ILLUMINA, INC. v.
`THE TRUSTEES OF COLUMBIA UNIVERSITY
`IN THE CITY OF NEW YORK
`
`Case IPR2018‐00291 (Patent 9,718,852)
`Case IPR2018‐00318 (Patent 9,719,139)
`Case IPR2018‐00322 (Patent 9,708,358)
`Case IPR2018‐00385 (Patent 9,725,480)
`Case IPR2018‐00797 (Patent 9,868,985)
`Illumina’s Demonstratives
`For Oral Hearing
`Judge James A. Worth
`Judge Michelle N. Ankenbrand
`Judge Brian D. Range
`March 5, 2019
`
`DEMONSTRATIVE EXHIBIT – NOT EVIDENCE
`
`
`
`
`
`Columbia Ex. 2017
`Illumina, Inc. v. The Trustees
`of Columbia University in the
`City of New York
`IPR2020-01177
`
`

`

`ILLUMINA, INC. v.
`THE TRUSTEES OF COLUMBIA UNIVERSITY
`IN THE CITY OF NEW YORK
`
`Case IPR2018‐00291 (Patent 9,718,852)
`Case IPR2018‐00318 (Patent 9,719,139)
`Case IPR2018‐00322 (Patent 9,708,358)
`Case IPR2018‐00385 (Patent 9,725,480)
`Case IPR2018‐00797 (Patent 9,868,985)
`Illumina’s Demonstratives
`For Oral Hearing
`Judge James A. Worth
`Judge Michelle N. Ankenbrand
`Judge Brian D. Range
`March 5, 2019
`
`DEMONSTRATIVE EXHIBIT – NOT EVIDENCE
`
`Illumina Ex. 1139
`
`Illumina v. Columbia
`IPR2018-00291, -00318, -00322,
`-00385, and -00797
`
`

`

`Columbia disputes two limitations
`
`What i claimed i :
`1. An adenine deoxyribonucleotide analogue having the
`stnicture:
`
`0
`0
`0
`II
`II
`II
`o·-P-0-P - 0-P - O~
`I
`I
`I
`o·
`o·
`
`wherein R fc;)I represents a small, chemically cleavable
`chemicafg?oup capping the oxygen at the 3' position of
`the deoxyribose of the deoxyribonucleotide analogue,
`~
`does not interfere with recognition of the analogue
`as a ubstrate by a DNA polymerase, (c) is table during
`a
`po ymera
`reac 1011, an
`ketone group;
`wherein OR i not a methoxy group or an ester group;
`wherein the covalent bond between the 3'-oxygen and R
`is stable during a DNA polymerase reaction;
`wherein tag represents a detectable fluorescent moiety;
`wherein Y represent a chemically cleavable, chemical
`linker which (a) does not interfere with recognition of
`the analogue as a substrate by a DNA polymerase and
`(b) is stable during a DNA polymerase reaction; and
`wherein the adenine deoxyribonucleotide analogue:
`i) is recognized as a su bstrate by a DNA polymerase,
`ii) is incorporated at the end of a growing strand of DNA
`during a DNA polymera e reaction,
`iii) produces a 3'-0H group on the deoxyribose upon
`cl avag of R,
`iv) no longer includes a tag on the base upon cleavage of
`Y, and
`v) is capable of forming hydrogen bonds with thymine or
`a thymine nucleotide analogue.
`
`2
`
`DEMONSTRATIVE EXHIBIT – NOT EVIDENCE
`
`wherein R l(a)lrepresents a small, chemically cleavable,
`chemical group capping the oxygen at the 3' position of
`the deoxyribose of the deoxyribonucleotide analogue,
`l(b)ldoes not interfere with recognition of the analogue
`as a substrate by a DNA polymerase,
`
`Ex. 10011 at 34:2‐35:4
`1All citations are to exhibits and papers from 
`IPR2018‐00291 unless otherwise indicated.
`
`

`

`Columbia’s incorrect arguments
`
`• Tsien does not disclose the 3’‐O‐allyl capping group
`• Metzker would have led a POSA to believe that 3’‐
`O‐allyl is inefficiently incorporated
`• It was not possible to cleave the allyl ether under 
`SBS‐compatible conditions
`
`3
`
`DEMONSTRATIVE EXHIBIT – NOT EVIDENCE
`
`Reply at 
`6‐22
`
`

`

`Tsien’s 3’‐O‐allyl capping group
`
`30
`
`A wide variety of hydroxyl blocking groups are
`25 cleaved selectively using chemical procedures other than
`base hydrolysis. 2,4-Dinitrobenzenesulfenyl groups are
`cleaved rapidly by treatment with nucleophiles such as
`thiophenol and thiosulfa~e (Letsinger et al . , 1964).
`Allyl ethers are cleaved bJ· treatment with HgrII) in
`acetone/water (Gigg and Warren, 1968).
`Tetrahydrothiofuranyl ethers are removed under neutral
`conditions using Ag(I) or Hg(Il) (Cohen and Steele, 1966;
`Cruse et al., 1978). Th~se protecting groups, which are
`stable to the conditions used in the synthesis of dNTP
`analogues and in the sequence incorporation steps, have(cid:173)
`some advantages over groups cleavable by base hydrolysis -
`deblocking occurs only when the specific deblocking
`reagent is present and premature deblocking during
`incorporation is minimized.
`
`35
`
`-
`
`4
`
`DEMONSTRATIVE EXHIBIT – NOT EVIDENCE
`
`Ex. 1013 at 24:25‐25:3
`Petition at 21‐22
`
`

