 215)); see also Ex.
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`1017 [Taylor] at p. 2 (stating that “PLL...present amine functional groups
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`….”)
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`43. The creation of amine groups on the surface of a solid support is useful for
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`the immobilization of nucleic acids and other molecules to the support
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`(including non-porous solid support). Ex. 1017 [Taylor] at p. 2. The
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`cationic nature of PLL makes it an attractive molecular coating for the
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`adhesion of negatively charged biomolecules, such as DNA, as described in
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`detail in ¶ 53 below. See, e.g., Ex. 1006 [Fish] at p. 535, left. col., first full ¶
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`(stating that PLL is “a positively charged polymer”); see also Ex. 1018
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`[Aotsuka] at p. 160 (discussing that it is reasonable to apply PLL for
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`coupling with strongly charged antigens such as dsDNA and that bonding of
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`dsDNA and PLL is ionic).
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`44. Fish discloses successful immobilization of ssDNA (a mixture of poly-dA
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`and poly-dC as well as denatured calf thymus DNA) to the PLL-coated
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`microtitration trays. Ex. 1006 [Fish], Abstract; p. 536, left col., first two full
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`¶¶; Figure 1 (Description).
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`45. Fish’s data in Fig. 1 confirm that the ssDNA (the poly dA + poly dC, and the
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`denatured calf thymus DNA) was bound to the PLL coated wells. Figure 1
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`at p. 539 of Ex. 1006 shows the results obtained after nuclease S1 treatment
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`of the three different types of immobilized DNA listed for two different
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`patients (see the second col. of Fig. 1 under the heading “Nucleic Acid.” S1
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`digests ssDNA but not double-stranded DNA. Ex. 1006, p. 538, right col., ¶
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`U.S. Patent No. 7,064,197
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`1. The empty and black bars in the last col. of Fig. 1 show the amount of
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`antibody bound to the DNA in the wells without S1 treatment (-) and with
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`S1 treatment (+), respectively. Ex. 1006, p. 539 Fig. 1. The longer the bar,
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`the higher the level of bound antibody. After addition of serum to the wells
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`and the anti-DNA antibodies in the serum are allowed to bind to the
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`immobilized DNA, the wells are washed to remove antibodies that are not
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`bound to the immobilized DNA and those that remain bound to the DNA are
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`detected with a second, labeled anti-Ig antibody that bind to remaining
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`antibodies. Ex. 1006, p. 536, left col., third full ¶ and ¶ bridging the cols.; p.
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`538, right col., first ¶.
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`46. The data in the last col. of Fig. 1 show that in the wells having bound
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`double-stranded poly dA-T for both patients (O.N. and B.E.) (see the top
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`two bars in the first row (poly dA-dT)), there was virtually no difference in
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`binding with or without S1 treatment. Id. (The first row for each patient
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`shows approximately equal length empty and black bars (equal anitbody
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`binding) extending to about 200). That was the expected result, since S1
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`does not digest double-stranded DNA and all of the immobilized DNA
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`would still be available for binding after S1 treatment.
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`47.
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`In the wells with immobilized single-stranded nucleic acid, for both patients,
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`there was significantly less antibody binding to the immobilized single-
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`U.S. Patent No. 7,064,197
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`stranded nucleic acid with S1 treatment than without S1 treatment. This is
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`shown by the data in the second and third row (“poly dA + dC” and
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`“denatured DNA”) for each patient, which show shorter black bars (S1
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`treated) than empty bars (no S1 treatment). That is the expected result for
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`ssDNA, since S1 digests ssDNA. After S1 treatment which digests single-
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`strands, less immobilized ssDNA would be available for antibody binding.
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`48. Those results prove that ssDNA must have been immobilized to the PLL-
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`coated wells. Because the wells are washed after the antibody is allowed to
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`bind to immobilized DNA, the antibody would have been washed away
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`during the experiment if there was no DNA available for binding.
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`Furthermore, there would have been no difference in antibody binding with
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`or without S1 treatment if immobilized ssDNA was not present.
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`49. The data in Figures 3 and 4 of Fish, which show binding of antibodies to
`
`immobilized ssDNA, also proves the presence of immobilized ssDNA. Ex.
`
`1006, p. 539, left col., first full ¶; p. 540, right col.- Fig. 3 (data for the
`
`single-stranded immobilized denatured calf thymus DNA); p. 541, left col.-
`
`Fig. 4 (data for the immobilized single-stranded poly dA and poly dC). Fish
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`describes measures that were taken to block non-specific antibody binding
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`directly to the surface of wells, and that allowed Fish to conclude that the
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`data in Figs. 3 and 4 show binding of antibodies to immobilized ssDNA.
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`U.S. Patent No. 7,064,197
`Declaration of Dr. Norman Nelson
`Ex. 1006, p. 537, left col., fourth line under Table 2, through the paragraph
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`bridging the cols. on p. 537.
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`50. Specifically, Fish disclosed that a solution of 2% bovine gamma globulin
`
`(BGG) and 1% bovine serum albumin (BSA) was added to the wells to
`
`block the antibodies from binding non-specifically to the wells. Id. at p.
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`536, left col., third full ¶. Non-specific binding to the wells means antibody
`
`binds to the surface of the wells rather than to DNA immobilized on the
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`wells. Thus, Fish accurately reported that the data in Figs. 3 and 4 was due
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`to binding of the antibodies to immobilized ssDNA (specific binding).
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`Those data provide further evidence that ssDNA in Fish bound effectively to
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`the PLL-coated wells of the microtitration trays.
