Joris Vermeesch et al.Download PDFPatent Trials and Appeals BoardMar 20, 202014430293 - (D) (P.T.A.B. Mar. 20, 2020) 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. 14/430,293 03/23/2015 Joris Vermeesch 20150088-04 1020 22878 7590 03/20/2020 Agilent Technologies, Inc. Global IP Operations 5301 Stevens Creek Blvd Santa Clara, CA 95051 EXAMINER KIM, YOUNG J ART UNIT PAPER NUMBER 1637 NOTIFICATION DATE DELIVERY MODE 03/20/2020 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): Agilentdocketing@cpaglobal.com ipopsadmin@agilent.foundationip.com PTOL-90A (Rev. 04/07) UNITED STATES PATENT AND TRADEMARK OFFICE BEFORE THE PATENT TRIAL AND APPEAL BOARD Ex parte JORIS VERMEESCH, THIERRY VOET, FEMKE HANNES, JEROEN VAN HOUDT, and GREGORY MAES Appeal 2018-006184 Application 14/430,293 Technology Center 1600 Before DONALD E. ADAMS, FRANCISCO C. PRATS, and JAMIE T. WISZ, Administrative Patent Judges. WISZ, Administrative Patent Judge. DECISION ON APPEAL Appeal 2018-006184 Application 14/430,293 2 STATEMENT OF THE CASE Pursuant to 35 U.S.C. § 134(a), Appellant1 appeals from the Examiner’s decision to reject claims 1 and 3–14. We have jurisdiction under 35 U.S.C. § 6(b). We AFFIRM. CLAIMED SUBJECT MATTER The Specification describes “a method and system providing a rapid discovery, validation and assessment of genetic variations or chromosomal disorders throughout the whole genome . . . in samples containing low amounts of target nucleic acids, such as relatively small analytes, such as few or single cells or free-flowing tumor or fetal nucleic acids.” Spec. 1. Claim 1, the only independent claim, is illustrative of the claimed subject matter and is reproduced below: 1. A method for analysis of target nucleic adds, the method comprising: (i) providing a sample comprising up to 200 pg of nucleic acids, (ii) generating a reduced representation library of said nucleic acids by a method comprising: fragmenting said nucleic acids; ligating adaptors to said fragments; and selecting a subset of said adaptor-ligated fragments, (iii) sequencing said reduced representation library, and (iv) identifying variants in target nucleic adds in the sample by analyzing results obtained by said sequencing. 1 We use the word “Appellant” to refer to “applicant” as defined in 37 C.F.R. § 1.42. Appellant identifies the real party in interest as Agilent Technologies, Inc. Appeal Br. 1. Appeal 2018-006184 Application 14/430,293 3 Appeal Br. 9 (Claims App.). REJECTIONS The Examiner rejected claims 1 and 3–14 under 35 U.S.C. § 103 as obvious over Elshire,2 in view of Spits.3 ISSUES AND ANALYSIS The Examiner finds that Elshire discloses a method for analysis of target nucleic acids comprising a) providing a sample wherein target nucleic acids are present in low amounts; b) generating a reduced representation library by i) fragmenting target nucleic acids, ii) ligating adapters to said fragments, and iii) selecting a subset of adaptor-ligated fragments; c) sequencing said reduced representation library; and d) identifying variants in the target nucleic acids. Final Act. 4–6 (citing Elshire 2, 4, 8, Figure 2). With respect to the limitation of “selecting a subset of adaptor-ligated fragments,” the Examiner finds that this limitation is disclosed in Elshire and its incorporation of Bentley. 4 Ans. 11–14. According to the Examiner “the selection of a subset of the adaptor ligated DNA fragments [occurs] by virtue of the step wherein the amplified adaptor ligated DNA fragments are applied to the flow cell of the DNA sequencer as discussed in Elshire.” Ans. 11–12 (citing Elshire 4). Specifically, the Examiner finds that, in Elshire, in 2 Robert Elshire et al., A Robust, Simple Genotyping-by-Sequencing (GBS) Approach for High Diversity Species, PLos One, Vol. 6(5), 1–10 (2011) (“Elshire”). 3 C. Spits et al., Optimization and Evaluation of Single-Cell Whole-Genome Multiple Displacement Amplification, Human Mutation, 27(5), 496–503 (2006) (“Spits”). 4 Bentley et al., Accurate whole human genome sequencing using reversible terminator chemistry,” Nature, Vol. 456, 53–59 (2008). Appeal 2018-006184 Application 14/430,293 4 each of the wells of the 96-well plate, a plurality of DNA inserts are flanked by two adaptor sequences. Id. at 12. According to the Examiner, “[t]hese adapter pair ligated DNA inserts are then pooled and amplified using the same primer pair for all of the adapter pair ligated DNA inserts from all of the samples in the pool.” Id. As found by the Examiner, the resulting amplification products are loaded to a sequencing reaction platform, the Genome Analyzer of Illumina as described in Bentley as shown below (modified by the Examiner to include the diagonal arrow). Portion of Figure 1 of Bentley showing the formation of a clonal single- molecule array. Id. at 13. According to the Examiner, “[a]s it can be seen, the pair of adapter ligated DNA insert is then hybridized to the immobilized primer on a solid surface of the flow cell (‘DNA fragments are generated ... joined to a pair of oligonucleotides ... ligated products are amplified using two oligonucleotide primers, resulting in double-stranded blunt-ended material ... DNA fragments prepared in a are denatured and single strands are annealed to complementary oligonucleotides on the flowcell surface.’