`

`Dr. Menchen’s Testimony
`
`5
`
`6
`
`7
`
`8
`
`9
`
`10
`
`11
`
`12
`
`13
`
`14
`
`15
`
`16
`
`17
`
`Q.
`
`And there Tsien is
`
`referring to allyl ethers; is that
`
`correct? That ' s how a person of
`
`ordinary skill in the art would
`
`understand this disclosure?
`
`MR. SCHWARTZ: Objection,
`
`form.
`
`A.
`
`So a person with ordinary
`
`skill in the art would understand
`
`that the
`
`that allyl ethers in
`
`general had some advantages, that
`
`they wouldn't be cleaved by base
`
`hydrolysis when deblocking occurs.
`
`5
`
`DEMONSTRATIVE EXHIBIT – NOT EVIDENCE
`
`Ex. 1113 at
`326:5‐17
`
`Reply at
`5
`
`

`

`3’‐O‐allyl meets all claim requirements for ‘R’
`
`Dr. Ju’s testimony
`
`a. Only a
`
`limited number of 3'-0 capping groups meet
`
`the
`
`standard of "small" along with
`
`the other structural and
`
`I estimate the
`functional features recited in the claim.
`number of such groups would be less than 10 and 2 examples
`
`of such groups were provided.
`
`Ex. 1022 at
`¶22a
`
`Dr. Menchen’s testimony
`
`22
`
`23
`
`24
`
`25
`
`2
`
`3
`
`Q .
`
`At some point it was
`
`demonstrated that allyl would work
`
`as a capping group for the claimed
`
`inventions of the Ju patents,
`
`correct?
`
`A .
`
`That's -- that's correct.
`
`6
`
`DEMONSTRATIVE EXHIBIT – NOT EVIDENCE
`
`Ex. 1112 at 
`296:22‐297:3
`
`Reply at 8;
`Petition at 22, 24, 25
`
`

`

`“Allyl” means “‐CH2‐CH=CH2”
`
`IUPAC Definition
`3.5-The names of univalent radicals derived from unsaturated acyc1ic
`hydrocarbons have the endings " -enyl ,, , " -ynyl ", " -dienyl ", etc., the
`positions of the double and triple bonds being indicated where necessary . .
`The carbon atom with the free valence is numbered as I.
`
`Examples:
`Ethynyl
`2•Propynyl
`l•Propenyl
`2-Butenyl
`1,3-Butadienyl
`2-Pentenyl
`2-Pen ten-4-yny 1
`
`CH==C-
`CH==C-CH 2-
`CH3-CH=CI-I-.
`CH 3-CH =CH-CH2-
`CH2=CH-Cl-I=CH(cid:173)
`CH 8-CH 2-CH=CH-CH2-
`CH==C-CH=CH-CH2-
`
`Exceptions:
`The following names are retained :
`Vinyl (for ethenyl)
`Allyl (for 2-propenyl)
`Isopropenyl (for 1-methylvinyl)
`
`CH 2=CH(cid:173)
`CH2=CH-CH2-
`CH 2=C-
`(for unsubstituted
`I
`radical only)
`CH3
`
`7
`
`DEMONSTRATIVE EXHIBIT – NOT EVIDENCE
`
`Ex. 1099 at 13
`Reply at 4
`
`

`

`Greene and Wuts
`
`Allyl Ether (Allyl-OID: CH2=CHCH2-OR (Chart 1)
`
`The use of allyl ethers for the protection of alcohols is common in the literature
`on carbohydrates because allyl ethers are generally compatible with the various
`methods for glycoside fonnation. 1 Obviously~ the allyl ether is not compatible
`with powerful electrophiles such as bromine and catalytic hydrogenation, but it
`is stable to moderately acidic conditions (1 N HCI, reflux, l O h).2 The ease of
`fonnation, the many mild methods for its cleavage in the presence of numerous
`other protective groups, and its general stability have made the ally 1 ether a
`mainstay of many orthogonal sets. The synthesis of perdeuteroallyl bromide and
`its use as a protective group in carbohydrates has been reported. The perdeutero
`derivative has the advantage that the allyl resonances in the NMR no longer
`obscure other, more diagnostic resonances, such as those of the anomeric carbon
`in glycosides. 3 The use of the allyl protective group primarily covering carbohy-
`drate chemistry has been reviewed.4
`·
`
`8
`
`DEMONSTRATIVE EXHIBIT – NOT EVIDENCE
`
`Ex. 1101 at 67;
`Reply at 4, 19
`
`

`

`Gigg and Warren
`
`The Allyl Ether as a Protecting Group in Carbohydrate Chemistry.
`Part II la
`By Roy Gigg • and C. D. Warren, National Institute for Medical Research, Mill Hill, London N.W.7
`
`Ex. 1046 at title
`
`The 2-methylallyl ether (XLIX) of 1,2:5,6-di-O-iso(cid:173)
`propylidene-n-glucofuranose was prepared and the rates
`of isomerisation of this compound and of the allyl ether
`(LI) were compared. The allyl ether was isomerised
`about twenty times more quickly than compound
`(XLIX) which was converted into the crystalline ether
`(L).
`~ re have shown 10 that y-substituted allyl ethers are
`eliminated to give dienes by treatment with potassium
`t-butoxide in dimethyl sulphoxide and this has been
`confirmed by others. 25 The action of these basic con(cid:173)
`ditions on the 3-methylallyl ( crotyl} ether (LII) 26 of
`1,2:5,6-di-O-isopropylidene-n-glucofuranose was investi(cid:173)
`gated.
`
`9
`
`DEMONSTRATIVE EXHIBIT – NOT EVIDENCE
`
`0
`I
`O-CMe2
`(XLIX) R = CH2-C(Me)=CH 2
`(L) R = CH=CMe2
`(LI) R = CH2-CH=CH2
`(LII) R = CH2-CH=CH-CH3
`
`Ex. 1046 at 1906‐07
`Reply at 5; 
`Sur‐reply at 9
`
`

