T 0127/14 (DNA SEQUENCING BY OLIGONUCLEOTIDE EXTENSION/SOLEXA) 01-12-2020

Summary of Facts and Submissions

I. European patent No. 0 871 646 is based on European patent application No. 96912800.8 (published as International patent application WO 1996/033205; hereinafter "the patent application"), and was opposed on the grounds of Articles 100(a), (b) and (c) EPC. The opposition division maintained the patent in amended form. It was held that the main request contravened Article 123(2) EPC, the first auxiliary request contravened Article 83 EPC, the second auxiliary request lacked novelty, and the third auxiliary request complied with the requirements of the EPC.

II. The patent proprietor and the opponent (appellant I and II respectively) lodged appeals against the decision of the opposition division.

III. With their statement of grounds of appeal, appellant I submitted a main request and a first to fourth auxiliary requests.

IV. Both appellants replied to each other's appeals. Appellant I submitted a fifth and sixth auxiliary requests.

V. The parties were summoned to oral proceedings.

In reply thereto, both appellants withdrew their previous requests for oral proceedings.

The oral proceedings were cancelled.

VI. Claims 1 and 11 of the main request read as follows:

"1. A method for identifying a sequence of nucleotides in a polynucleotide, the method comprising the steps of:

(a) extending an initializing oligonucleotide along the polynucleotide by ligating an oligonucleotide probe thereto to form an extended duplex which is not capable of ligating to a further oligonucleotide probe;

(b) identifying one or more nucleotides of the polynucleotide;

(c) regenerating an extendable terminus on said extended duplex; and

(d) repeating steps (a)-(c) until the sequence of nucleotides is determined.

11. A method for determining the nucleotide sequence of a polynucleotide, the method comprising the steps of:

(a) providing a template comprising the polynucleotide;

(b) providing an initializing oligonucleotide which forms a duplex with the template adjacent to the polynucleotide;

(c) annealing an oligonucleotide probe to the template adjacent to the initializing oligonucleotide;

(d) ligating the oligonucleotide probe to the initializing oligonucleotide to form an extended duplex which is not capable of ligating to a further oligonucleotide probe;

(e) identifying one or more nucleotides of the polynucleotide by a label on the ligated oligonucleotide probe;

(f) regenerating an extendable terminus on said oligonucleotide probe; and

(g) repeating steps (c) through (f) until the nucleotide sequence of the polynucleotide is determined."

Dependent claims 2 to 10 and 12 to 15 define specific embodiments of the methods of claims 1 and 11 respectively.

VII. Claims 1 and 11 of the third auxiliary request differ from claims 1 and 11 of the main request in that the method of claim 1 step (a) and of claim 11 step (c) require the oligonucleotide probe to have a blocking moiety and in that the method of claim 1 step (b) requires identification of one or more nucleotides of the polynucleotide by a label on the ligated oligonucleotide probe.

VIII. Claims 1 and 11 of the fourth auxiliary request are identical to claims 1 and 11 of the third auxiliary request except that the method of claim 1 step (b) requires identification of one or more nucleotides of the polynucleotide by a fluorescent label on the ligated oligonucleotide probe, and in that the method of claim 11 step (e) requires identification of one or more nucleotides of the polynucleotide by a fluorescent label on the ligated oligonucleotide probe.

IX. The following documents are cited in this decision:

D1: WO95/04169 (publication date 9 February 1995)

D2: US 5403708 (publication date 4 April 1995);

D3: WO93/05183 (publication date 18 March 1993).

X. The submissions made by appellant I, insofar as relevant to the present decision, may be summarised as follows:

Main request

Article 123(2) EPC

Claims 1 and 11 did not comprise added subject-matter for the reasons set out in the decision under appeal in item 2.2.1.1.

Novelty (Article 54 EPC)

Document D1 described a reagent and a method of using it for sequencing a polynucleotide using mass spectrometry. The reagent was a bivalent linker associated with a nucleotide on one side and a reporter group on the other side of the linker such that the nucleotide at each position within the reagent was labelled "with a unique tag" (see page 5, lines 23-24; Fig.4 and 5). The reagent comprising an oligonucleotide sequence (analyte) and a detectable tag portion was generated by repetitive addition of both nucleotides and reporter groups. Each reporter group in the tag portion identified a nucleotide and its position in the oligonucleotide portion (analyte) of the reagent (see Fig.1 and 5). The nucleic acid sequence determination by progressive ligation of example 1 was the only embodiment potentially relevant for novelty (see Figure 4 and page 14, lines 31 to page 16, line 35). The first step of the method required capture of a target polynucleotide by an immobilised initializing oligonucleotide (see Figure 4 and page 15, line 23). The oligonucleotide portion of the reagent hybridising to the target polynucleotide at a position adjacent to the initializing oligonucleotide along the sequence of the target polynucleotide (see page 15, lines 34-36 and Figure 4) was then ligated to the initializing oligonucleotide. The subsequent cleavage of the tag moiety and "analysis of the cleaved tag" by mass spectrometry allowed the sequence of nucleotide of the oligonucleotide of the ligated reagent to be determined, revealing thereby the nucleotide sequence of the hybridised section of the target polynucleotide (see page 16, lines 7-9).