[)]” Id. (citing Bentley 12). Appeal 2018-006184 Application 14/430,293 5 The Examiner concludes: In other words, the all of the amplified products produced in the pooled amplification process of Elshire et al., when being sequenced, are loaded to a flow cell containing a limiting amount of primers which are immobilized on the surface. Such process results in a portion of the amplified, adapter pair-ligated DNA inserts not being annealed to the limiting amount of immobilized primers and thus become washed out. What remains therefore, on the sequencing platform, will be a subset of the adaptor-pair ligated DNA inserts, the subset of which are then sequenced in the sequencing reaction. Id. Appellant disagrees that the claim term “selecting a subset of said adaptor-ligated fragments” encompasses annealing a portion of Elshire’s adaptor pair-ligated inserts to immobilized primers on the flow cell of a sequencing instrument and washing out the inserts which are not annealed. Reply 3. According to Appellant, such a construction ignores that the “selecting” step in claim 1 is part of the generation of a reduced representation library of nucleic acids. Id. Appellant argues that “[i]f the selecting step merely requires loading all of the adaptor-ligated fragments to a flow cell, then a reduced representation library would not be generated prior to sequencing.” Id. at 3–4. Appellant further argues that the Examiner’s interpretation is inconsistent with the Specification which indicates that a reduced representation library would be loaded on a flow cell, not all fragments from a sample because “[t]he advantage of a reduced representation library is not present if all fragments from a sample are loaded into a sequencing instrument.” Id. at 4. Appeal 2018-006184 Application 14/430,293 6 According to Appellant, the Examiner’s Answer improperly cites new findings in stating that, in Elshire, a portion of the amplified, adapter pair- ligated DNA inserts not being annealed to the limiting amount of immobilized primers become washed out. Id. (citing Ans. 13). Appellant asserts that the Examiner cites no evidence for these findings. Id. Having considered Appellant’s arguments in support of claim 1, we are not persuaded of any reversible error in the Examiner’s rejection of claim 1. We agree with the Examiner that Elshire discloses selecting a subset of adaptor-ligated fragments through its reference to Bentley. This selection occurs by annealing the amplification products of Elshire to complementary oligonucleotide primers on the flow-cell surface as described in Bentley. This selection of fragments is similar to what is described in the Specification in which amplification using a selective primer only amplifies a subset of adaptor-ligated fragments, namely those to which the selective primer hybridizes with sufficient stringency to allow its elongation. Spec. 11:17–32. Furthermore, although Appellant argues that “[i]f the selecting step merely requires loading all of the adaptor-ligated fragments to a flow cell, then a reduced representation library would not be generated prior to sequencing,” the selecting/annealing step described in Elshire does occur before sequencing. Ans. 3–4. With regard to Appellant’s argument that the Specification “indicates that a reduced representation library would be loaded on a flow cell, not all fragments from a sample,” these limitations are not recited in the claims. Id. at 4. Appellant may not import limitations from its Specification into the claims. See CollegeNet, Inc. v. ApplyYourself, Inc., 418 F.3d 1225, 1231 (Fed. Cir. 2005) (“In examining the specification Appeal 2018-006184 Application 14/430,293 7 for proper context” it is improper to “import limitations from the specification into the claims.”). With regard to the limitation of providing a sample comprising up to 200 pg of nucleic acids, the Examiner concedes that Elshire does not disclose this limitation but finds that this deficiency is cured by Spits. Final Act. 7. Specifically, the Examiner finds that Spits recognizes situations wherein the amount of available genomic DNA is scarce and states: In some fields of current genetic research such as forensic analysis, prenatal or preimplantation diagnosis, or oncogenetics, DNA analysis has to be performed on small pools of cells or even single cells. The limited amount of genomic DNA (gDNA) is a restrictive factor in these tests. To overcome this difficulty, several methods of whole-genome amplification (WGA) have been developed, providing sufficient template for numerous genetic analysis. Id. at 7–8 (citing Spits 496) (emphasis added). The Examiner also finds that a single cell would necessarily comprise less than 200 pg of DNA. Ans. 15. According to the Examiner, it would have been prima facie obvious to one of ordinary skill in the art at the time of the invention to combine the teachings of Elshire and Spits. Final Act. 