`

`Metzker
`
`Improvements to the Sanger protocols are being sought to meet
`the increasing demands of large scale sequencing of whole
`genomes (14). We and others (15-18) have independently
`conceived a radically different, gel-free alternative to the Sanger
`scheme for DNA sequencing. This metlioo, called the Base
`Addition Sequencing Scheme (BASS), is based on novel
`nucleotide analogs that terminate DNA synthesis. BASS involves
`repetitive cycles of incorporation of each successive nucleotide,
`in situ monitoring to identify the incorporated base, and
`deprotection to allow the next cycle of DNA synthesis, (Figure
`1). Compared to Sanger sequencing, BASS has two major
`advantages: base resolution would not require gel electrophoresis
`and there is a tremendous capacity for simultaneous analyses of
`multiple samples. The complete scheme demands nucleotide
`analogs that are tolerated by polymerases, spectroscopically
`distinct for each base, stable during the polymerization phase,
`and deprotected efficiently under mild conditions in aqueous
`solution. These stringent requirements are formidable obstacles
`for the design and synthesis of the requisite analogs.
`
`REFERENCES
`15. Tsien, R. Y., Ross, P., Fahnestock, M., and Johnston, A. J., PCT number
`WO 91/06678, 'DNA sequencing. ', filed: October, 26, 1990, published: May
`16, 1991.
`DEMONSTRATIVE EXHIBIT – NOT EVIDENCE
`
`Ex. 1016 at 4259, 4267; Petition at 23, 62
`
`10
`
`

`

`Metzker
`
`9 9 9
`HCrf-0-f-O-f~~denine
`0-0-0-
`
`'r-1
`
`where P' =
`
`OP'
`
`;,i
`~
`
`'>'1(CH3 ~C"2 15
`i o~'()
`
`0
`2
`
`"2N
`
`3
`
`4
`
`A
`
`IA
`
`1
`
`;;,! 0
`
`, ~
`
`A
`
`N02
`5
`
`6
`9 9 9
`HO-f-O-f-0-f~°'JThyrnine
`0-
`0-
`0-
`
`7
`
`'r-1
`
`OCH3
`8
`Figure 2. Chemical structures of the 3 '-modified-nucleotides. Details of the
`chemical syntheses are described in the Materials and Methods section.
`
`11
`
`DEMONSTRATIVE EXHIBIT – NOT EVIDENCE
`
`Ex. 1016 at 4260
`Petition at 59
`
`

`

`Dr. Menchen’s 1998 patent
`
`United States Patent 119J
`Henson et al.
`
`( 111 Patent um her:
`(45 ] Date or Patent:
`
`6,111,116
`Aug. 29, 2000
`
`[54( OIRF.N7,(lRH0f>AMI NF. l)Y F.S
`
`[7S]
`
`lnvcnlor.;: Scott C. Benso n; J oe Y. L Lam;
`Ste,·en Mlcl1a el Menchen, all uf Fuster
`C i1y, Calif.
`
`[73) m;signee: Tito Perkin-Elmer Corporation, Foster
`L'ily, Calif.
`
`Sun c t al., ··Syn1hcsis o r f luorina rccl Fluorcsccins,'' J _ Org.
`Chem. 62(19) 6469-6475 (1997).
`
`Pri11wry F.xam;11er--:Z.inn a Northing1on Oavi.
`A rrom ey, Agelll, or Firm-Paul 0. Grossman; Alex Andrus
`ABSTRACT
`[S7]
`
`Dihcn:,.orhoda minc compounds hav ing lhc s1ructurc
`
`f21) Appl. Nn.: 09/ 199,402
`
`[22]
`
`riled:
`
`Nu,·. 24, 1998
`
`Rclllll'<l U.S. Application Dnta
`
`37. The compound of claim 1 wherein the nucleoside,
`nucleotide or analog comprises the structure:
`
`B-D
`
`***
`
`Exem(cid:173)
`plary sugar analogs include but are not limited to 2'- or
`3'-modifications where the 2'- or 3'-position is hydrogsn,
`hydroxy, alkoxy, e.g., methoxy, ethoxy, allyloxy,
`isopropoxy, butoxy, isobutoxy and phenoxy, ammo or
`alkylamino, fluoro, chloro and bromo.
`
`wherein D is a dibenzorhodamine dye, B is a nucleobase;
`and B is connected to D by a linkage;
`W 1
`is OH, H, F or a group capable of blocking
`polymerase-mediated template polymerization;
`is OH, H, F or a group capable of blocking
`W 2
`polymerase-mediated template polymerization; and
`W 3 is OH, monophosphate, diphosphate, triphosphate, or
`phosphate analog.
`
`Note:  3’‐allyloxy is 3’‐O‐allyl
`Ex. 1119 at ¶26
`Ex. 1112 at 49:25‐50:6
`
`12
`
`DEMONSTRATIVE EXHIBIT – NOT EVIDENCE
`
`Ex. 1091 at 
`5:56‐61, 27:35‐52
`
`Reply at 
`6‐7
`
`