Document D1 was however non-enabling, because the technical detail disclosed in document D1 necessary to practice the invention was insufficient.

Although document D1 disclosed a method where steps c) to f) were repeated to determine the nucleotide sequence of a longer target polynucleotide, it remained silent how this repetition had to be achieved. The embodiment on pages 14 to 16 failed to disclose how the cleavage of the tag was carried out and how the extended duplex was returned to conditions enabling another ligation, in particular how the extended duplex is transferred from a dry state, required for the tags' cleavage from the reagent, back into solution for the next ligation step to occur (see page 12, lines 21-23 and page 45, lines 13-20).

Document D1 did not describe furthermore 1) how to determine the sequence of one or more nucleotides of a target polynucleotide based on the sequence of the oligonucleotide used in the reagent; 2) how to prepare a library of labelled oligonucleotide reagents for use in the sequencing method, and 3) how to perform multiple repeated sequencing steps.

The progressive ligation method of document D1 established only that the complete tag moiety was "recovered and analysed" as an indication of the sequence of a first part of a target nucleic acid (see page 16, lines 9-12). The analysis of the tag moiety could however lead to ambiguities if it was fragmented (see page 14, paragraph 1). There was no indication of how a complete tag moiety allowed to determine the identity of the nucleotide at each position of the oligonucleotide portion of the reagent and of how the one or more nucleotides of the target polynucleotide was/were determined.

There was no disclosure how to determine which reporter was present at which specific position in the complete tag moiety to determine the identity of one or more nucleotides of a target polynucleotide. Although example 19 described the detection of a sequence of five thymine residues and a separate sequence of ten adenosine residues using an attached tag (see page 46, lines 12-33), the mass spectrometric analysis of the two tags associated with a specific oligonucleotide would not allow the skilled person to determine the nucleotide sequence of a polynucleotide, as required by claims 1 and 11.

There was no hint in document D1 that the tag synthesis technique described on page 7 or Figure 3 had to be used in the progressive ligation method described on pages 15-16 and Figure 4 (see decision T 305/87 of 1 September 1989). Even if both passages were nevertheless combined the coupling efficiency would cause each hexamer to be linked to a nested set of seven partial synthesis tags each containing a hexameric nucleotide sequence labelled with 0, 1, 2, 3, 4, 5 or 6 reporter groups (see page 7, paragraph 2) which would prevent the skilled person from deconvolving the partial synthesis products to ascertain the polynucleotide's sequence.

Last, document D1 did not establish how to prepare a library of labelled oligonucleotide reagents for use in a progressive ligation sequencing method. If the coupling efficiency for both the nucleotide and the corresponding reporter to a bivalent linker occurred at 100%, then a library of 4096 hexamers would be enough. If the coupling efficiencies occurred at 100% for the nucleotide and occurred only at 80% for the label, then a library of 28672 labelled hexanucleotides would be needed, as each specific hexanucleotide was linked with a set of tags consisting of up to 6 reporter groups. Hence, the skilled person had to use a large volume comprising a complex mixture of labelled hexamers with only a hope to determine the target polynucleotide's sequence.

Third auxiliary request

Article 123(2) EPC

Claims 1 and 11 did not comprise added subject-matter for the reasons set out in the decision under appeal in item 4.2.1.

Novelty (Article 54 EPC)

The third auxiliary request was not anticipated by the content of document D1 for the same reasons as outlined for the main request.

Fourth auxiliary request

Article 123(2) EPC

Claims 1 and 11 did not comprise added subject-matter for the reasons set out in the decision under appeal in item 5.1.2.

Novelty (Article 54 EPC)

The fourth auxiliary request was not anticipated by the content of document D1, for the same reasons as outlined for the main request.

Inventive step (Article 56 EPC)

The subject-matter of claims 1 and 11 involved an inventive step for the reasons set out in the decision under appeal in item 5.2.

The patent and document D3 related to methods that did not require high resolution electrophoretic separations of DNA fragments (see patent application on page 3, document D3, page 1, lines 6-9). Document D3 represented therefore a suitable starting point.

