Case 1:19-cv-01681-CFC-SRF Document 1-8 Filed 09/10/19 Page 1 of 9 PageID #: 197
`
`
`
`
`EXHIBIT 8
`
`Columbia Ex. 2031
`Illumina, Inc. v. The Trustees
`of Columbia University
`in the City of New York
`IPR2020-01177
`
`

`

`Case 1:19-cv-01681-CFC-SRF Document 1-8 Filed 09/10/19 Page 2 of 9 PageID #: 198
`
`U.S. Pat. No. 10,407,459
`
`1. An adenine deoxyribonucleotide analogue having the
`structure:
`
`EXHIBIT 8
`
`Infringement by Illumina’s nucleotide
`analogues in the Accused Kits
`Bentley et al., Nature, Vol. 456:53-59 (2008) (“Bentley”) (Exhibit 4)
`Supplementary Information to Bentley (Exhibit 5)
`Milton et al., WO 2004018497 A2 (2004) (Exhibit 12)
`Illumina’s sequencing chemistry uses adenine deoxyribonucleotide
`analogues.
`“To ensure base-by-base nucleotide incorporation in a stepwise manner,
`we used a set of four reversible terminators, 3′-O-azidomethyl 2′-
`deoxynucleoside triphosphates (A, C, G and T), each labelled with a
`different removable fluorophore (Supplementary Fig. 1a).” Ex. 4 at 53.
`
`0
`0
`0
`II
`II
`11
`o-- i- o- r- o- ,- 0
`o·
`o·
`
`o-
`
`Ex. 5 at 14, Fig. 1a. Although only the structure for a thymine
`deoxyribonucleotide analogue is provided, Bentley indicates that the A, C,
`and G nucleotides have the same general structure. Ex. 4 at 53.
`On information and belief, Illumina uses an adenine deoxyribonucleotide
`
`1
`
`

`

`Case 1:19-cv-01681-CFC-SRF Document 1-8 Filed 09/10/19 Page 3 of 9 PageID #: 199
`
`analogue with a linker attached to the 7-deaza position of the adenine as
`shown in the claimed structure. Ex. 12 at 83, compound 32 (showing a
`linker attached to the 7-deaza position of an adenine).
`As shown in Fig. 1a of Supplementary Information to Bentley, Illumina
`uses an azidomethyl group (CH2N3) at position R (highlighted below).
`The azidomethyl group is a small chemically cleavable, chemical group,
`does not contain a ketone group, and is not a –CH2CH=CH2 group.
`
`wherein R (a) represents a small, chemically cleavable,
`chemical group capping the oxygen at the 3′ position of
`the deoxyribose of the deoxyribonucleotide analogue,
`(b) does not interfere with recognition of the analogue as
`a substrate by a DNA polymerase, (c) is stable during a
`DNA polymerase reaction, (d) does not contain a ketone
`group, and (e) is not a –CH2CH=CH2 group;
`
`wherein OR is not a methoxy group or an ester group;
`
`a
`
`Yo
`
`;__
`
`~o '
`
`N~o.._ _,,,....._ _ I'
`
`'
`
`-
`
`N~~ Q
`~ A-__,,O~ o
`~ , y---'
`
`o
`
`0~
`
`O'-../ N,
`
`0
`
`Ex. 5 at 14, Fig. 1a. Illumina uses these modified nucleotides for
`sequencing. Ex. 4 at 53 (“We sequenced DNA templates by repeated
`cycles of polymerase-directed single base extension.”). Since Illumina
`uses these nucleotides successfully in a polymerase reaction to
`accomplish DNA sequencing, it follows that the azidomethyl group does
`not interfere with recognition of the analogue as a substrate by a DNA
`polymerase and is stable during a DNA polymerase reaction.
`
`Illumina uses an azidomethyl group to cap the 3′-OH of its nucleotide to
`form an OR moiety. This OR moiety is not a methoxy group or an ester
`group.
`
`wherein the covalent bond between the 3′-oxygen and R As Illumina uses nucleotides with an azidomethyl group “[t]o ensure
`base-by-base nucleotide incorporation in a stepwise manner,” it follows
`
`2
`
`