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`51. Figure 1 also demonstrates that the denatured calf thymus DNA remained in
`
`single-stranded form following immobilization. This means that the single
`
`complementary strands of the calf thymus DNA that was bound to the PLL
`
`did not anneal to each other. Thus, if complementary single-stranded
`
`nucleic acids had been provided in a hybridizing solution, the bound calf
`
`thymus DNA would have been accessible for hybridization. If the single
`
`strands of the denatured calf thymus DNA had annealed to each other to
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`form double strands, treatment with the nuclease would not have diminished
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`the antibody binding results that are clearly shown in Fig. 1 (third row for
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`U.S. Patent No. 7,064,197
`Declaration of Dr. Norman Nelson
`each patient which shows a long empty bar (without S1 treatment) and a
`
`very short black bar (with S1 treatment)).
`
`52. With respect to binding of double-stranded DNA, Fish reports that poly dA-
`
`dT did not bind to polyvinyl surfaces without PLL treatment. Ex. 1006
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`[Fish] at p. 536, right col., second full ¶ (titled “The effect of PLL treatment
`
`on DNA surface binding.”). The binding of single-stranded DNA also
`
`would be facilitated by the PLL coating because both single- and double-
`
`stranded DNA have negative charges that ionically interact with the positive
`
`charges of the PLL.
`
`53. The DNA in Fish is immobilized to the microtitration tray wells via one or
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`more of the amines on the surface of the PLL coated trays. It is my
`
`understanding that it is known that the amine groups of PLL form non-
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`covalent bonds with nucleic acids via ionic interactions between the positive
`
`charges of the amine groups and the negative charges of the phosphate
`
`groups in the DNA backbone. Ex. 1001 [the ’197 Patent] at 8:57-60 (stating
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`that alkylamine treated surfaces are suitable for immobilizing negatively
`
`charged polyelectrolytes); see also Ex. 1020 [Diehl patent publication] at ¶
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`[0019] (“For the ionical binding, use is made of the fact that nucleic acids
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`are generally negatively charged. By providing positive charges on the
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`surface of the carrier, binding between the negatively charged nucleic acids
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`U.S. Patent No. 7,064,197
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`and the positively charged surface of the carrier can be achieved by an
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`interaction of the charges. For this purpose, glass surfaces coated with
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`compounds providing positive charges, e.g. coated with poly-L-lysine and/or
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`aminosilane, are used. Such activated slides are well-known in the art.”).
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`54. Fish does not explicitly discuss the immobilized single-stranded DNA is in
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`hybridizable form, because the Fish assay did not involve hybridization of
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`such ssDNA. I understand that the term “hybridizable form” does not
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`require a showing of actual hybridization of two strands. Rather, it means
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`that the single-stranded nucleic acid is capable of binding through Watson
`
`Crick base pairing.
`
`55. Single-stranded DNA immobilized onto a solid support via the PLL method
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`used in Fish is necessarily in hybridizable form. This is clearly
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`demonstrated in subsequent research papers that utilize the PLL
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`immobilization method. For example, Diehl (Ex. 1021) discloses coating of
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`microscope slides with PLL, followed by the binding of single-stranded
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`DNA to the PLL-coated slides, and then subsequent hybridization of the
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`immobilized single-stranded DNA to complementary sequences that are
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`labeled with fluorescent probes. Ex. 1021, pp. 1-2. Diehl also discloses that
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`the microscope slides with the hybridized DNA were imaged using a
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`ScanArray 5000 unit to detect the fluorescent signals that show
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`U.S. Patent No. 7,064,197
`Declaration of Dr. Norman Nelson
`hybridization to the immobilized DNA. Id. at p. 2. Diehl discloses strong
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`fluorescent signals at the target-positive spots of the array (see id. at Figure
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`1). Those signals clearly demonstrate that the immobilized DNA is in
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`hybridizable form. Without hybridization, there would be no signal.
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`56. The PLL coating method used by Fish is similar to the method used in
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`Diehl. Diehl cites a Stanford laboratory protocol1 for the PLL coating
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`method. See Ex. 1021 at 1, right col, first sentence under the heading
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`“Fabrication of microarrays.” The Stanford laboratory protocol states that
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`PLL was purchased from Sigma (product # P8920). Ex. 1032. Sigma
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`reports that the Sigma PLL (product #P8920) was at a weight/volume
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`concentration of 0.10 % (w/v) in water (Ex. 1033). This means that the
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`concentration of purchased PLL was 1000 (cid:541)g/mL. The Stanford protocol
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`shows diluting PLL ten-fold (70 mL PLL + 70 mL PBS + 560 mL water (70
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`mL of a total volume of 700 mL), which yields a 100 (cid:541)g/mL solution of
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`PLL. Thus, Diehl coated the microscope slides with a 100 (cid:541)g/mL solution
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`of PLL using the Stanford protocol cited at page 1 of Ex. 1021.
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`57. Fish coats the microtitration tray wells with PLL by incubating them in a 50
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`(cid:541)g/mL solution of PLL in Tris-HCL buffer. Ex. 1006, p. 536, left col., first
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`1 http://cmgm.stanford.edu/pbrown/protocols/1_slides.html (last visited March 25,
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`2016).
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`two full ¶¶. A slide coated with 50 (cid:541)g/mL solution of PLL necessarily binds
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`single-stranded DNA in hybridizable form, which is evidenced by the
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`binding of single-stranded DNA to solid supports coated with the 100 (cid:541)g/mL
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`solution of PLL as demonstrated by Diehl.
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`58.
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`If there would have been any impact on the capability of the ssDNA to
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`hybridize caused by PLL co
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