`

`Dr. Menchen’s 1999 patent
`
`, 12) United States Patent
`Lam ct al.
`
`( 10) Patent o.:
`(45) Date of Patent:
`
`US 6,248,884 Bl
`Jun. 19, 2001
`
`lex Andrus
`(74) Alwrm:y, l\g(?nJ, or Firm
`AIISTRACT
`
`(57)
`
`Extended rbodamine compounds exhibiting favorable lluo(cid:173)
`rcsccncc cbaractcristi{".S bav iag the s truc ture
`
`Particularly preferred nucleosides/tides of the present
`invention are shown below wherein
`
`B-1:-D
`
`l)F,I) RHOllA nm: COMPO
`(54) F:XTF,
`USEFUL AS FL ORESCENT lAilELS
`
`l)S
`
`(75)
`
`lnveDLors: J oe Y. L. Lum , Castro Valky; cull C.
`!Jenso n, Alame<la; Steven M.
`Menchen. Fremont, all of CA (US)
`
`(73) As.signee: T h,, l'c rkln-~lnrnr C orp ora tio n , Fos1 r
`Ci1y, CA (US)
`
`( • )
`
`oticc:
`
`Subject to any disclaimer, the term of this
`patent is extended or adju sted under 35
`U.S.C. !54(b) by O days.
`
`(21 ) Appl. o.: 09/325,243
`
`(22) Filed:
`
`Jun. 3, 1999
`
`***
`
`Exemplary modified
`pentose portions include but are not limited to 2'- or
`3'-modifications where the 2'- or 3'-position is hydrogen,
`hydroxy, alkoxy, e.g., methoxy, ethoxy, allyloxy,
`isopropoxy, butoxy, isobutoxy and phenoxy, azido, amino or
`alkylamino, fl.uoro , chloro, bromo and the like.
`***
`Nucleotide terminators also include reversible nucleotide
`terminators (Metzker et al., Nucleic Acids Research) 22(20):
`4259 (1994)).
`
`B is a nucleoside/tide base, e.g., uracil, cytosine,
`deazaadenine, or deazaguanosine; W 1 and W 2 taken sepa(cid:173)
`rately are H or a group capable of blocking polymerase(cid:173)
`mediated template-directed polymerzation, e.g., -H, fluo(cid:173)
`rine and the like; W 3 is OH, or mono-, di- or triphosphate or
`phosphate analog; D is a dye compound of the present
`invention; and Lis a covalent linkage linking the dye and the
`nucleoside/tide .
`
`13
`
`DEMONSTRATIVE EXHIBIT – NOT EVIDENCE
`
`Ex. 1092 at 10:40‐45, 11:22‐24, 52:17‐35
`Reply at 6‐7
`
`

`

`Dr. Menchen’s Testimony
`
`8
`
`9
`
`10
`
`11
`
`12
`
`13
`
`Q.
`
`I didn ' t mean to exclude
`
`anything.
`
`My question to you is in
`
`your
`
`' 116 patent you proposed
`
`allyloxy as a synthetic modification
`
`at the 3 ' location ; is that correct?
`
`A.
`
`That ' s correct.
`
`Ex. 1112 at
`64:8‐13
`
`14
`
`DEMONSTRATIVE EXHIBIT – NOT EVIDENCE
`
`14
`
`15
`
`16
`
`17
`
`18
`
`19
`
`20
`
`21
`
`22
`
`23
`
`Q .
`
`I ' m just saying if it was
`
`some
`
`the mere fact that you can
`
`make something if you knew it would
`
`be completely disruptive of DNA
`
`sequencing , you wouldn ' t have listed
`
`it here , how about that? Let me ask
`
`the easy question.
`
`A.
`
`I don't think -- yeah .
`
`If
`
`we knew it was going to fail you
`
`probably wouldn't make it .
`
`Ex. 1112 at
`80:14‐23
`
`Reply at 
`6‐7
`
`

`

`Dr. Menchen’s Testimony
`
`5
`
`6
`
`7
`
`8
`
`9
`
`10
`
`11
`
`12
`
`13
`
`Q.
`
`How come you still
`
`identified allyl groups as a
`
`suitable modifier for 3' position in
`
`your patents?
`
`A.
`
`Because that's what Metzker
`
`calls it. But he definitely refers
`
`to the structure of propenyl in that
`
`paper.
`
`He calls it allyl in the
`
`paper.
`
`Ex. 1112 at
`189:5‐13
`
`15
`
`DEMONSTRATIVE EXHIBIT – NOT EVIDENCE
`
`Reply at
`15
`
`

`

`Hiatt
`
`United States Patent [19J
`Hiatt et al.
`
`[11] Patent Number:
`(45] Date of Patent:
`
`5,808,045
`*Sep. 15, 1998
`
`[5~] COMPOSITIO S FOR ENZYME
`CATALYZED TEMPLATE-INDEPENDENT
`C IU:ATION OF PIIOSPIIOlll ESTF,R no ns
`USI (; PROTECTl•:I) NlJCLEOTIDES
`
`[75]
`
`l11 v1;utor:,: Andrew C. Hiatt, 660 Torrnn1.:1; SL.,
`San Diego, Calif. 92 103; Flnyd Rose,
`Del Mar, Ca lif.
`
`[73]
`
`Assignees: Andrew C. Hiatt, San Diego; Floyd D.
`Rose, Del Mar, both of Calif.
`
`Notice:
`
`The term of this patent shall not extend
`beyond 1bc cxpira1ion date of Pat. No.
`5,763,594.
`
`t\ppl. No.: 4!!6,!!97
`
`Filed:
`
`Jun. 7, 1995
`
`Related U.S. Appl1cat1on Data
`
`121 I
`[22]
`
`1631
`
`5,348,868
`.'i,16?.,866
`.'i,167,(l66
`5,380,833
`5,436,143
`
`9/1994 Reddy el al. .
`11 / 1')<)4 Arno ld , Jr ..
`t Jrcda ct al. .
`11 / 1994
`1/1995 Urdea .
`7/ L995 Hymann el al. .
`
`FOREIC.N PATENT DOCU M ENTS
`
`55-38324
`
`3/1980
`
`Japan .
`
`OTHER PUBLICATIO S
`
`Bollum, Fed Proc. Soc. Exp. Biol. Med., 17, 193 (1958).
`Deng and Wu, Meth. Fnzymol., 100: 96-116 (1983).
`Kaufmann et al. , liur. J. Bioch.em, 24:4-11 (1971).
`Himon aud Gumport, Nucleic Acid Res., 7:453--464 (1979).
`Modak, Biochemistry, 17, 3116-3120 (1978).
`am.I Uhl1;nblX:k, Hiochemisl ry,
`England
`vo l.
`17,
`11 :2069-2076 ( I 978).
`C:hang ancl Hollu m, Rinchemist,y, vol. rn, 3::B6- .'i42
`(1971) .
`Beoncll et al., Biochemistry, vol. 12, 20:3956-3960 (1973).
`Kosse! and Roycboudmiy, Eur J. Biochem., 22:271-276
`(1971).
`Flun-el et al., Biochem. Biophys. Acta, 308:35-40 (1973).
`
`Conlinualiou-in-parl of Ser.
`abandoned.
`
`o. 300,484, Sep. 2, 1994,
`
`***
`An alternative type of removable blocking moiety utilizes
`an ether linkage which forms the structure nucleotide-3'-
`0-R'. In this instance R' 1 can be methyl, substituted
`meythyl, ethyl, substituted ethyl, butyl, allyl, cinnamyl,
`benzyl, substituted benzyl, anthryl or silyl.
`
`16
`
`DEMONSTRATIVE EXHIBIT – NOT EVIDENCE
`
`Ex. 1106 at 11:55‐59
`See also Ex. 1106 at 12:39, 30:8; Reply at 6
`
`

