Ex Parte Rank et alDownload PDFPatent Trial and Appeal BoardNov 14, 201711982465 (P.T.A.B. Nov. 14, 2017) Copy Citation United States Patent and Trademark Office UNITED STATES DEPARTMENT OF COMMERCE United States Patent and Trademark Office Address: COMMISSIONER FOR PATENTS P.O.Box 1450 Alexandria, Virginia 22313-1450 www.uspto.gov APPLICATION NO. FILING DATE FIRST NAMED INVENTOR ATTORNEY DOCKET NO. CONFIRMATION NO. 11/982,465 10/31/2007 David Rank 105-001105US 9130 57770 7590 11/16/2017 Pacific Biosciences of California, Inc. 1305 O' Brien Drive MENLO PARK, CA 94025 EXAMINER GROSS, CHRISTOPHER M ART UNIT PAPER NUMBER 1639 NOTIFICATION DATE DELIVERY MODE 11/16/2017 ELECTRONIC Please find below and/or attached an Office communication concerning this application or proceeding. The time period for reply, if any, is set in the attached communication. Notice of the Office communication was sent electronically on above-indicated "Notification Date" to the following e-mail address(es): PATENTS@PACB.COM PTOL-90A (Rev. 04/07) UNITED STATES PATENT AND TRADEMARK OFFICE BEFORE THE PATENT TRIAL AND APPEAL BOARD Ex parte DAVID RANK, JONAS KORLACH, YUE XU, STEPHEN TURNER, JEFFERY WEGENER, DANIEL ROITMAN, and JOHN LYLE Appeal 2017-001578 Application 11/982,4651 Technology Center 1600 Before ULRIKE W. JENKS, RICHARD J. SMITH, and DAVID COTTA, Administrative Patent Judges. COTTA, Administrative Patent Judge. DECISION ON APPEAL This is an appeal under 35 U.S.C. § 134 involving claims directed to a method of identifying a sequence of a nucleic acid molecule. The Examiner rejected the claims on appeal as anticipated under 35 U.S.C. § 102(b). We reverse. 1 According to Appellants, the real party in interest is Pacific Biosciences of California, Inc. App. Br. 2. Appeal 2017-001578 Application 11/982,465 STATEMENT OF THE CASE The Specification discloses that “[t]here are a wide range of analytical operations that may be benefited from the ability to analyze the reaction of individual molecules, relatively small numbers of molecules or molecules at relatively low concentrations.” Spec. 13. The Specification further discloses that a number of approaches have been described for analyzing reactions involving small numbers of molecules including by using “optical confinement techniques” “to ascertain signal information only from a relatively small number of reactions, e.g., a single molecule, within an optically confined area.” Id. ^ 4. One method of optical confinement is to use a zero mode waveguide (“ZMW”). Id. 132. ZMWs are “generally characterized by the existence of core surrounded by a cladding, where the core is dimensioned such that it precludes a substantial amount of electromagnetic radiation that is above a cut-off frequency from propagating through the core.” Id. 133. “As a result, when illuminated with light of a frequency below the cutoff frequency, the light will only penetrate a short distance into the core, effectively illuminating only a small fraction of the core’s volume.” Id. The Specification discloses that reactions outside the illuminated portion of a ZMW may “impact the reaction of interest or the monitoring of that reaction, by affecting reaction kinetics through depletion of reagents, increasing concentration of products, [or] contributing to signal background noise levels.” Id. 135. The Specification thus teaches that “it may be further desirable that reactions of interest be reduced or even eliminated from other regions outside of the observation volume, e.g., on the overall substrate 2 Appeal 2017-001578 Application 11/982,465 housing ZMWs, the cladding layer, etc., both inside and outside of the observation volume.” Id. The Specification discloses that the present invention is directed to “methods for localizing individual molecules within a particular space or volume, such that the spatial individuality of the molecule may be exploited, e.g., chemically, optically, electrically, or the like.” Id. ^31. “[Selective and preferential deposition and/or immobilization of the reaction components within the observation volume [of a ZMW] are particular advantages of the invention.” Id. ]f 35. Claims 41, 42, 57—59, 61, and 64—66 are on appeal. Claim 41 is illustrative and reads as follows: 41. A method of identifying a sequence of a nucleic acid molecule, comprising: providing nucleic acid polymerase/template/primer complexes within discrete observation regions on a substrate