`

`Case 1:19-cv-01681-CFC-SRF Document 1-8 Filed 09/10/19 Page 4 of 9 PageID #: 200
`
`is stable during a DNA polymerase reaction;
`
`that the bond between the 3′-oxygen and R is stable during a DNA
`polymerase reaction. Ex. 4 at 53.
`
`wherein tag represents a detectable fluorescent moiety; As shown in Fig. 1a below, Illumina’s modified nucleotides contain a
`fluorescent moiety tag (highlighted below).
`
`{I
`
`HN
`
`0
`
`'
`
`Y N~O
`A
`~ 0
`'t)
`
`II
`0
`
`H
`
`~
`
`N,
`1·
`o~o
`
`0
`
`~o "~~ Q
`lSJJ "
`'('--"
`
`0
`
`wherein Y represents 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
`
`O'-../N,
`
`Ex. 5 at 14, Fig. 1a.
`
`As shown in Fig. 1a below, Illumina’s modified nucleotides use a
`chemically cleavable linker (highlighted below).
`
`{I
`
`Ex. 5 at 14, Fig. 1a. Illumina has indicated that this linker is chemically
`cleavable using tris(2-carboxyethyl)phosphine (TCEP). Ex. 4 at 53.
`
`As Illumina “sequenced DNA templates by repeated cycles of
`polymerase-directed single base extension” using nucleotides with this
`
`3
`
`

`

`Case 1:19-cv-01681-CFC-SRF Document 1-8 Filed 09/10/19 Page 5 of 9 PageID #: 201
`
`wherein the adenine deoxyribonucleotide analogue:
`
`i)
`
`ii)
`
`iii)
`
`iv)
`
`v)
`
`is recognized as a substrate by a DNA
`polymerase,
`
`is incorporated at the end of a growing strand of
`DNA during a DNA polymerase reaction,
`
`produces a 3′-OH group on the deoxyribose upon
`cleavage of R,
`
`no longer includes a tag on the base upon
`cleavage of Y, and
`
`is capable of forming hydrogen bonds with
`thymine or a thymine nucleotide analogue.
`
`2. An adenine deoxyribonucleotide analogue having the
`structure:
`
`linker, it follows that this linker (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. Ex. 4 at 53 (emphasis added); Ex. 5 at 33,
`Figure S13 (showing “Illumina sequence reads” identifying the A base
`resulting from incorporation by a DNA polymerase of an adenine
`deoxyribonucleotide analogue having a label attached via a chemically
`cleavable, chemical linker); Ex. 12 at 83, compound 32.
`As discussed above, as Illumina “sequenced DNA templates by repeated
`cycles of polymerase-directed single base extension” using nucleotide
`analogues, including adenine deoxyribonucleotide analogues, it follows
`that these nucleotide analogues are recognized as a substrate by a DNA
`polymerase, are incorporated at the end of a growing strand of DNA
`during a DNA polymerase reaction, and are capable of forming hydrogen
`bonds with thymine or a thymine nucleotide analogue.
`
`Illumina also discloses using tris(2-carboxyethyl)phosphine (TCEP) “to
`remove the fluorescent dye [i.e. tag] and side arm from a linker attached
`to the base and simultaneously regenerate a 3′ hydroxyl group ready for
`the next cycle of nucleotide addition (Supplementary Fig. 1b).” Ex. 4 at
`53. Thus, Illumina’s sequencing chemistry results in an adenine
`deoxyribonucleotide that produces a 3′-OH group on the deoxyribose
`upon cleavage of R and no longer includes a tag on the base upon
`cleavage of Y.
`
`Illumina’s sequencing chemistry uses adenine deoxyribonucleotide
`analogues.
`“To ensure base-by-base nucleotide incorporation in a stepwise manner,
`we used a set of four reversible terminators, 3′-O-azidomethyl 2′-
`deoxynucleoside triphosphates (A, C, G and T), each labelled with a
`
`4
`
`

`

`Case 1:19-cv-01681-CFC-SRF Document 1-8 Filed 09/10/19 Page 6 of 9 PageID #: 202
`
`0 II
`I
`0-- P-
`o-
`
`0 -
`
`o
`~-
`1-
`o·
`
`o
`II
`0 - P-
`!-
`
`0
`
`wherein R (a) represents a small, chemically cleavable,
`chemical group capping the oxygen at the 3′ position of
`the deoxyribose of the deoxyribonucleotide analogue,
`(b) does not interfere with recognition of the analogue as
`a substrate by a DNA polymerase, (c) is stable during a
`DNA polymerase reaction, and (d) does not contain a
`ketone group;
`
`different removable fluorophore (Supplementary Fig. 1a).” Ex. 4 at 53.
`
`a
`
`Ex. 5 at 14, Fig. 1a. Although only the structure for a thymine
`deoxyribonucleotide analogue is provided, Bentley indicates that the A, C,
`and G nucleotides have the same general structure. Ex. 4 at 53.
`On information and belief, Illumina uses an adenine deoxyribonucleotide
`analogue with a linker attached to the 7-deaza position of the adenine as
`shown in the claimed structure. Ex. 12 at 83, compound 32 (showing a
`linker attached to the 7-deaza position of an adenine).
`As shown in Fig. 1a of Supplementary Information to Bentley, Illumina
`uses an azidomethyl group (CH2N3) at position R (highlighted below).
`The azidomethyl group is a small chemically cleavable, chemical group,
`and does not contain a ketone group.
`
`{l
`
`5
`
`

