T 0289/96 (Detecting sequences/ZENECA) 11-02-1999

Formal admissibility under Article 123(2) and (3) EPC

1. The sole claim request on file consists of method claims 1 to 9 as granted against which no objections under Article 100(c) EPC have been raised. The amended description pages 2, 6, 7, 10 and 11 have been adapted to this limited set of claims. By these amendments no new matter has been generated. The appellants had no objections thereto. The formal requirements of Article 123(2) and (3) EPC are met.

Sufficiency of disclosure (Article 83 EPC)

2. Article 83 EPC requires that the invention be disclosed in a manner sufficiently clear and complete for it to be carried out by a person skilled in the art. Article 84 EPC specifies inter alia that the claims, which define the matter for which protection is sought, should be supported by the description. In the European case law there are several examples of cases in which questions of sufficiency and support were decided (cf eg T 409/91 (supra), T 694/92, OJ EPO 1997, 408). These, however, are questions which have to be examined in each case on its own merits. There is general agreement that, as pointed out eg in decision T 409/91 (supra, cf point 3.5 of the reasons), the purpose of the requirement of support by the description, insofar as its substantive aspect is concerned, and of the requirement of sufficient disclosure is the same, namely to ensure that the extent of protection conferred by the granted claims is justified by the actual technical contribution to the art. This implies inter alia, firstly, that a claim may not encompass subject-matter which is not sufficiently disclosed within the meaning of Article 83 EPC as it cannot be performed without undue burden and, secondly, that a claim should contain all the essential features of the invention which are necessary to meet the requirement of sufficient disclosure. These are exactly the issues raised by the appellants in the present case.

3. The description of the patent in suit reveals that the invention stems from the realisation that "...by selecting the nucleotide sequence of an oligonucleotide primer appropriately it is possible to selectively achieve primer extension of either a sequence containing a suspected variant nucleotide or the corresponding sequence containing the normal nucleotide or to prevent such primer extension thus substantially simplifying the detection procedures necessary." (cf page 3, lines 16 to 19).

4. Accordingly, the skilled person is taught by the description a method whereby, when the presence or absence of one or more variant nucleotide sequence has to be detected in a sample, this is treated, together or sequentially, with appropriate nucleoside triphosphates, an agent for polymerisation of the nucleoside triphosphates and a diagnostic primer for a diagnostic portion of a target base sequence under hybridising conditions, and then the presence or absence of the suspected variant nucleotide is detected based on the presence or absence of an extension product. Guidance is provided on how to design the nucleotide sequence of a suitable primer: its length may be from about 5 to 50 bp; it should be substantially complementary to the diagnostic portion of the target sequence, the 5' or 3' terminal nucleotide of the primer being either complementary to the suspected variant nucleotide or to the corresponding normal nucleotide, so that an extension product of the primer is synthesised only when the terminal nucleotide of the primer is complementary to the corresponding nucleotide in the target base sequence, and not when the terminal nucleotide of the primer is not complementary to the corresponding nucleotide in the target base sequence (cf page 3, lines 20 to 32). The reader is also told that "any extension product obtained may if desired be amplified by the polymerase chain reaction (PCR) as described in ..." (emphasis added) (cf page 3, lines 41 to 48). Details about the agent for polymerisation which may be used are given on page 7, lines 50 to 56. The description provides the definition of the different terms, information about the optimal length of the primer, an outline of the preferred embodiments, a table pointing to the relevant mutations in a series of known genetic disorders to be taken into account in designing the respective diagnostic primers, and examples in which the applicability of the approach as described is shown with reference to the figures (NB: as noted by the appellants, some of the figures in the printed patent specification are not as clear as those originally filed with the application. However, this is a practical problem linked to the reproduction of the document which has nothing to do with sufficiency of disclosure. Access to the original figures is possible through inspection of the file).

In all the examples, save Example 5, an amplification step is used. Example 5 is a model example in which two cloned DNA sequences are used, one containing a "normal" sequence and the other containing the "variant" sequence. This example reports the detection of the product when a matched primer is used in accordance with the approach described in the specification without amplification.

5. The wording of claim 1 (see Section II supra) outlines the same operational steps as the basic teaching of the patent in suit. It characterises the invention in the broadest outline as disclosed in the patent specification.

6. The claim does not need to include technical details which are manifest in the light of the overall disclosure or common general knowledge, such as eg the need for a control reaction, indications in respect of the length of the primer, nor features which characterise particular embodiments.

The amplification step is presented in the patent specification as an additional step (cf page 3, lines 41 to 45 of the patent specification) and no need is seen to include it in the main claim. In respect of the appellants' objection that without this operational step the method of claim 1 does not work in a real-life diagnostic situation, it is observed that:

- Claim 1 is generally directed to a method for detecting the presence or absence of at least one variant nucleotide in one or more nucleic acids contained in a sample, which does not necessarily always mean a clinical sample where the target sequence can be present in very small amounts together with many other interfering substances so as to render amplification desirable, if not necessary. As a matter of fact, the method of claim 1 is meant to be broadly applicable also to simpler samples (cf Example 5) or to technical situations where the target sequence has undergone previous isolation or enrichment;

- The skilled person is able, on the basis of the description and of common technical knowledge to recognise technical situations in which amplification is necessary (possibly the great majority of the real-life diagnostic situations) and those in which amplification is not necessary. No undue burden is placed on the skilled person by leaving this option open, as done by the wording of claim 1.

The same rationale applies also to the use of a second primer which constitutes a further embodiment of the general approach outlined in claim 1 (cf claim 3).