The method of claim 1 differed from the method of document D3 in that a ligase instead of a polymerase was used to extend the initializing oligonucleotide (see page 3, lines 15-19 and page 9, lines 23-25). The identity of the attached nucleotide was identified by a reporter that could be a fluorescent label (see page 7, line 6). The use of a ligase rather than a polymerase in a sequencing method allowed longer target polynucleotides to be sequenced and prevented problems associated with the use of polymerases e.g. fidelity, processivity and polymerization rate (see page 2, lines 43-56).

Starting from the closest prior art document D3, the technical problem to be solved by the patent was therefore to provide an improved sequencing method which can be used to accurately sequence longer target polynucleotides.

Document D2 disclosed a nucleic acid sequencing method using a ligation reaction to extend a primed template (see column 2, lines 23-61). After a single round of ligation, the identity of the extension oligonucleotide was determined by separating the ligation products using gel electrophoresis (column 2, lines 56-61; column 4, lines 1-6; column 8, lines 5-9) to identify one or more nucleotides of the nucleic acid. It did not contemplate any mechanism for identifying one or more nucleotides of the nucleic acid without using gel electrophoresis.

Thus, starting from the method described in document D3 (see page 1, lines 6-9; page 2, lines 27-29), a skilled person seeking to provide an improved sequencing method, would not have turned to the method of document D2.

Without prior knowledge of the invention claimed the skilled person faced with the technical problem identified above would not have modified the iterative sequencing method of document D3 to include the ligation reaction used in the method of D2, as the gel separation step was mandatory in document D2 whereas it was excluded in document D3.

If the teaching of document D3 were combined with the method of document D1, the skilled person would have used the bivalent reagent and mass tag label disclosed in document D1 and would definitely not have arrived at a method of claim 1 using a fluorescent label.

XI. The submissions made by appellant II, insofar as relevant to the present decision, may be summarised as follows:

Main request

Amendments (Article 123(2) EPC)

The subject-matter of claims 1 and 11 differed from the claims of the patent application in that they specified that the extended duplex was not capable of ligating to a further oligonucleotide probe, and included an additional step of regenerating an extendable terminus on the oligonucleotide probe. These features were only mentioned in the context of a method where a blocking moiety was present and/or which comprised the step of removing the chain-terminating moiety and extending the oligonucleotide probe with a nucleic acid polymerase in the presence of one or more labelled chain-terminating nucleotide triphosphates.

There was no disclosure on page 11 at lines 15 to 17 of the patent application that the prevention of multiple extensions occurred when an extended duplex itself was incapable of ligating to a further oligonucleotide probe. The method of the patent application referred to an oligonucleotide probe that had a blocking moiety preventing multiple probe ligations on the same template in a single extension cycle, which was however not mentioned in claims 1 and 11 (see page 3 lines 30-33,page 7 line 25, page 12, line 5 and page 15, line 2). The patent application failed to disclose a method defined by the specific steps of claims 1 and 11, comprising the function of being incapable of ligating to a further oligonucleotide probe in combination with the general step of regenerating an extendable terminus on the extended duplex in the absence of the blocking moiety. Thus, the subject-matter of claims 1 and 11 contravened Article 123(2) EPC.

Novelty (Article 54 EPC)

Document D1 described a method of nucleic acid sequencing by sequential ligation (see section beginning on page 14 at line 31 and page 15 lines 1 to 10). The method comprised the steps (a) to (d) of claim 1: hybridising the nucleic acid sequence to be determined or target nucleic acid with an immobilised oligonucleotide (an initialising oligonucleotide), hybridising an oligonucleotide adjacent to the immobilised oligonucleotide (the oligonucleotide probe), and ligating the adjacent oligonucleotide. The oligonucleotide comprised a tag moiety which was analysed to provide an indication of the sequence it hybridized to as set out in step (f) (see page 15, line 12 and page 16, lines 5 to 10). The removal of the linker according to page 16, lines 10 to 14, regenerated an extendable terminus on the extended duplex for another ligation step, which meant that said duplex was not capable of being ligated to any further oligonucleotide probe prior to removal of said linker. The identification of one or more nucleotides of the polynucleotide sequence and the repetition step (d) of claim 1 were described on page 15, lines 12 to 14 and page 16 line 19 of document D1. Hence, the subject-matter of claim 1 was not novel.

Document D1 disclosed all the individual steps of the method of claim 11 as well (see page 14, line 31 to page 16, line 25; especially page 15, lines 1-2).

The "primer" oligonucleotide was chosen to be part of the vector sequence comprising the polynucleotide of interest, wherein the initialising oligonucleotide hybridises to the vector sequence adjacent to the polynucleotide sequence of interest, as claimed in step (b) of claim 11 (see page 15, line 20 and lines 26 to 27). The steps (c), (d) and (e) of claim 11, describing the identification of a nucleotide of the polynucleotide by a tag on the ligated oligonucleotide probe, were disclosed on page 15, lines 3 to 9 and page 16, lines 9 to 10. The regeneration of an extendable terminus and the repetition of method steps (c) to (f) were disclosed on page 16, lines 10 to 14, and line 19. Thus, document D1 disclosed the method steps (f) and (g) of claim 11 too.

