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`- MAIWALD
`- t r —
`fNT^LLECTUAL
`— PROPERTY
`
`Munched
`DQ sseldorf
`
`Etisenhol Etisenstrade 3
`80335 Munched
`T +49 (89) 7472860
`F +49 {89}776424
`E mfa@maiwald.eu
`H www.maiwaid.eu
`
`Dr. Dirk Buhier
`Partner
`buehter@maiwald.eu
`
`MAIW ALD ' EHsenstraflo 3 ■ 80335 M iinchen
`Europaisches Patentamt
`
`80298 Munchen
`
`Official Ref.:
`Patentee:
`Opponent:
`
`EP 15 195 765.1 / EP 3 034 627
`The Trustees of Columbia University in the City of New York
`(LLUMINAfNC.
`
`Munich. 29 October 2019
`
`Our Ref.
`
`I09472EPOP/DB
`
`In the name and on behatf of
`
`liiumina, Inc.
`5200 liiumina Way
`San Diego, CA 92122 USA
`
`we herewith file an opposition against the European Patent EP 3 034 627 B1
`entitled "MASSIVE PARALLEL METHOD FOR DECODING DNA AND RNA”,
`which was granted to "The Trustees of Columbia University in the City of New
`York". The mention of the grant of the patent was published on 30 January 2019.
`
`We authorize the deduction of the opposition fee from our deposit account no.
`28 000 741.
`
`Columbia Ex. 2075
`Illumina, Inc. v. The Trustees
`of Columbia University in the
`City of New York
`IPR2020-00988, -01065,
`-01177, -01125, -01323
`
`

`

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`A. REQUESTS................................................................................................................. 3
`B.
`CITED DOCUMENTS................................................................................................... 3
`C.
`THE OPPOSED PATENT............................................................................................... 5
`
`I.
`
`It.
`
`1.
`2.
`3.
`4.
`
`1.
`
`Background...............................................................................................................................5
`Structure of DNA................................................................................................. 5
`Syn th esis of DNA.................................................................................................6
`Sanger DNA sequencing...................................................................................... 9
`DNA sequencing by synthesis............................................................................11
`The ALLEGED INVENTION....................................................................................................................14
`The independent claims.................................................................................... 14
`1.1
`Independent claim 1 ......................................................................................................14
`1.2
`Independent claim 3 ......................................................................................................16
`1.3
`Independent claim 5 ......................................................................................................16
`The 3'-OH capping group and the specification ofEP'627...................................16
`2.
`Further comments............................................................................................19
`3.
`D. GROUNDS OF OPPOSITION...................................................................................... 21
`
`1 i.
`
`2.
`3.
`
`The subject matter of the granted claims is not directly and unambiguously disclosed by
`1.
`THE APPLICATION AS FILED (ARTICLE 100(c) EPC)......................................................................................... 21
`Claim 1............................................................................................................. 21
`1.
`1.1
`Feature 9a on its own as well as its combination with Features 9b and 9c creates new
`subject matter which is not originally disclosed in in the application as filed..........................21
`1.2
`Feature 1 is not originally disclosed in the application as filed.................................. 26
`1.3
`Feature 2b is not originally disclosed in in the application as filed............................ 27
`1.4
`The combination of Features 3-6 is not originally disclosed in in the application as
`filed
`27
`Claims 3 and 5 ..................................................................................................28
`The dependent claims....................................................................................... 28
`Insufficiency of disclosure (Article 100(b) EPC, A rticle 83 EPC)..........................................29
`"Small" chemically deavable chemical moiety...................................................29
`Cleavability of the 3'-OH capping group............................................................ 34
`Summary on sufficiency of disclosure................................................................36
`Lack of inventive step (Article 1 0 0 (a) EPC, Article 56 EPC).................................................37
`Lack of inventive step of independent claim 1....................................................37
`1.1
`Lack of inventive step of the subject matter of claim 1 in view of D4 (Tsien) in
`combination with 07 (Prober), D2 (Hobbs i], D3 (Hobbs ii), or 04 (Rosenbium)......................37
`1.2
`Lack of inventive step of the subject matter of claim 1 in view of D13 (Dower) in
`combination with 07 (Prober) and D14 (Metzker)......................................................................46
`1.3
`Lack of inventive step of the subject matter of claim 1 in view of D15 (Stempie) in
`combination with 07 (Prober) and D9 (Metzker)........................................................................ 52
`Lack of inventive step of independent claims 3 and 5 ........................................ 53
`2.