`

`The criteria disclosed in Tsien and Columbia’s patents
`
`Tsien
`
`Columbia’s patents
`
`Blocking Groups and Methods for Incorporation
`The coupling reaction generally employs
`3'hydroxyl-blocked dNTPs to prevent inadvertent extra
`additions.
`The criteria for the successful use of
`3'-blocking groups include:
`(1)
`the ability of a polymerase enzyme to
`accurately and efficiently incorporate the dNTPs carr ying
`the 3"-blocking groups into the cDNA chain,
`(2)
`the availability of mild conditions for
`rapid and quantitative debl ocking, and
`( 3)
`the ability of a polymerase enzyme· to
`reinitiate the cDNA synthesis subsequent to the deblocking
`stage .
`
`Ex. 1013 at
`20:24‐21:3
`
`1. The Sequencing by Synthesis Approach
`Sequencing DNA by synthesis involves the detection of
`the identity of each nucleotide as it is incorporated into the
`growing strand of DNA in the polymerase reaction. The
`fundmnental requiren1ents for such a system to work are: (1)
`the availability of 4 nucleotide analogues (aA, aC, aG, aT)
`each labeled with a unique label and containing a chemical
`moiety capping the 3'-0H group ; (2) the 4 nucleotide
`analogues (aA aC aG, aT) need to be efficiently and
`faithfully incorporated by DNA polymerase as tem1inators
`in the polymerase reaction; (3) the tag and the group capping
`the 3'-0H need to be removed with high yield to allow the
`incorporation and detection of the next nucleotide; and ( 4)
`the growing strand of DNA should survive the washing,
`detection m1d cleavage processes to remain mmealed to the
`DNA tern late.
`
`17
`
`DEMONSTRATIVE EXHIBIT – NOT EVIDENCE
`
`Ex. 1001 at
`21:2‐18
`
`Petition at 24, 36;
`Reply at 1, 8, 18
`
`

`

`Dr. Menchen’s Testimony
`
`5
`
`6
`
`7
`
`8
`
`9
`
`10
`
`11
`
`12
`
`13
`
`6
`
`7
`
`8
`
`9
`
`10
`
`11
`
`12
`
`13
`
`Q.
`
`Why don ' t you go through
`
`them one at a
`
`time and state whether
`
`you think the fundamental
`
`requirements for sequencing by
`
`synthesis disclosed in Ju are the
`
`same as those described in the prior
`
`art. Why don t you go through -- do
`
`i t by number and break i t down, that
`
`will be easier .
`
`***
`
`Start with two again .
`
`Two ,
`
`' The four nucleotide
`
`Q .
`
`A.
`
`analogs , AA , AC , AG , AT , need to be
`
`efficiently and faithfully
`
`incorporated by DNA polymerase as
`
`terminators of the polymerase
`
`reaction. ' That s
`
`a common
`
`requirement.
`
`Ex. 1112 at
`258:5‐259:13
`
`18
`
`DEMONSTRATIVE EXHIBIT – NOT EVIDENCE
`
`2
`
`3
`
`4
`
`Q. Whats a high yield in SBS
`
`according to you?
`
`A. Well, quantitative.
`
`Ex. 1112 at
`124:2‐4; id. at 138:4‐9
`
`Reply at
`8, 18
`
`

`

`Columbia relied on Metzker
`
`BACKGROUND OF THE INVENTION
`***
`
`If small
`chemical moieties that can be easily cleaved chemically with
`high yield can be used to cap the 3'-OH group, such
`nucleotide analogues should also be recognized as substrates
`for DNA polymerase. It has been reported that 3'-O(cid:173)
`methoxy-deoxynucleotides are good substrates for several
`polymerases (Axelrod et al. 1978). 3'-O-allyl-dATP was also
`shown to be incorporated by Ventr( exo-) DNA polymerase
`in the growing strand of DNA (Metzker et al. 1994).
`
`19
`
`DEMONSTRATIVE EXHIBIT – NOT EVIDENCE
`
`Ex. 1001 at 3:22‐30; 
`Petition at 23, 24, 59‐62, 65‐66, 72
`
`

`

`Dr. Menchen’s Testimony
`
`6
`
`7
`
`8
`
`9
`
`10
`
`11
`
`12
`
`13
`
`14
`
`15
`
`16
`
`17
`
`18
`
`Q. What did Ju teach about the
`
`incorporation efficiency of the
`
`allyl groups?
`
`MR. SCHWARTZ: Objection,
`
`form.
`
`A.
`
`He taught that they should
`
`be incorporated efficiently.
`
`Q.
`
`Did he teach how that
`
`should be done?
`
`MR. SCHWARTZ: Objection,
`
`scope.
`
`A.
`
`Yeah
`
`I
`
`I don't -- I don't
`
`remember seeing that.
`
`20
`
`DEMONSTRATIVE EXHIBIT – NOT EVIDENCE
`
`Ex. 1112 at
`284:6‐18
`
`Reply at
`9, 23
`
`