7. As regards the alleged misleading information about the polymerases which can be used, the board considers that, as it is a fundamental teaching of the patent specification that elongation should take place only when the 5' or 3' terminal nucleotide of the primer matches the relevant nucleotide of the target sequence (cf points 4 and 5 supra), the skilled person is thereby unambiguously instructed to avoid the use of polymerases with proofreading activity.

8. For these reasons, the board considers that the information provided in the description of the patent specification is sufficiently clear and complete to enable a skilled person to carry out the method as claimed without undue burden. The requirements of Article 83 EPC are therefore satisfied.

Inventive step (Article 56 EPC)

9. In the board's judgement, the closest prior art is represented by document (9) which is also cited in the body of the specification (cf page 2, line 27 to page 3, line 12). This document, like the patent in suit, is concerned with a method for detecting the presence or absence of at least one nucleotide variation in one or more nucleic acids contained in a sample. The sample is repeatedly treated with primers (one for each strand of each nucleic acid), nucleotide triphosphates, and an agent for polymerization of the triphosphates, and then denatured, in a process wherein the sequence containing the nucleotide variation, if present, is amplified. The primer or primers are selected so as to be substantially complementary to each nucleic acid strand containing each different variation, such that the extension product synthesized from one primer, when it is separated from its complement, can serve as a template for synthesis of the extension product of the other primer. The detection step involves either the use of a labelled sequence-specific oligonucleotide probe and/or the use of a specific restriction endonuclease and/or use of direct sequencing methods on the amplified DNA.

10. In the board's opinion, document (6), selected by the appellants as the closest prior art, is no more relevant than document (9), which also refers to it (cf sentence bridging pages 1 and 2 of document (9)), for the reasons given hereinafter:

The document in question relates essentially to the diagnosis of sickle cell anemia by an oligomer restriction method the sensitivity of which is enhanced by way of amplification of ß-globin DNA sequences by use of primers and DNA polymerase. The appellants have drawn the board's attention to the description of the primer PC04 (cf ibidem, Figure 1) which, in their view, is used to distinguish between the ß- and -globin genes based on a mismatch at the 3' terminus. However, apart from the fact that in Figure 1 more than one nucleotide difference between the ß- and -globin genes is indicated, no particular emphasis being placed on the mismatch in question, nothing in the text of the document points specifically to the mismatch at the 3' end of PC04 as being in any way significant for selective detection of the ß- or -gene based on primer elongation. Primer PC04, which is also described in document (9), is only one of the two primers used to amplify the ß-globin gene segment containing the relevant restriction site in view of the oligomer restriction analysis.

11. In view of document (9), the underlying technical problem is defined as being the finding of an alternative, possibly simpler, method for detecting at least one single base difference in nucleic acids.

12. As a solution thereto, the claims at issue propose a method based on the determination of the elongation or non-elongation of a primer so designed that its 5' or 3' terminal nucleotide is either complementary to the suspected variant nucleotide or to the corresponding normal nucleotide in the target sequence. The examples show that, as mismatches at the 3' end prevent the elongation by polymerases, the method can indeed be used for detection of variant nucleotides in nucleic acids.

13. It has been argued by the appellants that, as the skilled person was aware of the fact that base pair matching was necessary for polymerisation to occur (cf documents (2) or (3)), the skilled person would have readily arrived at the claimed method, especially in the light of the prior art knowledge on how a genetic disease could be spotted by using an oligonucleotide with a mismatch (cf documents (5) and (6)) as well as in the light of the knowledge that selective primer elongation could be achieved with eg Taq polymerase (cf document (21)).

14. Various documents in the art dealt with methods for detecting specific nucleotide variations in nucleic acids, in particular in relation to genetic diseases. Documents (6) and (9) were among them. Their contents have already been discussed above (cf points 9 and 10). Nothing in these two documents would have provided a hint in the direction of the method claimed as both of them were essentially dealing with the amplification of the nucleotide sequence containing the variation to be detected in view of the subsequent detection step, be that hydridisation with a labelled probe or restriction analysis etc.

Document (5) was also known to the skilled person. This document proposed a method of detection based on forming a hybrid between a labelled probe and the target sequence in a position adjacent to the mutated nucleotide, adding a nucleotide derivative which, if complementary to the mutated base, protects the probe from digestion, and observing the presence or absence of the label attached to the target. This document explicitly draws the reader's attention to the need to use faithful polymerases free of exonuclease activity, like the calf thymus DNA polymerase (cf page 10, lines 24 to 36). The essential objective of document (5) is to ensure the elongation of the probe up to the mutation site and the insertion at this site of a nucleotide derivative which protects the probe from subsequent digestion with eg an exonuclease. From this teaching, alone or in combination with that of documents (6) or (9), the skilled person would not have readily derived the idea of a method based on the achievement or prevention of selective primer extension beyond the mutation site of either a sequence containing a suspected variant nucleotide or the corresponding sequence containing the normal nucleotide.

14. It is true that the skilled person knew from the art that for the polymerisation step to occur pairing has to be present between the primer terminus and the template (cf documents (2) and (3)) and that, in case of a mismatch, several polymerases would exert a proofreading function (cf document (2)). However, this prior art, alone or in combination with documents (9) or (6), would not have suggested using a polymerase lacking the proofreading activity in order to detect a nucleotide variation in a target sequence based on the elongation or non-elongation of a specifically designed primer.

15. The skilled person was aware of the availability of various polymerases, among them of the thermostable Taq polymerase of document (21) which was used therein to improve the amplification step in the framework of the analysis of nucleotide sequences. However, nothing also in the latter document indicated to the skilled person the possibility of taking advantage of its lack of proofreading activity in a detection method based on selective primer extension.

16. For these reasons, in the board's judgement the method according to claims 1 to 9 at issue involves an inventive step.