`Lack of inventive step of the dependent claims.................................................. 53
`3.
`FURTHER COMMENTS............................................................................................. 54
`CONCLUSION........................................................................................................... 55
`
`1.
`2.
`3.
`111.
`1.
`
`E.
`F.
`
`2
`
`

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`INTELLECTUAL
`PROPERTY
`
`A.
`
`Requests
`
`1
`
`It is herewith requested that the patent EP 3 034 627 B1 {in the following
`also referred to as 'the opposed patent” or “EP’627”) be revoked in its
`entirety on the basis of
`
`• Article 100 {a) EPC {the subject matter of the opposed patent is not
`patentable under Articles 56 EPC);
`
`• Article 100 (b) EPC {the opposed patent does not disclose the
`invention in a manner sufficiently clear and complete for it to be
`carried out by a person skilled in the art according to Article 83 EPC);
`and
`
`• Article 100 (c) EPC (the subject-matter of the opposed patent extends
`beyond the content of the application as filed contravening Article
`123(2) EPC and beyond the content of the earlier application as Hied
`contravening Article 76(1) EPC.
`
`2 Should the Opposition Division not be in a position to grant the above
`request to revoke the opposed patent in its entirety, orai proceedings are
`requested as an auxiliary measure.
`
`B.
`
`Cited documents
`
`3 The following prior art documents are cited in the context of the opposition
`grounds:
`
`D1:
`
`D2:
`
`D3:
`
`Alberts et al.: "Molecular Biology of the Cell", Third Edition,
`Garland Publishing Inc., New York (1994), pp. 98-103
`
`US 5,608,063 {Hobbs I), published on March 4, 1997
`
`US 5,151,507 {Hobbs II), published on September 29, 1992
`
`D4: WO 91/06678 A1 {"Tsien")
`
`D5: M. B. Welch et at., Chem. Eur. J. 1999, 5(3), pp. 951-960
`
`D6:
`
`B. B. Rosenbium et al., Nucleic Acids Research 1997, 25(22), pp.
`4500-4504
`
`D7:
`
`J. M. Prober et at., Science 1987, 238(4825), pp. 336-341
`
`D8: B. Canard et al., PNAS 1995, 92, pp. 10859-10863
`
`3
`
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`D9:
`
`R. Gigg et at., Journal of the Chemical Society 1968, 1903-1911
`
`D10: N. Ramzaeva et al., Helvetica Chimica Acta 1995, 1083-1090
`
`D11: Seela et al., Bioorganic & Mechanical Chemistry Letters 1995,
`5:3049-3052
`
`D12: N. Ramzaeva et al.. Helvetica Chimica Acta 1997, 80:1809-1822
`
`D13: US 5,547,839 {„Dower‘'}
`
`D14: M. L. Metzker et al., Nucleic Acids Research 1994, 22{20), pp.
`4259-4267
`
`D15: WO 00/53805 A1 {“Stempte’1)
`
`4 The opposed patent EP 3 034 627 B1 has a filing date of 5 October 2001
`and claims priority of two US applications filed on 6 October 2000 and 26
`June 2001. Documents D1 to D15 were published before the priority date
`of the opposed patent, and therefore constitute state of the art under
`Article 54{2) EPC.
`
`5
`
`Further, reference is made to the fotlowing evidence submitted during the
`examination proceedings of the opposed patent by the Proprietor.
`
`D16: Dectaration of the inventor Jingyue Ju, Ph. D. as filed on 12th
`March 2018
`
`6 We also refer to the decision by the Examining Division rejecting the
`parent application EP 1 790 736 A2:
`
`D17: Decision of the Opposition Division dated 23 March 2015
`regarding EP 1 790 736 A2.
`
`7 D18 provides an overview of decisions by the USPTO PTAB revoking US
`counterparts to EP’627.
`
`4
`
`

`

`MAIWAL.D
`
`INTELLECTUAL
`PROPERTY
`
`C.