`

`Ju provides no new incorporation or cleavage chemistry
`
`Wniteb ~tates QCou11 of ~peals
`for tbe jfeberal QCtrcuit
`
`TRUSTEES OF COLUMBIA UNIVERSITY IN THE
`CITY OF NEW YORK,
`Appellant
`
`v .
`
`ILLUMINA, INC.,
`Appellee
`
`20 14-1547
`
`However, as already
`explained, if novel and nonobvious chemistry was needed
`to practice the claimed inventions, Dr. Ju would have
`been obligated to disclose this chemistry in the patent.
`See 35 U.S. C. § 112(1) (2000).
`
`21
`
`DEMONSTRATIVE EXHIBIT – NOT EVIDENCE
`
`Ex. 1008 at 31;
`Petition at 36‐37, 73; Reply at 2, 9, 23
`
`

`

`3’‐O‐allyl dATP efficiently competes with natural dATP
`Metzker
`
`concentration dependent. Thus, minimum dNTP concentrations
`that gave efficient incorporation, but no apparent misincorporation
`were first defined in this assay. These dNTP concentrations were
`then used to determine the minimum ddNTP concentration that
`yielded complete termination. Pfu (exo- ) DNA polymerase was
`excluded from
`the Oligo-template assay since a ddNTP
`concentration that yielded complete termination for this enzyme
`
`[1] by AMV-RT. In addition to termination, some readthrough
`was also observed due to the presence of contaminating dA TP.
`All RP-HPLC purified 3 '-modified-dA TPs ( compounds [ 1] - [7])
`showed approximately 1 % dA TP contamination, and these trace
`levels could not be removed by subsequent RP-HPLC.
`3'-0-Methyl-dATP [1] wasfilso incorporated ByM=MuLV(cid:173)
`RT and inhibited DNA syntheses by rTth and VentR® (exo - )
`
`Table 2. Activity matrix of RP-HPLC purified 3'-protecting dNTPs challenged against commercially available polymerases
`3 ___ _.,~~ ~~TY
`
`AMV-Kl M-MuLV-RT
`
`.iuenow
`fragment
`
`Sequenase(!) BstDNA
`polymerase
`
`Amphiaq® VentR(exo·)<!l
`DNA
`DNA
`polymerase ~ lymerase
`
`rTthDNA
`polymerase
`
`-
`-
`-
`-
`-
`-
`Inhibition Termination Termination• Termination•
`
`-
`-
`-
`
`-
`-
`-
`
`Inhibition
`
`Inhibition
`
`Termination•
`
`-
`-
`-
`
`Inhibition•
`
`-
`-
`-
`-
`-
`-
`
`-
`-
`-
`-
`
`-
`
`-
`
`-
`-
`-
`-
`
`-
`-
`-
`-
`
`Inhibition
`
`Inhibition Termination Termination Termination Termination
`
`(except compound (8))
`
`(1) 0-methyl
`
`(2)0-acyl
`
`(3) D-allyl
`
`(ti 0-tetrahydropyran
`
`(5) 0-(4-nitrobenzoyl)
`
`[6) 0-(2-aminobenzoyl)
`
`(7) 0-(2-nitrobenzyl)
`
`[8) 3'-0-methyl-dTTP
`
`-
`
`-
`
`-
`
`-
`
`Termination Termination•
`-
`-
`-
`
`-
`
`-
`-
`-
`-
`-
`
`All compounds were assayed at a final concentration of 250 µM according to the conditions specified in Table 1. • - ' means
`no activity was detected, 'Termination' means that the termination bands mimic ddNTP termination bands, and 'Inhibition'
`means the rate of DNA synthesis is reduced in a nonspecific manner. '*' means the activity was incomplete at a final concentration
`of 250 µM .
`
`22
`
`DEMONSTRATIVE EXHIBIT – NOT EVIDENCE
`
`Ex. 1016 at 4263
`Petition at 23; Reply at 9‐10
`
`

`

`Metzker and Columbia disclose the same polymerases
`
`Metzker
`
`Table 2. Activity matrix of RP-HPLC purified 3'-protecting dNTPs challenged against commercially available polymerases
`--:·~ ~ ~ .. ATP
`3'-
`(except compound [8))
`
`AmpliTaq® VentR(exo•)Q\
`DNA
`DNA
`polymerase polymerase
`
`rTthDNA
`polymerase
`
`AMV-Kl M-MuLV-RT
`
`ow
`fragment
`
`SequenaseW BstDNA
`polymerase
`
`Inhibition
`
`Inhibition•
`
`- -
`
`Inhibition
`
`Termination•
`
`-
`-
`-
`
`[1] 0-methyl
`
`Termination Termination•
`
`[2] 0-acyl
`
`[3] 0-allyl
`
`-
`
`-
`
`-
`
`-
`
`-
`-
`-
`
`-
`-
`-
`
`-
`-
`
`-
`
`Columbia’s Patents
`Possible DNA polymerases include Thermo Sequenase,
`Taq PS DNA polymerase, T7 DNA polymerase, and Vent
`( exo-) DNA polymerase.
`
`Ex. 1016 at 
`4263
`
`23
`
`DEMONSTRATIVE EXHIBIT – NOT EVIDENCE
`
`Ex. 1001 at 
`22:4‐6
`
`Reply at
`9
`
`