Since the extended duplex, resulting from the ligation of an initialising oligonucleotide with an adjacent template oligonucleotide probe, composed of an oligonucleotide moiety, a bivalent linker and a tag moiety, would expose an hydroxyl group or phosphate group only after the linker associated with the tag moiety was removed, said tag moiety linked to the oligonucleotide probe preventing multiple ligations was a chain-terminating moiety at a terminus distal to the initialising oligonucleotide. Thus, document D1 anticipated the subject matter of claims 2 and 12 as well.

Third auxiliary request

Article 123(2) EPC

Claims 1 and 11 added subject-matter beyond the content of the patent application.

Claims 1 and 11 of the third auxiliary request differ from claims 1 and 11 of the main request in that the method of claim 1 step (a) and of claim 11 step (c) require the oligonucleotide probe to have a blocking moiety and in that the method of claim 1 step (b) requires identification of one or more nucleotides of the polynucleotide by a label on the ligated oligonucleotide probe.

Amended claims 1 and 11 infringed Article 123 (2) EPC because the blocking moiety was not at a terminal position of an oligonucleotide and was not specified to first prevent multiple oligonucleotide extensions during a single extension cycle to occur and to be capable, when removed, of regenerating an extendable terminus on the extended duplex (see page 3, line 31, of the patent application).

Since the scope of claim 1 was broader than that of claim 2, which specified that the blocking moiety was a chain terminating moiety at a terminus distal to said initialising oligonucleotide, claim 1 embraced oligonucleotide probes with a blocking moiety at any position. For the above reasons, the third auxiliary request contravened Article 123(2) EPC.

Novelty (Article 54 EPC)

Appellant II repeated the arguments already put forward in the context of the main request.

Fourth auxiliary request

Article 123(2) EPC

The objection under Article 123(2) EPC raised against the third auxiliary request was repeated.

Inventive step (Article 56 EPC)

Document D3 disclosed a method with repetitive cycles in which an initialising probe was extended and contacted with a medium containing a modified nucleotide and an enzyme to attach the modified nucleotide to the 3'-hydroxyl terminus of the primer attached to each template. The modified nucleotides comprised a blocking group and a reporter group which may be a fluorescent dye (see page 6, lines 29 to 30). The blocking group prevented the addition of further nucleotides to the primer (see page 3,lines 18 to 19). The blocking group was removed to regenerate an extendable terminus to allow the method steps to be repeated until the DNA or RNA was sequenced (see page 3, lines 22 to 26, page 11, line 27). The blocking group could be removed by acid/base or light treatment. Thus, it provided an iterative process comprising the steps of extending an oligonucleotide, incorporating a blocking moiety to prevent further extension, an analysis and identification step of the extended sequence, a regeneration of an extendable terminus, and a repetition of these steps.

The difference between the method of document D3 and the method of claim 1 was the use of a probe having more than one nucleotide instead of using an extension reaction adding single nucleotides.

The use of a ligase instead of a polymerase allowed the sequencing of long target polynucleotides and of sequences more distant from the initialising target to be identified. The nucleotides of the probe served as spacers to ensure that the next ligation will take place at a predetermined site or number of bases along the template (see patent application on page 8, first paragraph).

Starting from the teaching of document D3 and based on the technical effect ascribed to the difference identified above, the technical problem to be solved was to provide an alternative method allowing downstream sequences to be queried.

The solution to this problem was to use a ligation probe instead of the single nucleotide extension reaction as described in document D3.

The use of oligonucleotide probes as spacers to identify sequences distant from an initialising probe was well known in the art. The use of ligation reactions to distinguish between matched and mismatched sequences was equally well known.

Thus, the use of oligonucleotide probes in a ligation reaction instead of a polymerase extension reaction to effectively walk along a sequence at intervals of N nucleotides in order to determine sequences distant from the initial oligonucleotide was a matter of routine and an obvious choice for the skilled person (see document D2, col.4, line 32 et seq.; Figure 2).

The same iterative process using oligonucleotide probes ligated one by one on a polynucleotide template was described in document D1 (see beginning of page 15).

Thus, the method of claim 1 lacked an inventive step over document D3 in combination with either document D2 or D1.

XII. Appellant I requests that the decision under appeal be set aside and that the patent be maintained on the basis of the main request or one of the first to sixth auxiliary requests.

XIII. Appellant II requests that the decision under appeal be set aside and that the patent be revoked. They request the first, second, fifth and sixth auxiliary requests not to be admitted into the appeal proceedings.