`
`The opposed patent
`
`s The opposed patent pertains to a specific aspect of DNA sequencing by
`synthesis {see below).
`
`9
`
`10
`
`In the following, we will set forth some principles underlying DNA
`sequencing in general and particularly DNA sequencing by synthesis as
`they were known before the priority date.
`
`It is respectfully submitted that this wilt help appreciate the subsequent
`arguments that the claims as granted are not disclosed by the application
`as filed, that the subject matter of the claims cannot be worked across the
`scope of the claims by the person skilled in the art without an undue
`burden and that the claimed subject matter is obvious over the prior art.
`
`t.
`
`1.
`
`Background
`
`Structure of DNA
`
`11 DNA consists of two complementary strands that wind around one
`another to form a double helix.1 The strands of DNA are made up of
`individual deoxyribonucteotides {also referred to as ‘'nucleotides’1), which
`are composed of deoxyribose (i.e. a sugar with five carbon atoms, that
`lacks the 2’-OH group that ribose normally contains), a nucleobase {also
`referred to as “nitrogenous base”), and a phosphate group. There are four
`different nucleotides in DNA, which differ from each other by their
`nitrogenous bases: adenine {A), cytosine (C), guanine (G), and thymine
`{T). The nucleotides in each strand are linked by their phosphate groups,
`which in each case attach the 5’ carbon atom of the deoxyribose of one
`nucleotide to the 3’ carbon atom of the deoxyribose of the next nucleotide,
`to form the sugar-phosphate backbone of the DNA strand as shown in
`Figure 1 below.
`
`12 The two complementary strands assemble together by base-pairing with
`the formation of hydrogen bonds between the bases, where C pairs with
`G and A pairs with T, as also shown in Figure 1 below. The C-G and A-T
`pairs are also commonly referred to as Watson-Crick base pairs.
`
`1 See D1, pp. 98-102.
`
`5
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`MAIWAL.D
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`INTELLECTUAL
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`Nitrogenous bases:
`5.
`easas® Adenine
`
`Hydrogen bonds
`
`(a)
`
`(b)
`
`Figure 1: Structure of DNA. {a) The double helix comprised of
`complementary base pairs heid together by a sugar-phosphate backbone,
`(b) The hydrogen bonding between the four different nucleotides.
`
`2.
`
`Synthesis of DNA
`
`13
`
`In the cell, DNA is synthesized during replication by DNA polymerase, an
`enzyme which uses a singte-stranded DNA molecule as a template to
`synthesize new DNA strands that are complementary to the template.2
`DNA polymerase synthesizes new DNA strands by attaching individual
`nucleotides to the end of the DNA strand being synthesized (see Figure
`2, below). This attachment links the deoxyribose of one nucleotide to the
`deoxyribose of another nucleotide via a phosphate linkage in a specific
`direction to form the sugar-phosphate backbone of the newly synthesized
`DNA strand (see Figure 1, above): the 5' carbon atom of one deoxyribose
`is connected via a phosphate group to the 3’ carbon atom of another
`deoxyribose.3 Thus, the DNA strand is produced in 5l -> 3l direction.
`
`2 See D1, pp. 98-102.
`3 The 3' and 5' carbon atoms of the deoxyribose are labelled on the dNTPs in
`Figures 1 and 3.
`
`6
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`MAIWAL.D
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`INTELLECTUAL
`PROPERTY'
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`Figure 2: DNA synthesis during replication.
`
`14 The nucleotides used by DNA polymerases to synthesize DNA are
`deoxyribonucieotide triphosphates (also referred to as 'dNTPs”). Figure 3
`beiow shows the structures of the four dNTPs (where 'N' in dNTP is A, C,
`G, or T, corresponding to the four nucleotides of DNA, i.e. dATP, dCTP,
`dGTP, and dTTP) that are used by DNA polymerase in synthesizing the
`new strand of DNA.
`
`NH«
`
`j
`
`Deoxyadenosine-5'
`triphosphate
`
`Deoxycytidine-5' triphosphate
`
`Deoxyguanosine-5’
`triphosphate
`
`Deoxythymidine-5'
`triphosphate
`
`• OH
`
`0 O 0
`
`OK Q,H OH
`
`0
`{ T
`
`i
`
`p
`
`■OH
`
`q
`q
`O-F’-O-p-C.