`

`Vent polymerase incorporation of labeled nucleotides
`
`0
`
`0 NH~t
`'i'r'i'
`I/ 0 , r 0-r-0-r-0-r-0 ·
`0- 0- O-
`""'-'(
`
`NO
`
`OH
`
`m=l for Cy3-4-dUTP
`m=2 for CyS-4-dUTP
`
`Ex. 1040 at 
`3228
`
`24
`
`DEMONSTRATIVE EXHIBIT – NOT EVIDENCE
`
`1 a-1 d
`
`N f:IH2 [o,,....__,,o....,,,..._o,,....__,,o....,,,..._o,,....__,,o....,,,..._NHR
`
`~~
`
`3-H09P30~
`Hd
`
`2a-2b
`
`1 a X = 0, R = H2 +.
`1b X = 0, R = 3.
`1c X = NH2, R = H2 •.
`1 d X = NH2, R = 3.
`2a X = 0, R = H2 +.
`2b X = 0, R = 3.
`
`dUTP-22.
`Biotin-36-dUTP.
`dCTP-22.
`Biotin-36-dCTP.
`dc7 ATP-22.
`Biotin-36-dc7 ATP.
`Ex. 1041 at 
`4833
`
`Petition at 30;
`Reply at 17, 22‐23
`
`

`

`B.
`
`Bst DNA polymerase
`I
`
`Amphiaq® DNA polymerase
`I
`
`<el
`I
`I
`
`I
`I
`I
`C
`I G
`~ (cid:141) Termination
`G
`I G
`I T
`G
`I G
`
`Metzker
`
`rTth DNA polymerase
`
`VentN_® (exo-) D A polymerase
`
`I
`
`...
`
`I
`
`•
`
`I
`I
`I
`
`I
`I
`~ I
`
`I
`I
`I
`
`I
`I
`I
`
`~ (cid:141) Termination
`
`C
`G
`
`G
`G
`T
`G
`G
`
`1 2 3 4 5 6 17 8 9
`Lr
`'-,--1 J
`I
`ddlTP
`3'-0-methyl-dlTP
`
`~ ~ ·
`
`1 2 3 4 s 6 l 7 s 9
`I
`ddlTP
`3'-0-methyl-dlTP
`
`1 2 3 4 5 617 8 9
`1 2 3 4 5 ~ 7 8 9
`L,-J l. ~ I
`L,-J
`.t...,-J _.
`I
`I
`ddTTP
`ddTTP
`3'-0-methyl~TIP
`3'-0-methyl~TTP
`
`Figure 4. Incorporation of 3'-O-methyl-dTTP by Bst, AmpliTaq® , rTth , and
`VentR® (exo-) DNA polymerases.
`(B) Conditions for the Oligo-template assay were used for
`Bst, AmpliTaq® , rTth , and VentR®
`(exo-) DNA polymerases. Lane 1
`contained no dNTPs or ddNTPs. Lanes 2-7 contained dA TP, dCTP and ddGTP.
`In addition, lanes 3-5 contained (Bst) 0.1 µM, 0.5 µM and 2.5 µM ddTTP;
`(AmpliTaq®) 1.0 µM, 5.0 µMand 25 µM ddTTP; (rTth) and (VentR® (exo-))
`4 µM, 20 µM, and 100 µM ddTTP, respectively; lane 6 contained dTTP; and
`lanes 7-9 contained (Bst) 4 µM, 20 µM, and 100 µM of 3'-O-methyl-dTTP;
`(AmQliTa ® ), (rTth ), and (Vent ® (exo-)) 20 µM, 100 ~M, and 500 µM of
`3 '-O-methyl-dTTP, respectively.
`DEMONSTRATIVE EXHIBIT – NOT EVIDENCE
`
`Ex. 1016 at Fig 4(B);  Reply at 13
`
`25
`
`

`

`Metzker 1998
`
`(cid:141)
`
`G
`A
`G
`T (cid:141)
`G
`G
`
`R · d hrough
`p· sen · 0 '
`
`TP)
`
`ddATP
`'-0 rneth 1 d
`termin ti n
`
`TP
`
`----=-
`
`-
`
`-
`
`A B y
`
`·rp
`
`H
`
`Following en(cid:173)
`zymatic mop-up, the incorporation of
`3'-0-methyl-dATP by AMV-RT shows
`co1nplete tenninatio without natural
`nucleotide read-through (Figure 3, lanes
`B).
`
`26
`
`DEMONSTRATIVE EXHIBIT – NOT EVIDENCE
`
`Ex. 2131 at 816;  Reply at 12, 14
`
`

`

`Metzker’s Reaction Time
`
`Polymerase incorporation assays
`***
`For each reaction, 5 µL aliquots of the annealed primer(cid:173)
`template samples were dispensed into separate tubes containing
`5 µL mixtures of each enzyme and nucleotides in their specific
`buffers. The final buffer conditions, concentrations of nucleotides,
`enzymatic units, and incubation temperatures are given in Table
`1. The reactions were incubated for 10 min. and then stopped
`by the addition of 5 µL of stop solution containing 98% D.I.
`formamide, 10 mM EDTA, pH 8.0, 0.025% bromophenol blue,
`and 0.025% xylene cyanol.
`
`76.
`
`Finally, a skilled artisan would have appreciated that extending the
`
`reaction tin1e would have likely increased the degree of incorporation, and
`
`termination, for a particular 3 '-O-1nodified dNTP. This is underscored by the fact
`
`that Metzker 1994 appears to run the incorporation reactions for 10 minutes. Ex.
`
`1016 (Metzker 1994) at 4262 (reaction time for Table 1 termination is 10 minutes).
`
`27
`
`DEMONSTRATIVE EXHIBIT – NOT EVIDENCE
`
`Ex. 1016 at 4262; Ex. 1119 ¶76;  Reply at 13
`
`

