T 0902/94 (DNA amplification/Baylor College of Medicine) 06-03-1998

I. European patent application No. 89 310 424.0 published under No. 0 364 255 with the title " Multiplex genomic DNA amplification for deletion detection" was refused by the Examining Division in a decision dated 12 April 1994 and posted on 6 June 1994.

Originally filed claim 1 read as follows:

"1. A method for simultaneously detecting deletions at a plurality of DNA sequences, comprising the steps of:

treating DNA to form single-stranded complementary strands;

adding a plurality of paired oligonucleotide primers, each pair specific for a different sequence, one primer of each pair substantially complementary to a part of the sequence in the sense-strand and the other primer of each pair substantially complementary to a different part of the same sequence in the complementary anti-sense strand;

annealing the plurality of primers to their complementary sequences;

simultaneously extending said plurality of annealed primers from each primer's 3' terminus to synthesize an extension product complementary to the strands annealed to each primer, said extension products, after separation from their complement, serving as templates for the synthesis of an extension product from the other primer of each pair;

separating said extension products from said templates to produce single-stranded molecules;

amplifying said single stranded molecules by repeating, at least once, said annealing, extending and separating steps; and

identifying said amplified extension products from each different sequence."

Dependent claims 2 to 4 related to further specific features of the plurality of genomic DNA sequences to be detected. Dependent claims 5 and 6 related to the properties and sequences of the plurality of paired primers to be used in the case of X-linked muscular dystrophy. Dependent claim 7 related to the nature of the tissue, the sequences to be tested should be derived from. Dependent claim 8 related to a plurality of specific paired primers to be used in the simultaneous detection of a plurality of genomic DNA sequences. Dependent claims 9 to 17 related to specific DNA sequences from the gene responsible for muscular dystrophy.

II. The decision of the Examining Division was taken on the basis of a set of claims 1 to 18 filed on 12 April 1994. Claim 1 differed from originally filed claim 1 in that the wording "plurality" was replaced by "at least three" and the expression "all primers being subjected to the same conditions" was added to the second characterising feature of the claim. A new claim 9 was introduced which related to further possible embodiements of the method of claim 1. Claims 9 to 17 were renumbered claims 10 to 18.

III. The grounds for refusal were that "the subject-matter of claims 1 to 5 does not meet the requirements of Article 54 EPC. The subject-matter of claim 9 lacks an inventive step under Article 56 EPC. The subject-matter of claims 10 to 18 does not meet the requirements of Article 84 EPC." A further ground for refusal was lack of unity a posteriori (Article 82 EPC),(see point 3 of the decision).

IV. The reasons given for the decision were as follows:

The method of claims 1 to 5 was not new because it was already disclosed in

Document (3) American Journal of Human Genetics, vol. 43, no. 3 suppl. (1988) Abstract no. (0711) 3.2

where the detection of multiple deletions at the Duchenne muscular dystrophy locus via multiplex genomic DNA amplification (PCR) was performed by combining several sets of primers in a single reaction.

Claim 9 related to the same method as in claim 1, specifying the technical parameters of the extension step, its subject-matter being thus novel over the teachings of document (3). These technical parameters were, however, known from the skilled person at the filing date. Inventive step was, thus, lacking.

Claims 10 to 18 lacked clarity in that they related to DNA sequences which only partly included but were not defined primers to be used in the claimed method.

There was lack of unity a posteriori between the claims relating to the method of claim 1 being performed on different genes, as this method which constituted the link between these claims was not novel.

V. The Appellant lodged an appeal against this decision, paid the appeal fee and submitted a statement of grounds for the appeal.

VI. A communication was sent by the Board according to Article 11(2) of the Rules of Procedure of the Boards of Appeal, setting out the Board's provisional, non binding opinion.

VII. The Appellant sent some further submissions together with affidavits.

VIII. In the course of oral proceedings, the Appellant filed a new set of eight claims as new only request.

Claim 1 of this last set of claims 1 to 8 read:

"1. A method for simultaneously detecting in a sample target DNA sequences, comprising the steps of:

providing in a common reaction vessel the sample in single-stranded form and pairs of oligonucleotide primers, each pair specific for a different sequence, one primer of each pair substantially complementary to a part of the sequence in the sense-strand and the other primer of each pair substantially complementary to a different part of the same sequence in a complementary anti-sense strand;

annealing the pairs of primers to their complementary sequences;

simultaneously extending said pairs of annealed primers from each primer's 3' terminus to synthesize an extension product complementary to the strands annealed to each primer, said extension products, after separating from their complement, serving as templates for the synthesis of an extension product from the other primer of each pair;

separating said extension products from said templates to produce single-stranded molecules of the target sequences;

amplifying said single stranded target sequences by repeating, at least once, said annealing, extending and separating steps; and

identifying whether said amplified extension products have been synthesized from each different sequence, as a measure of the presence or absence of each target sequence, characterized in that:-

the method is adapted to detect simultaneously more than two target sequences by utilising more than two pairs of oligonucleotide primers."

Dependent claims 2 to 8 corresponded to originally filed claims 2 to 8, except for the fact that the expression "a plurality of" was deleted from claims 2, 5, 6 and 8.

IX. The Appellant argued as follows:

With regard to Article 123(2) EPC:

New claim 1 differed from originally filed claim 1 in that the term "plurality" had been replaced by the term "more than two". Although this last expression was not to be found expressis verbis in the application as originally filed, the content of the application left the person skilled in the art in no doubt that the claimed method was to be performed on more than two genomic sequences, with more than two pairs of primers. Evidence for this was to be found, for example, on page 1, line 29, on page 3, line 25 to 32, on page 14, line 24 to 26. In example 8, three different primer pairs were used to screen three different genetic loci. On page 13 line 30, it was contemplated to simultaneously screen four deficiencies. The requirements of Article 123(2) EPC were, thus, fulfilled.

With regard to Article 54 EPC:

Document (3) which was considered by the Examining Division as detrimental to novelty was not enabling and, thus, should not be taken into consideration. Indeed, although it disclosed that the PCR reaction could be carried out starting from a multiplicity of pairs of primers, it did not provide any instructions on how to proceed. In particular, the necessity to use primers with about the same melting temperature, as well as the need to use a high amount of enzyme and to increase the time of reaction were not mentioned. The subject-matter of claims 1 to 8 was, thus, novel.

X. The Appellant requested that the decision under appeal be set aside, and that a patent be granted on the basis of the new set of claims 1 to 8 filed as new only request at the oral proceedings.