`OH OH
`
`o O
`
`H
`
`HO-
`
`oh
`
`0 O 0
`C—F*—
`
`6 h
`
`o h
`
`Figure 3: Structure of deoxyribonucieotide triphosphates (dNTPs; from
`top to bottom, dATP, dCTP, dGTP, and dTTP).
`
`7
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`MAIWAL.D
`INTELLECTUAL
`PROPERTY
`
`15 DNA synthesis by DNA polymerase is a template-dependent process;
`the same hydrogen bonds that mediate the stability of the DNA double
`helix control how a polymerase copies a template. For example, when
`DNA polymerase encounters a G in a template, it binds dCTP and
`incorporates a C, whereas when it encounters an A in the template, it
`binds dTTP and incorporates a I (see below Figure 4 and above Figure
`2). To start the DNA synthesis, an oligonucleotide is used as a so-called
`primer, from which the DNA strand is enzymatically extended in 5' -> 3’
`direction.
`
`Figure 4: Template driven incorporation of dNTPs by DNA polymerase.
`
`16 With the incorporation of each dNTP into the newiy synthesized DNA
`strand, a pyrophosphate (two phosphate groups linked together, also
`referred to as “PPi") is released and one phosphate (the so-called a-
`phosphate) remains to link the nucleotides to each other to form the sugar-
`phosphate backbone of the DNA strand (see below Figure 5). The release
`of PPi and its subsequent hydrolysis provides the energy for the extension
`and makes the incorporation of the nucleotide into the DNA strand
`essentially irreversible:
`
`8
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`

`MAIWAL.D
`INTELLECTUAL
`PROPERTY
`
`m 2
`
`G
`
`*
`
`CH,
`
`T
`
`Figure 5: Newly synthesized DNA strand with free 3’-0H group.
`
`17 The 3’-OH group of the last nucleotide added to the growing DNA strand
`(circled in red in the above Figure 5) is essential for the incorporation of a
`further nucleotide by the DNA polymerase via reaction with the a-
`phosphate of the incoming dNTP and release of pyrophosphate. As the
`incorporated dNTP itself also contains a 3’-OH group, further extension of
`the DNA strand is possible.
`
`3.
`
`Sanger DNA sequencing
`
`18 The so-catted Sanger method was one of the first sequencing methods
`used for large-scale sequencing before the priority date of the opposed
`patent.
`
`19 The Sanger method is based on the termination of DNA synthesis by
`using
`2',3’-dideoxyribonucleotide-5’-triphosphates
`(ddNTPs).
`The
`nucleotides are referred to by using the prefix “dideoxyribose" because
`they tack the 3’-OH that the deoxyribose of DNA would normally possess,
`i.e. ddNTPs lack both the 2’- and 3’-OH groups that ribose would normally
`possess (see below Figure 6):
`
`9
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`

`MAIWAL.D
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`INTELLECTUAL
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`0
`0
`0
`II
`II
`[I
`HO—P—0 —P—0 —P—0
`I _
`I _
`i _
`\
`0
`0
`0
`
`Figure 6: Structure of ddNTP used in Sanger sequencing.
`
`20 As the ddNTPs contain no 3'-0H group, the DNA strand cannot be
`extended further. Once a ddNTP has been incorporated into a growing
`DNA strand, termination occurs specifically at positions where that ddNTP
`is incorporated. Thus, if an oiigonucleotide-primed DNA template is
`enzymatically extended in 5' -> 3' direction in the presence of a mixture of
`ait four dNTPs and a small amount of one ddNTP, namely ddATP, ddTTP,
`ddGTP, or ddCTP, the ddNTP will be occasionally incorporated into the
`growing DNA strand leading to termination of strand extension at a
`position determined by the respective ddNTP. By running four such
`reactions in parallel, a population of DNA strands of different lengths is
`obtained ait of which end either with A, T, G, or C. These DNA strands of
`different lengths are then resolved by gel electrophoresis and the
`sequence can be determined from the obtained length pattern if, e.g., the
`chain-terminating ddNTPs are labeled with detectable markers (see below
`Figure 7).