`

`Case 1:19-cv-01681-CFC-SRF Document 1-8 Filed 09/10/19 Page 7 of 9 PageID #: 203
`
`wherein OR is not a methoxy group, an ester group, or
`an allyl ether group;
`
`wherein the covalent bond between the 3′-oxygen and R
`is stable during a DNA polymerase reaction;
`
`Ex. 5 at 14, Fig. 1a. Illumina uses these modified nucleotides for
`sequencing. Ex. 4 at 53 (“We sequenced DNA templates by repeated
`cycles of polymerase-directed single base extension.”). Since Illumina
`uses these nucleotides successfully in a polymerase reaction to
`accomplish DNA sequencing, it follows that the azidomethyl group does
`not interfere with recognition of the analogue as a substrate by a DNA
`polymerase and is stable during a DNA polymerase reaction.
`
`Illumina uses an azidomethyl group to cap the 3′-OH of its nucleotide to
`form an OR moiety. This OR moiety is not a methoxy group, an ester
`group, or an allyl either group.
`
`As Illumina uses nucleotides with an azidomethyl group “[t]o ensure
`base-by-base nucleotide incorporation in a stepwise manner,” it follows
`that the bond between the 3′-oxygen and R is stable during a DNA
`polymerase reaction. Ex. 4 at 53.
`
`wherein tag represents a detectable fluorescent moiety; As shown in Fig. 1a below, Illumina’s modified nucleotides contain a
`fluorescent moiety tag (highlighted below).
`
`a
`
`A
`0Z0--- 0
`
`0
`
`'
`
`HN
`
`Y N~O
`'t)
`
`II
`0
`
`H
`
`~
`
`N,
`1·
`o ~o
`
`0
`
`~o "~~ Q
`~ " ~
`
`0
`
`O......_,,..N,
`
`Ex. 5 at 14, Fig. 1a.
`
`wherein Y represents a chemically cleavable, chemical
`
`As shown in Fig. 1a below, Illumina’s modified nucleotides use a
`
`6
`
`

`

`Case 1:19-cv-01681-CFC-SRF Document 1-8 Filed 09/10/19 Page 8 of 9 PageID #: 204
`
`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
`
`chemically cleavable linker (highlighted below).
`
`Ex. 5 at 14, Fig. 1a. Illumina has indicated that this linker is chemically
`cleavable using tris(2-carboxyethyl)phosphine (TCEP). Ex. 4 at 53.
`
`As Illumina “sequenced DNA templates by repeated cycles of
`polymerase-directed single base extension” using nucleotides with this
`linker, it follows that this linker (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. Ex. 4 at 53 (emphasis added); Ex. 5 at 33,
`Figure S13 (showing “Illumina sequence reads” identifying the A base
`resulting from incorporation by a DNA polymerase of an adenine
`deoxyribonucleotide analogue having a label attached via a chemically
`cleavable, chemical linker); Ex. 12 at 83, compound 32.
`As discussed above, as Illumina “sequenced DNA templates by repeated
`cycles of polymerase-directed single base extension” using nucleotide
`analogues, including adenine deoxyribonucleotide analogues, it follows
`that these nucleotide analogues are recognized as a substrate by a DNA
`polymerase, are incorporated at the end of a growing strand of DNA
`during a DNA polymerase reaction, and are capable of forming hydrogen
`bonds with thymine or a thymine nucleotide analogue.
`
`7
`
`wherein the adenine deoxyribonucleotide analogue:
`
`i)
`
`ii)
`
`is recognized as a substrate by a DNA
`polymerase,
`
`is incorporated at the end of a growing strand of
`DNA during a DNA polymerase reaction,
`
`iii)
`
`produces a 3′-OH group on the deoxyribose upon
`
`

`

`Case 1:19-cv-01681-CFC-SRF Document 1-8 Filed 09/10/19 Page 9 of 9 PageID #: 205
`
`cleavage of R,
`
`iv)
`
`v)
`
`no longer includes a tag on the base upon
`cleavage of Y, and
`
`is capable of forming hydrogen bonds with
`thymine or a thymine nucleotide analogue.
`
`Illumina also discloses using tris(2-carboxyethyl)phosphine (TCEP) “to
`remove the fluorescent dye [i.e. tag] and side arm from a linker attached
`to the base and simultaneously regenerate a 3′ hydroxyl group ready for
`the next cycle of nucleotide addition (Supplementary Fig. 1b).” Ex. 4 at
`53. Thus, Illumina’s sequencing chemistry results in an adenine
`deoxyribonucleotide that produces a 3′-OH group on the deoxyribose
`upon cleavage of R and no longer includes a tag on the base upon
`cleavage of Y.
`
`8
`
`