Ex Parte Makarov et al

Patent Trial and Appeal BoardNov 19, 2012

11367046 (P.T.A.B. Nov. 19, 2012)

UNITED STATES PATENT AND TRADEMARK OFFICE __________ BEFORE THE PATENT TRIAL AND APPEAL BOARD __________ Ex parte VLADIMIR L. MAKAROV, EMMANUEL KAMBEROV, and BRENDAN J. TARRIER __________ Appeal 2011-000599 Application 11/367,046 Technology Center 1600 __________ Before DONALD E. ADAMS, ERIC GRIMES, and ERICA A. FRANKLIN, Administrative Patent Judges. GRIMES, Administrative Patent Judge. DECISION ON APPEAL This is an appeal under 35 U.S.C. § 134 involving claims to a method of amplifying GC-rich DNA molecules, which the Examiner has rejected as obvious. We have jurisdiction under 35 U.S.C. § 6(b). We reverse. STATEMENT OF THE CASE The Specification states that “CpG dinucleotides play a very special role in human and other mammalian organisms by providing a target for Appeal 2011-000599 Application 11/367,046 2 DNA methylation. . . . Methylation of promoter CpG islands plays an important role in the regulation of gene expression.” (Spec. 1, ¶ 4.) Claims 1-13, 21-28, and 36-43 are on appeal. Claim 1 is representative and is directed to a method of amplifying GC-rich DNA molecules by providing a mixture of GC-rich and GC-poor DNA molecules, subjecting them to conditions that preferentially denature GC-poor but not GC-rich molecules, attaching an adaptor to the molecules that remain double-stranded, and treating them by either: (a) digesting with a methylation-sensitive restriction enzyme to cleave non-methylated DNA molecules, and PCR amplifying the uncleaved (methylated) molecules, or (b) digesting with a methylation-specific restriction enzyme to cleave methylated DNA molecules, and PCR amplifying the uncleaved (unmethylated) molecules. The full text of claim 1 is reproduced in Exhibit 10 in the Evidence Appendix attached to the Appeal Brief.1 The claims stand rejected under 35 U.S.C. § 103(a) as follows: • Claims 1-13, 21-28, 36-39, and 41-43 based on Bellizzi,2 either of Page3 or Rabinovitch,4 and Huang5 (Answer 4) and • Claim 40 based on Bellizzi, either of Page or Rabinovitch, Huang, and Martienssen6 (Answer 6). 1 The Examiner indicated that the proposed amendment in the Evidence Appendix was entered. (Answer 2.) 2 Dina Bellizzi et al., A procedure for cloning genomic DNA fragments with increasing thermoresistance, 219 GENE 63-71 (1998). 3 Page et al., US 5,871,920, Feb. 16, 1999. 4 Rabinovitch, US 5,814,444, Sept. 29, 1998. 5 Huang, US 6,605,432 B1, Aug. 12, 2003. 6 Martienssen, US 2004/0132048 A1, July 8, 2004. Appeal 2011-000599 Application 11/367,046 3 I. The Examiner has rejected all of the claims on appeal, except for claim 40, as obvious based on Bellizzi, Page or Rabinovitch, and Huang (Answer 4). The Examiner finds that Bellizzi discloses a method that includes subjecting DNA molecules to conditions that denature GC-poor regions but not GC-rich regions (id.) and selecting the GC-rich regions by cloning them (id. at 5). The Examiner finds that Page and Rabinovitch both disclose ligating an adaptor to DNA fragments and amplifying them by PCR (id.). The Examiner finds that Huang discloses separating methylated from non-methylated GC-rich DNA fragments by digesting with a methylation- sensitive restriction enzyme and amplifying the undigested fragments (id.). The Examiner concludes that it would have been obvious to modify Bellizzi’s method “by substituting a PCR-adaptor ligation step and subsequent PCR amplification step for the direct cloning step of Bellizzi” (id.) because a skilled worker would have understood that Page’s or Rabinovitch’s method would also be selective for double-stranded DNA (id. at 6). The Examiner also concludes that “[t]he skilled artisan would have been further motivated to use methylation-sensitive restriction enzymes because Huang disclosed this as a means of distinguishing between methylated and non-methylated GC-rich fragments, or CpG islands” (id.). Appellants argue that, while Bellizzi discloses “a scheme for obtaining progressively GC-rich DNA fractions, using progressively increasing temperatures” (Appeal Br. 5), the purpose of the claimed method is “to not only to selectively amplify thermo-resistant DNA, but also selectively amplify thermo-resistant DNA that is either enriched for Appeal 2011-000599 Application 11/367,046 4 methylated (step (a)) or non-methylated DNA (step (b)). Not only is Belizzi [sic] silent as to PCR amplification, or selective isolation of methylated or non-methylated DNA.” (Id. at 6.) Appellants also argue that “nowhere does Huang teach or suggest the desirability of enriching the GC-rich DNA fraction prior to treatment with the methylation-sensitive enzymes and PCR amplification” (id.) and that “no reason can be articulated, based on a consideration of Huang, as to why one of skill would in any way be motivated to pre-treat