`
`PCD in preecfiu of fluDreacent, chnln<tennlnttlng mteleotldea
`
`Fluorescent fragment? detected by laser and represented on a chromate pram
`
`Figure 7: Scheme depicting characteristic steps of Sanger sequencing.
`
`10
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`MAIWAL.D
`iNTEUECRJAt.
`PROPERTY
`
`21 A major advance in Sanger sequencing was to use chain-terminating
`ddNTPs in which fluorescent labels were attached at the 5-position of the
`pyrimidine bases (T and C) or at the 7-position of 7-deazapurine bases (A
`and G) 4 Some of the chain terminating ddNTPs of D2 are reproduced
`betow to ittustrate the attachment of labet groups through tinkers to the 5-
`position of T and C or the 7-position of 7-deazapurine versions of A and
`G:
`
`these positions and using
`in
`22 Modifying pyrimidines and purines
`deazapurines instead of purines allowed for efficient recognition and
`incorporation by DNA polymerase.5 However, the requirement for size
`separation by electrophoresis as part of the analytical scheme remained
`an important conceptual limitation of the Sanger method, in particular for
`high-throughput applications where many sequences are obtained in
`parallel.
`
`4.
`
`DNA sequencing by synthesis
`
`throughput sequencing
`this background, alternative high
`23 Against
`approaches were discussed before the priority date of the opposed patent
`
`* See e.g. D2, column 16, tl. 15-18; column, 17, t. 61 - column 18, 1.13 and
`molecules depicted at columns 26 and 27.
`5 See D2, column, 17, t. 61 - column 18, I. 13; and D3, column 27, t. 52 - column
`28, i. 2.
`
`n
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`

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`MAIWAL.D
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`INTELLECTUAL
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`which avoid the need for size separation by electrophoresis but altow for
`parallel sequencing of DNA molecules.
`
`24 The approaches considered included DNA sequencing by synthesis
`(SBS) methods which rely on the synthesis of new complementary DNA
`strands by DNA polymerase using modified dNTPs that are labelled with
`a cleavable base-specific detectable label and contain a cteavable 3’-
`O-capptng group.
`
`25 Thus, instead of ddNTPs lacking the 3’-OH group, the SBS methods use
`modified dNTPs, wherein the 3’-OH group is capped, so that the growing
`DNA strand cannot be extended further once the modified dNTP has been
`incorporated. However, in contrast to the ddNTPs as used in the Sanger
`method, the 3’-OH group of the modified dNTPs used in the SBS methods
`can be cleaved after incorporation, such that a further extension of the
`DNA strand is possible after a de-capping step. The de-capping step is
`typicatty performed after identification of the incorporated nucleotide
`through the base-specific detectable label, which may then be cleaved
`together with the capping group.
`
`26 A typical reaction cycle of such SBS methods thus generatty comprises
`the following steps:
`
`(i)
`
`(ii)
`
`(iii)
`
`the appropriate modified dNTP by DNA
`incorporation of
`polymerase in a template-driven process;
`
`identification of the newly added base through the base-specific
`detectable label; and
`
`removal of the base-specific detectable label and regeneration of
`a free 3’-OH terminus by removal of 3'-OH capping group.
`
`27 Subsequently the next reaction cycle begins by adding new modified
`dNTPs. By repeating this cyclic scheme, one can determine the sequence
`of a template DNA molecule by synthesizing the complementary strand.0
`
`20 With respect to the subsequent sections, we emphasize that it was known
`that the 3’-position of (modified) dNTPs is very close to the amino acid
`residues in the active site of DNA polymerase, and that DNA polymerase
`is therefore sensitive to bulky modifications at this position.6 7 Further, it
`was known, inter alia from the above-mentioned work of D2 and D3 on
`
`6 This SBS approach is disclosed inter alia in D4 (Tsien) which will be discussed in
`more detail in the context of inventive step (see section D.111.1).
`7 This is reflected by D4 when discussing the requirements for a blocking group on
`p. 20, i. 25 - p. 25,1. 33.