`

`Dr. Menchen’s Testimony
`
`19
`
`20
`
`21
`
`22
`
`23
`
`24
`
`25
`2
`
`3
`
`4
`
`5
`
`6
`
`7
`
`8
`
`9
`
`13
`
`14
`
`15
`
`16
`
`17
`
`18
`
`Q.
`
`A.
`
`Q.
`
`Are you an enzymologist?
`
`No, I'm not.
`
`Do you know which
`
`polymerases would work with ally!
`
`just based on your technical
`
`expertise in the field?
`
`MR. SCHWARTZ:
`scope.
`
`Objection,
`
`A.
`
`Yeah.
`
`I'm not -- I don't
`
`have the background to -- to say
`
`that or determine that .
`
`Q.
`
`Do you have any idea which
`
`enzymes
`
`would work with MOM?
`
`MR. SCHWARTZ:
`
`Same objection .
`
`A.
`
`Q.
`
`I
`
`I have no idea.
`
`Have you personally worked
`
`with Vent(exo-) DNA polymerase?
`
`MR . SCHWARTZ: Objection to
`
`form.
`
`A.
`
`I have never worked with
`
`any polymerase myself .
`
`28
`
`DEMONSTRATIVE EXHIBIT – NOT EVIDENCE
`
`2
`
`3
`
`4
`
`5
`
`6
`
`7
`
`8
`
`9
`
`10
`
`11
`
`12
`
`13
`
`14
`
`15
`
`16
`
`Q.
`
`My question isn't that. My
`
`question
`
`is whether it's meaningful
`
`information?
`
`A.
`
`Q .
`
`I can ' t comment on that.
`
`Why not?
`
`MR . SCHWARTZ: Objection to
`
`scope to all those questions.
`
`Just
`
`give me a chance to object
`
`Dr. Menchen.
`
`THE WITNESS: Okay .
`
`A.
`
`Because I'm
`
`I'm not a
`
`crystallographer and I'm not an
`
`expert on polymerases.
`
`So I can't
`
`really evaluate, yeah, that analysis
`
`myself.
`
`Ex. 1112 at 141:19‐142:9, 193:13‐18, 270:2‐16
`Reply at 11; Motion to Exclude at 2‐3, 5‐6
`
`

`

`5‐substituted pyrimidines and 7‐substituted 7‐deaza‐purines
`
`. The alkynylamino
`linker-containing nucleotides of this invention have
`distinct advantages such as: the small steric bulk of the·
`alkynylamino-linker minimizes pert~rbation of the nu-.
`cleotide; positioning the linker on the 5-position of PY·
`rimidine nucleotides and the 7-position of 7-deazapurine
`nucleotides eventually places the linker and reporter in
`the major groove when the nucleotide is incorporated
`into double-stranded DNA (this will serve to minimize
`interference with hybridization and other processes,
`which require that a double-stranded conformation be
`possible); and alkynylamino-nu~leotides with a reporter
`attached are excellent substrates for AMV reverse tran ...
`scriptase.
`
`Ex. 1029 at
`27:52‐65
`
`29
`
`DEMONSTRATIVE EXHIBIT – NOT EVIDENCE
`
`See also Ex. 1030 at 1087; Ex. 1031 at 3051; 
`Petition at 14
`
`

`

`Escalating efficiency expectations in mid‐to‐late 2000’s
`
`“The Race for the 
`$1000 Genome,” 
`Science, 311:1544, 
`2006 
`
`The race is on
`The first group to produce a technology capable
`of sequencing a human genome sequence for
`$1000 will get instant gratification, as well as
`potential future profits: In September 2003, the
`J. Craig Venter Science Foundation promised
`$500,000 for the achievement. That challenge
`has since been picked up by the Santa Monica,
`Calif omia- based X Prize Foundation, which is
`expected to up the ante to between $5 million and
`$20 million. But the competition really began in
`earnest in 2004, when the National Institutes of
`Health launched a $70 million grant program to
`support researchers working to sequence a
`complete mammal-sized genome initially for
`$100,000 and ultimately for $1000. That pro(cid:173)
`gram has had an "amazing" effect on the field,
`encouraging researchers to pursue a wide variety
`of new ideas, says Church. That boost in turn has
`led to a miniexplosion of start-up companies,
`each pursuing its own angle on the technology
`(see table, p. 1546).
`DEMONSTRATIVE EXHIBIT – NOT EVIDENCE
`
`30
`
`Ex. 1120 at 1544;
`Reply at 15
`
`

`

`Columbia relied on Kamal
`
`BACKGROUND OF THE INVENTION
`***
`However, the procedure to chemically cleave the methoxy
`group is stringent and requires anhydrous conditions. Thus,
`it is not practical to use a methoxy group to cap the 3'-OH
`group for sequencing DNA by synthesis. An ester group was
`also explored to cap the 3'-OH group of the nucleotide, but
`it was shown to be cleaved by the nucleophiles in the active
`site in DNA polymerase (Canard et al. 1995). Chemical
`groups with electrophiles such as ketone groups are not
`suitable for protecting the 3'-OH of the nucleotide in enzy(cid:173)
`matic reactions due to the existence of strong nucleophiles
`in the polymerase. It is known that MOM (- CH2OCH3 )
`and allyl (- CH2CH==CH2 ) groups can be used to cap an
`- OH group, and can be cleaved chemically with high yield
`(Ireland et al. 1986; Kamal et al. 1999).
`
`31
`
`DEMONSTRATIVE EXHIBIT – NOT EVIDENCE
`
`Ex. 1001 at 3:31‐44; 
`Petition at 23, 32, 50, 60
`
`

`

`Columbia repeatedly relied on Metzker and Kamal
`
`The MOM
`(- CH20CH3 ) or allyl (- CH2CH=CH2) group is used to
`cap the 3'-0H group using well-established synthetic pro(cid:173)
`cedures (FIG. 13) (Fuji et al. 1975, Metzker et al. 1994).
`These groups can be removed chemically with high yield as
`shown in FIG. 14 (Ireland, et al. 1986: Kamal et al. 1999).
`The chemical cleavage of the MOM and ally} gr