Huang’s DNA to remove GC-poor sequences” (id. at 7). We agree with Appellants that the Examiner has not persuasively shown that the cited references would have made obvious a process meeting all of the limitations of claim 1. Bellizzi discloses a series of experiments on the helix-coil transition of DNA (Bellizzi 63, abstract). Bellizzi discloses that the experiments were intended to investigate “the arrangement of the more heat-resistant [regions] vis-à-vis the less resistant ones along a chromosomal DNA” and to provide “a better understanding of the physico- chemical parameters of the helix-coil transition” (id. at 64, left col.). Bellizzi does not indicate that the methylated or unmethylated state of CpG dinucleotides would have any effect on the results of its experiments. Bellizzi therefore does not support the Examiner’s conclusion that it would have been obvious to modify its method by treating its DNA with a methylation-sensitive restriction enzyme, because the Examiner has not provided any reason a skilled worker would have considered that to be a useful modification of Bellizzi’s method. Appeal 2011-000599 Application 11/367,046 5 For its part, Huang discloses “methods for detecting the presence or absence of methylated CpG islands within a genome utilizing a microarray based technology” (Huang, col. 1, ll. 16-18). Huang describes its method as follows: [Nucleic acid] fragments containing the CpG islands are then digested with a methylation-sensitive enzyme resulting in a digestion product comprising methylated CpG island loci. The digestion product is amplified and labeled to form amplicons which are used to screen a plurality of nucleic acid fragments affixed to a solid support. The presence or absence of labeled amplicons bound to the plurality of nucleic acid fragments of the screening array is then determined. (Id. at col. 3, ll. 49-57.) The Examiner points to Huang’s columns 12-14, which describe in more detail the above process (Answer 5). However, the Examiner has not pointed to any disclosure in Huang that its process would benefit from including a denaturation/renaturation step like that of Bellizzi, to preferentially remove GC-poor DNA. Thus, if the Examiner’s reasoning is based on modifying Huang’s method to include Bellizzi’s denaturation/renaturation step, he has not provided evidence or technical reasoning that is adequate to support a conclusion that such a modification would have been obvious to a skilled worker. In summary, the Examiner has not provided persuasive evidence or reasoning to support the conclusion that combining Bellizzi’s denaturation/renaturation process and Huang’s methylation-specific restriction enzyme treatment in the same procedure would have been obvious to a person of ordinary skill in the art based on the teachings of the cited references or the knowledge of those skilled in the art. We therefore Appeal 2011-000599 Application 11/367,046 6 reverse the rejection of claim 1, and dependent claims 2-13, 21-28, 36-39, and 41-43, based on Bellizzi, Page or Rabinovitch, and Huang. II. The Examiner has rejected claim 40 as obvious based on Bellizzi, either of Page or Rabinovitch, Huang, and Martienssen (Answer 6). Claim 40 is directed to the “method of claim 1, wherein the methylation-specific enzyme is McrBC” (Appeal Br., Exh. 10, page 5). McrBC is a restriction enzyme that cuts methylated, but not unmethylated, DNA (Spec. 20, ¶ 86). The Examiner’s rejection is based on a rationale similar to the rejection of claim 1, with substitution of a methylation-specific enzyme for Huang’s methylation-sensitive enzyme, because “Martienssen et al. disclosed that like methylation-sensitive restriction enzymes, methylation- specific restriction enzymes are also useful to distinguish and separate methylated and unmethylated DNA fragments” (Answer 7). However, as with the rejection of claim 1, the Examiner has not pointed to persuasive evidence or technical reasoning to show that it would have been obvious to a skilled worker to modify Bellizzi’s method to include a step of distinguishing between DNA fragments based on their methylated or unmethylated state. The Examiner has also not provided an adequate basis for concluding that it would have been obvious to modify Huang’s process (with substitution of Martienssen’s enzyme) to include Bellizzi’s denaturation/renaturation step. Because the Examiner has not made out a prima facie case of obviousness with respect to claim 40, we reverse the rejection of that claim as well. Appeal 2011-000599 Application 11/367,046 7 SUMMARY We reverse the rejection of claims 1-13, 21-28, 36-39, and 41-43 under 35 U.S.C. § 103(a) based on Bellizzi, either of Page or Rabinovitch, and Huang. We also reverse the rejection of claim 40 under 35 U.S.C. § 103(a) based on Bellizzi, either of Page or Rabinovitch, Huang, and Martienssen. REVERSED lp