`
`12
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`MAIWAL.D
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`INTELLECTUAL
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`chain-terminating ddNTPs of the Sanger method, that DNA-polymerase
`is far less sensitive to extensive modification by linkers and bulky
`fluorescent groups at the 5-position of pyrimidines and the 7-position of
`(deaza)purines.8
`
`29 We note that the opposed patent itself acknowledges that, in the context
`of SBS methods, the criticality of the butkiness of the cleavabte 3’-OH
`capping group for recognition by DNA polymerase was known and that
`DNA polymerase would on the other hand accept extensive modifications
`at the 5-position of pyrimidines and the 7-position of (deaza)purines:
`
`‘’[0005] More recent work in the literature exploring DNA sequencing
`by a synthesis method is mostly focused on designing and
`synthesizing a photocleavabie chemical moiety that is iinked to a
`the 3’-OH group of deoxynucleoside
`fluorescent dye
`to cap
`triphosphates (dNTPs) (Weich et a!. 1999). Limited success for the
`incorporation of the 3 '-modified nucleotide bv DNA polymerase is
`reported. The reason is that the 3’-position on the deoxvribose is
`very dose to the amino acid residues in the active site of the
`polymerase, and
`to
`the polymerase
`is
`therefore sensitive
`modification in this area o f the deoxvribose ring. On the other hand,
`it is known that modified DNA polymerases (Thermo Sequenase
`and Taq FS polymerase) are able to recognize nucleotides with
`extensive modifications with bulky groups such as energy transfer
`dyes at the 5-position of the pyrimidines (T and CJ and at the 7-
`po sit ion of purines (G and A) (Rosenbium et al. 1997, Zhu et al.
`1994). The ternary complexes of rat DNA polymerase, a DNA
`template-primer, and dideoxycytidine triphosphate (ddCTP) have
`been determined (Pelletier et al. 1994) which supports this fact. As
`shown in Figure 1, the 3-D structure indicates that the surrounding
`area of the 3'-position of the deoxyribose ring in ddCTP is very
`crowded, while there is ample space for modification on the 5-
`position the cytidine base." [emphasis added]
`
`30 D5 (Welch) of the above quotation stated:9
`
`“3'-0-B!ocked nucleoside triphosphates, wherein the 3'-protecting
`fluorescent, are synthetically
`group
`is both photolabile and
`accessible. However, they tend to be too big to fit into the active site
`of DNA polymerases as evidenced by the data from the activity
`screens and the moiecuiar-simuiation experiments."
`
`s See also, e.g., D4, in particular disclosure on reporter groups on p. 28, i. 5 - p. 29,
`I. 19.
`3 See D3, p. 966, left column, section ..Conclusions* 1'.
`
`13
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`MAIWAL.D
`INTELLECTUAL
`PROPERTY
`
`31 D6 (Rosenbtum) of the above quotation is concerned with providing new
`chain-terminating ddNTPs in which fluorescent labels were attached at
`the 5-position of the pyrimidine bases {T and C) or at the 7-position of 7-
`deazapurine bases {A and G). Its disclosure is thus comparable to D2 and
`D3.
`
`it.
`
`1.
`
`The atteged invention
`
`The independent claims
`
`32 Against this background, the SBS method claimed by the opposed patent
`(EP’627) is based on the use of modified dNTPs. each of which is tabetted
`with a different detectable label, such as a fluorescent dye or a mass tag
`{“Labet”), which is attached through a cleavabte tinker to the base of the
`dNTP. Further, the dNTPs comprise a chemically cteavable moiety (“R”)
`to cap the 3'-OH group of the deoxyribose.10
`
`1.1
`
`Independent claim 1
`
`33
`
`Independent claim 1 of EP’627 relates to:
`
`A method for simultaneously sequencing a plurality of different
`deoxyribonucleic acids,
`
`wherein the plurality of different deoxyribonucleic acids is covalently
`immobilized on a solid surface, and [Feature 1]
`
`wherein a sequencing method by synthesis comprising a plurality of
`cycles, each cycle having a plurality of steps, is simultaneously
`immobilized different
`applied
`to each of said covalently
`deoxyribonucleic acids, [Feature 2a]
`
`said sequencing method involving the detection of the identity of a
`plurality of nucleotide analogues incorporated into a plurality of
`growing strands of DNA hybridized to deoxyribonucleic acids,
`[Feature 2b]
`
`said method comprising:
`
`1C EP'627, paragraph [0006].