surface, and detecting sequential addition of nucleotides or nucleotide analogs in a template dependent manner to identify a sequence of incorporation of the nucleotides or nucleotide analogs in the observation regions; wherein the nucleotides or nucleotide analogs comprise at least one nucleotide analog that is labeled on a phosphate group, addition of which nucleotide analog results in release of a labeled polyphosphate; wherein the substrate comprises a cladding layer and a transparent layer; wherein the observation regions are disposed within zero mode waveguides that are disposed through the cladding layer to the transparent layer that forms the bottom of the zero mode waveguides; wherein either the polymerase or the template or the primer component of the nucleic acid polymerase/template/primer complexes is immobilized on the substrate surface; wherein density of the immobilized component on the bottom of the zero mode waveguides is 100 times or more greater than density of the immobilized component on the substrate surface other than the bottom of the 3 Appeal 2017-001578 Application 11/982,465 zero mode waveguides; and wherein detecting sequential addition of nucleotides or nucleotide analogs in a template dependent manner comprises providing illumination to and receiving electromagnetic radiation from the observation regions through the transparent layer. App. Br. 16. The Examiner rejected claims 41, 42, 57—59, 61, and 64—66 under 35 U.S.C. § 102(b) as anticipated by Levene.2 ANALYSIS Levene discloses a “zero-mode waveguide [ZMW], for highly efficient single-molecule analysis at high fluorophore concentrations and its application to enzyme analysis.” Levene 683. This is depicted in figure 1 (reproduced below). figure 1 of Levene depicts “[a]n apparatus for single-molecule enzymology using zero mode waveguides.” Id. Levene’s ZMWs “were manufactured as small holes in an 89-nm thick film of aluminum on fused silica cover slips.” Id. at 684. Levene discloses that enzymes, like polymerase, can be adsorbed onto the bottom of the waveguide to allow direct observation of single- 2 Levene et al., Zero-Mode Waveguides for Single-Molecule Analysis at High Concentrations, 299 Science 682—686 (2003) (“Levene”). 4 Appeal 2017-001578 Application 11/982,465 molecule enzymatic activity. Id. at 683, 685. The supporting materials for Levene disclose that “the enzyme bound nonspecifically to . . . fused silica” and that “[a]fter immobilization, unbound enzyme was washed away by extensive flushing with polymerization buffer.” Levene, Supplementary Materials, http://science.sciencemag.org/content/sci/suppl/2003/01/29/299. 5607.682. DC 1/Levene.SOM.pdf, (2003). The issue presented in this appeal is whether the preponderance of the evidence supports the Examiner’s determination that the density of the polymerase adsorbed at the bottom of the Levene’s waveguide is “100 times or more greater than the density” of any polymerase immobilized on substrate surfaces other than those at the bottom of the waveguides. In finding that Levene anticipated the pending claims, the Examiner concluded that polymerase bound to the silica substrate at the bottom of the Levene’s ZMWs but did not bind to (or was washed from) the aluminum that surrounds and forms the walls of Levene’s ZMWs, thus meeting the enzyme density limitation recited in claim 41. Ans. 5—6. As support, the Examiner points to the disclosure in Levene’s supporting materials that polymerase bound to the silica and “unbound enzyme was washed away.” Id. The Examiner points out that this interpretation is consistent with figure 1 of Levene (reproduced above), which “depict[s] a single enzyme molecule bound to the glass bottom of the ZMW with none shown elsewhere” and with the disclosure that Levene’s ZMWs detect the activity of single molecules. Id. at 6; see also, final Act.3 5 (“if the washing procedure did not successfully [remove] all excess Sequenase, then it would 3 Office Action mailed September 24, 2015 (“final Act.”). 