`
`
`
`
`EXHIBIT 7
`
`Columbia Ex. 2030
`Illumina, Inc. v. The Trustees
`of Columbia University
`in the City of New York
`IPR2020-01177
`
`
`
`Case 1:19-cv-01681-CFC-SRF Document 1-7 Filed 09/10/19 Page 2 of 9 PageID #: 189
`
`U.S. Pat. No. 10,407,458
`
`1. An guanine deoxyribonucleotide analogue having the
`structure:
`
`EXHIBIT 7
`
`Infringement by Illumina’s nucleotide
`analogues in the 4-Channel 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 guanine 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.
`
`HN
`I
`o
`o
`II
`II
`_
`O H2 r,A
`II
`O-P-0-P-
`I
`I O-P-0
`I o-
`o-
`o-
`
`OR
`
`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 a guanine deoxyribonucleotide
`analogue with a linker attached to the 7-deaza position of the guanine as
`shown in the claimed structure. Ex. 12 at 74, compound 24 (showing a
`
`1
`
`
`
`Case 1:19-cv-01681-CFC-SRF Document 1-7 Filed 09/10/19 Page 3 of 9 PageID #: 190
`
`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;
`
`linker attached to the 7-deaza position of a guanine).
`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 OR is 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;
`
`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.
`
`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
`
`2
`
`
`
`Case 1:19-cv-01681-CFC-SRF Document 1-7 Filed 09/10/19 Page 4 of 9 PageID #: 191
`
`wherein tag represents a detectable fluorescent moiety; As shown in Fig. 1a below, Illumina’s modified nucleotides contain a
`fluorescent moiety tag (highlighted below).
`
`polymerase reaction. Ex. 4 at 53.
`
`a
`
`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
`
`Ex. 5 at 14, Fig. 1a.
`
`As shown in Fig. 1a below, Illumina’s modified nucleotides use a
`chemically cleavable linker (highlighted below).
`
`a
`
`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
`
`3
`
`
`
`Case 1:19-cv-01681-CFC-SRF Document 1-7 Filed 09/10/19 Page 5 of 9 PageID #: 192
`
`wherein the guanine 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
`cytosine or a cytosine nucleotide analogue.
`
`2. An guanine deoxyribonucleotide analogue having the
`structure:
`
`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 G base
`resulting from incorporation by a DNA polymerase of a guanine
`deoxyribonucleotide analogue having a label attached via a chemically
`cleavable, chemical linker); Ex. 12 at 74, compound 24.
`As discussed above, as Illumina “sequenced DNA templates by repeated
`cycles of polymerase-directed single base extension” using nucleotide
`analogues, including guanine 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 cytosine or a cytosine 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 a guanine
`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 guanine 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.
`
`4
`
`
`
`Case 1:19-cv-01681-CFC-SRF Document 1-7 Filed 09/10/19 Page 6 of 9 PageID #: 193
`
`Tag
`
`a
`
`Yo ~ t
`"
`A
`~1,,.J
`
`r, ~•~,,,,.,.,J Q
`lQJ
`'
`y---'
`
`o
`
`0
`
`0
`0
`-o-U- _II
`~ H2
`I O P-O-P-0
`o-
`I _
`I
`0
`0-
`
`OR
`
`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;
`
`0~
`0......,,..,1.
`
`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 a guanine deoxyribonucleotide
`analogue with a linker attached to the 7-deaza position of the guanine as
`shown in the claimed structure. Ex. 12 at 74, compound 24 (showing a
`linker attached to the 7-deaza position of a guanine).
`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
`
`O
`
`N,
`
`,;t~:r/'-r.~,~~·'©l'r.~:~
`~ --i,,.J
`0 µ_
`
`0
`
`0-...../ N,
`
`Ex. 5 at 14, Fig. 1a. Illumina uses these modified nucleotides for
`
`5
`
`
`
`Case 1:19-cv-01681-CFC-SRF Document 1-7 Filed 09/10/19 Page 7 of 9 PageID #: 194
`
`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;
`
`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 ether 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).
`
`(l
`
`0
`
`A
`020_'.
`
`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
`linker which (a) does not interfere with recognition of
`
`As shown in Fig. 1a below, Illumina’s modified nucleotides use a
`
`6
`
`
`
`Case 1:19-cv-01681-CFC-SRF Document 1-7 Filed 09/10/19 Page 8 of 9 PageID #: 195
`
`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 G base
`resulting from incorporation by a DNA polymerase of a guanine
`deoxyribonucleotide analogue having a label attached via a chemically
`cleavable, chemical linker); Ex. 12 at 74, compound 24.
`As discussed above, as Illumina “sequenced DNA templates by repeated
`cycles of polymerase-directed single base extension” using nucleotide
`analogues, including guanine 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 cytosine or a cytosine nucleotide analogue.
`
`7
`
`wherein the guanine 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 1-7 Filed 09/10/19 Page 9 of 9 PageID #: 196
`
`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
`cytosine or a cytosine 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 a guanine
`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
`
`