`
`14
`
`

`

`(a) providing to the plurality of different deoxyribonucleic acids more
`than one nucleotide analogue, selected from the group consisting of
`aA, aC, aG, aT, and aU,
`
`wherein each nucleotide analogue is labeled with a unique label
`attached through a cleavabie linker to the base [Feature 3a]
`
`and contains a small chemically cleavabie chemical moiety capping
`the 3’-0H group, wherein said small chemically cleavabie chemical
`moiety is removable by chemical means, [Feature 3b]
`
`under conditions such that a plurality of growing strands are
`extended by incorporation of one nucleotide analogue per strand so
`as to create a plurality of extended growing strands of DNA using a
`DNA polymerase reaction, said incorporated analogues serving as
`terminators of the polymerase reaction; [Feature 3c]
`
`(b) detecting said unique label of said incorporated nucleotide
`analogues, so as to thereby identify 10,000 or more of the nucleotide
`analogues as having been incorporated into the plurality of growing
`strands; [Feature 4]
`
`(c) removing the label and removing by chemical means the small
`incorporated
`chemically cleavabie chemical moiety of said
`nucleotide analogues capping the 3’-0H group; and [Feature 5]
`
`(d) repeating the cycle of steps (a) through (c); [Feature 6]
`
`wherein the plurality of different deoxyribonucleic acids is covalently
`immobilized in a plurality of spots on a solid surface, wherein each
`spot comprises a plurality of the same deoxyribonucleic acid,
`[Feature 7]
`
`labels are dyes having a unique
`the unique
`and wherein
`fluorescence emission, and the unique fluorescence emission from
`a specific dye on the dye-labeied nucleotide analogues on each spot
`of the solid surface will reveal the identity of the incorporated
`nucleotide; [Feature 8]
`
`and wherein the small chemically cleavabie chemical moiety
`capping the 3'-0H group:
`
`(i) is a -CH2OCH3 group or a -CH 2CH =CH 2 group, or is as small as
`a -CH2CH=CH2group ora -CH2OCH3 group, [Feature 9a]
`
`(ii) does not contain a ketone group, [Feature 9b]
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`(Hi) when bound to the 3’-oxygen, does not form a methoxy group or
`an ester group, and [Feature 9c]
`
`(iv) forms a 3’-0H group on the deoxyribose upon cleavage of the
`small chemically cleavable chemical moiety capping the 3’-0H
`group; [Feature 9d]
`
`and wherein at least one of said incorporated nucleotide analogues
`is a 7-deaza adenine nucleotide analogue or 7-deaza guanine
`nucleotide analogue and said unique label is attached through a
`cleavable linker to a 7-position of deaza-adenine or deaza-guanine.
`[Feature 10]
`
`1.2
`
`Independent claim 3
`
`34
`
`Independent claim 3 relates to a plurality of different deoxyribonucleic
`acids comprising incorporated nucleotide analogues. The subject matter
`of claim 3 is further characterized by the Features as defined above in
`connection with claim 1T in particular Features 1 ,3a-bT 7, 8, 9a-cT and 10,
`wherein Feature 7 has been stightty reformulated. Moreover, claim 3
`contains the additional Feature that
`
`"greater than 10,000 spots are present on the solid support"
`[Feature 11]
`
`1.3
`
`Independent claim 5
`
`35
`
`Independent claim 5 relates to a method for sequencing a plurality of
`different deoxyribonucleic acids by synthesis, which is characterized by
`Features 1, 2b, 3a-c, 4, 7, 8, 9a-c, and 10 as defined in connection with
`claim 1, wherein Feature 3c has been slightly reformulated.
`
`2.