5 Appeal 2017-001578 Application 11/982,465 not be possible to observe single molecule kinetics such as disclosed in the article and the depiction in figure 1 would be inaccurate”). Appellants argue that polymerase was present not just on the silica bottoms of Levene’s ZMWs, but also on the aluminum surfaces that surround the ZMWs. App. Br. 9. Appellants rely on Korlach4 as support. According to Appellants, Korlach discloses that polymerase binds to untreated aluminum, even after it is washed to remove unbound polymerase. Id. at 9—10. As further support Appellants rely on the testimony of Dr. Korlach that “[t]he washing conditions [described in Levene] would . . . not disrupt nonspecific adsorption of the Sequenase enzyme elsewhere on the ZMW array.” Korlach Decl.5 3. Appellants argue that binding of polymerase to the aluminum surfaces surrounding Levine’s ZMW’s is not inconsistent with Levine’s teaching that its ZMWs can be used to detect the activity of single molecule kinetics because Levene speaks only to the presence or absence of enzyme in the observation volume of the ZMW. Appellants explain: Because illumination is effectively limited to a tiny observation volume at the bottom of the ZMW and fluorescence is effectively observed only from this observation volume, activity of a single polymerase molecule can be monitored as it incorporates fluorescently labeled nucleotides one at a time, 4 Korlach et al., Selective Aluminum Passivation for Targeted Immobilization of Single DNA Polymerase Molecules in Zero-Mode Waveguide Nanostructures, 105 Proc. Natl. Acad. Sci. 1176—81 (2008) (“Korlach”). Korlach was cited by Appellants as evidence that “various polymerase enzymes exhibit extensive nonspecific binding to metals, even after washing of the metal.” App. Br. 10. 5 Declaration of Dr. Jonas Korlach under 37 C.F.R. § 1.132, signed June 1, 2015 (“Korlach Decl.”). 6 Appeal 2017-001578 Application 11/982,465 despite the high concentration of fluorescently labeled nucleotides present in solution. App. Br. 7. Nor, Appellants assert, is binding enzyme to aluminum inconsistent with Figure 1 of Levene because Figure 1 is just “a schematic, intended to illustrate the principle of operation of a ZMW.” Reply Br. 2. As stated in In re Oetiker, 977 F.2d 1443, 1445 (Fed. Cir. 1992): “[T]he examiner bears the initial burden ... of presenting a prima facie case of unpatentability.” Appellants have persuaded us that the Examiner has not carried the burden of establishing that the claimed invention was anticipated by Levene. Levene discloses that Sequenase, a polymerase, binds to silica “nonspecifically.” Levene, Supplementary Materials, http://science. sciencemag.org/content/sci/suppl/2003/01/29/299.5607.682.DCl/Levene. SO M.pdf, (2003). Korlach establishes that, under circumstances like those described in Levene, phi29 polymerase may remain bound to aluminum, even after the aluminum is washed with a buffer. Korlach 1178—1179. In the absence of evidence to the contrary, these teachings suggest that another species of polymerase — the Sequenase disclosed in Levene — will also bind to aluminum. While the Examiner discounts the evidence provided by Korlach on the ground that Korlach used a different polymerase enzyme and a different washing buffer, it is the Examiner’s burden to establish a prima facie case of unpatentability. Under the Examiner’s anticipation rationale, this requires establishing that there was no polymerase bound to the aluminum component of Levene’s substrate. Based on the record before us, the Examiner has not established this. 7 Appeal 2017-001578 Application 11/982,465 Figure 1 of Levene, relied upon by the Examiner as not showing enzyme bound to aluminum, does little to support the Examiner’s anticipation rationale because, as Appellants point out, Figure 1 is only a schematic and does not speak to the presence of enzyme outside of the single ZMW depicted. Levene’s teaching that unbound enzyme is washed away is similarly not persuasive when considered together with Korlach’s example of a polymerase binding to aluminum. Finally, the Examiner’s assertion that it would not be possible to observe single molecule kinetics such as disclosed in the article is not supported by evidence of inoperability. While the Specification does disclose that localizing reactants within ZMWs is preferred and advantageous (see Spec. 135) the Examiner does not identify, and we do not find, evidence sufficient to establish that Levene’s ZMW’s would only be operable if the reactants were localized in the ZMW. Accordingly, we reverse the Examiner’s rejection of claims 41, 42, 57-59, 61, and 6A-66. SUMMARY For the reasons set forth herein we reverse the Examiner’s rejection of claims 41, 42, 57—59, 61, and 64—66 under 35 U.S.C. § 102(b) as anticipated by Levene. REVERSED 8 Copy with citationCopy as parenthetical citation