8. Specifically, the Examiner finds that While the samples employed by Elshire et al. may have contained more than 200 pgs of nucleic acids therein, given the motivation provided by Spits et al. which evidences the art’s desire to analyze from samples which contain less than 200 pg of nucleic acids therein (sample such as a single cell), one of ordinary skill in the art would have been motivated to combine the teachings of Elshire et al. with the teachings of Spits et al., so as to obtain samples which may contain less than 200 pgs of nucleic acids, and amplify the nucleic acids therein via WGA [to] provide enough starting amounts of genomic DNAs so as to perform the sequencing reaction of Elshire et al. Appeal 2018-006184 Application 14/430,293 8 Id. According to the Examiner, there would have been a reasonable expectation of success in combining the teachings of Elshire and Spits “given that WGA produced from [Spit’s] method would have yielded a plurality of nucleic acid molecules from which to generate [a] reduced representation library so as to perform the sequencing reaction as provided for by [Elshire]. Id. at 9. Appellant asserts that the cited references do not disclose a successful method for analysis of target nucleic acids using a sample comprising up to 200 pg of nucleic acids. Appeal Br. 6. According to Appellant, the Examiner’s citation to Spits does not mean that Elshire’s method can be employed with such low amounts of nucleic acid but, rather, reinforces Appellant’s contention that it is difficult to work with such low amounts of DNA. Id. at 7 (citing Spits 496). Appellant argues that the Examiner has not established a reasonable expectation of success in modifying Elshire according to Spits. The Examiner responds that Spits, which published in 2006, discloses a known amplification reaction called WGA (“whole genome amplification reaction”) and, therefore, WGA was well known in the art to successfully amplify from a single cell having such low amounts of starting DNA. Ans. 16. We are not persuaded by Appellant’s argument. As stated by the Examiner, the WGA amplification reaction disclosed in Spits was well- known by the application’s priority date. Furthermore, the Specification discloses use of this process, which is also recited in claim 14. See, e.g., 10:1–8. One of ordinary skill in the art would have been motivated to Appeal 2018-006184 Application 14/430,293 9 analyze small samples of nucleic acids (such as a single cell, which is less than 200 pg), based on the disclosure in Spits. One of skill in the art would also have had a reasonable expectation of success in being able to analyze such a small sample of nucleic acids given the well-known amplification procedure disclosed in Spits. With respect to claims 5 and 6, which require that the selection of a subset is performed using PCR-amplification, the Examiner finds that Elshire discloses that PCR is performed with primers which are complementary to the adaptor portions found on the adaptor-DNA-adaptor construct. Final Act. 6 (citing Elshire Figures 1, 2). As further explained by the Examiner the “selection process” as taught by Elshire is construed to include amplification of the pooled, adapter-ligated DNA inserts with primers which are then annealed to the sequencing platform, that is to say, the selection of a subset comprised both the amplification and the sequencing step as explicitly discussed by Elshire. Ans. 16. Appellant asserts that, while Elshire discloses PCR amplification, it “teaches that PCR amplification is used to increase the fragment pool” and does not disclose or suggest using PCR amplification to select a subset of adaptor-ligated fragments.” Reply 6 (citing Elshire 3, last paragraph). According to Appellant, “Elshire uses PCR primers to bind to all of the adaptor-ligated fragments.” Id. We are not persuaded by Appellant’s arguments. As discussed above, Elshire, along with its incorporation of Bentley, discloses that a subset of the adaptor-ligated fragments is selected by annealing the amplification products Appeal 2018-006184 Application 14/430,293 10 of Elshire to complementary oligonucleotide primers on the flow-cell surface as described in Bentley. For the reasons described herein and those already of record, we affirm the Examiner’s rejection of independent claim 1 under 35 U.S.C. § 103 as being obvious over Elshire in view of Spits. For the reasons described above, we also affirm the Examiner’s § 103 rejection of claims 5 and 6. Claims 3–4 and 7–14 are not argued separately, and, therefore, fall with claim 1. See 37 C.F.R. § 41.37(c)(1)(iv). CONCLUSION For the reasons described herein and those already of record, we affirm the Examiner’s rejection of claims 1, and 3–14. DECISION SUMMARY In summary: Claims Rejected 35 U.S.C. § Reference(s)/Ba sis Affirmed Reversed 1, 3–14 103 Elshire, Spits 1, 3–14 TIME PERIOD FOR RESPONSE No time period for taking any subsequent action in connection with this appeal may be extended under 37 C.F.R. § 1.136(a). See 37 C.F.R. § 1.136(a)(1)(iv). AFFIRMED Copy with citationCopy as parenthetical citation