`
`The 3’-OH capping group and the specification of EP’627
`
`36 The modified dNTPs according to the claims EP’627 may in essence be
`represented by the following structure:
`
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`PROPERTY
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`37 The labels which can be (bulky) fluorescent groups11 are attached to the
`base. As far as detection of purines (G, A} is concerned, the label is
`attached to the 7-position of deaza-adenine or deaza-guanine. The
`attachment of the label to the base, and more specificatty to the 7-position
`of deaza-guanines apparently is a tacit recognition of the warning in D5
`(Welch) that DNA polymerase will have a difficult time with bulky groups
`at the 3’-OH position* 12 and accounts for the disclosure of, e.g., D4 (Tsien),
`D2 (Hobbs i), and D6 (Rosenblum).13
`
`3S Claim 1 further defines the small chemically cleavable 3’-OH capping
`group to be a MOM group (-CH2OCH3), an altyt group (-CH2CH=CH2), or
`a group “as smatt as1’ these groups.14 In addition, the 3'-OH group must
`not
`
`•
`
`contain a ketone group, and,
`
`• when bound to the 3'-oxygen, form a methoxy group or an ester
`group.15
`
`39 Even if groups containing a ketone or forming a methoxy group or an ester
`group with the 3’-oygen would be “as small as" the MOM and the allyl
`group, they are thus not covered by claim 1 .
`
`40 The specification of EP'627 seems to provide an explanation for excluding
`3'-OH capping groups which contain a ketone, or which form a methoxy
`group or an ester group with the 3'-oxygen. It states:
`
`1- See granted claim 13 and paragraph [0075] of EP'627.
`12 See above margin nos. 29-30; and paragraph [0005] of EP'627.
`13 See above margin nos. 21-22, 31; and paragraph [0005] of EP’627. We also note
`that Feature 10 of claim 1, in tine with the mentioned prior art, requires to attach the
`label at the 7-position of deazapurines.
`14 See Feature 9a of claim 1. We note that this terminology is not disclosed in the
`application as filed. We will comment on this Feature particularly in section D.l. and
`D.tt. when addressing Art. 123(2), 76 (1) and 83 EPC.
`15 See Features 9b and 9c of claim 1.
`
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`70007] It is also desirable to use a photocieavabie group to cap the
`3’-0H group. However, a photocieavabie group is generally bulky
`and thus the DNA polymerase will have difficulty to incorporate the
`nucleotide analogues containing a photocieavabie moiety capping
`the 3’-OH group. If small chemical moieties that can be easily
`cleaved chemically with high yield can be used to cap the 3’-0H
`group, such nucleotide analogues should also be recognized as
`substrates for DNA polymerase, it has been reported that 3’-0 ­
`for several
`m e thoxy- de oxyn ucieo tid e s are good substrates
`polymerases (Axelrod et al. 1978). 3’-0-allyl-dATP was also shown
`to be incorporated by Ventr(exo-) DNA polymerase in the growing
`strand of DNA (Metzker et al. 1994). However, the procedure to
`chemicaiiy cleave the methoxy group is stringent and requires
`anhydrous conditions. Thus, it is not practical to use a methoxy
`group to cap the 3’-0H group for sequencing DNA by synthesis. An
`ester group was also explored to cap the 3 ’-0H group of the
`nucleotide, but it was shown to be cleaved by the nucleophiles in
`the active site in DNA polymerase (Canard et ai. 1995). Chemical
`groups with electrophiles such as ketone groups are not suitable for
`protecting the 3’-0H of the nucleotide in enzymatic reactions due to
`the existence of strong nucleophiles in the polymerase. It is known
`that MOM (-CH20CH3) and allyI (-CH2CH=CH2) groups can be
`used to cap an -OH group, and can be cleaved chemicaiiy with high
`yield (Ireland et al. 1986; Kama! et al. 1999).”
`
`41 These groups thus suffer from various disadvantages:
`
`• dNTPs with methoxy groups may not be suitable 3’-OH capping
`groups: even though they are incorporated by DNA polymerase
`and can be chemically cleaved, because the methoxy group
`atlegedty requires stringent chemical cleavage conditions.
`
`• dNTPs with ester groups may not be suitable 3-OH capping
`groups, even though they are incorporated by DNA polymerase
`and can be chemicaiiy cleaved, because they are also cieaved by
`DNA polymerase itself.
`
`• dNTPs with capping groups containing ketones may not be
`suitable 3’-0H capping groups because they interact with DNA
`polymerase.
`
`42 According to above-quoted paragraph [0007], the MOM group and allyl
`group apparently allow (i) for efficient incorporation of modified dNTPs by
`DNA polymerase, (ii) can be “easily" chemically cleaved, i.e. other than
`the methoxy group), (Hi) are not cleaved by DNA polymerase, i.e. other
`
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