DETAILED ACTION
Notice of Pre-AIA or AIA Status
1. The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Claim Rejections - 35 USC § 103
2. The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
3. This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
4. Claims 1-5 and 7-14 are rejected under 35 U.S.C. 103 as being unpatentable over Yeh (US Patent Application No. US 20190024127 A1, published 01-24-2019) in view of Raymond et al (US Patent Application No. US 20180245072 A1, published 08-30-2018) and Raghunath et al (United States Patent No. US8568982, issued 29 October 2013).
Regarding claim 1, Yeh teaches a method of amplifying cfDNA from a biological sample (i.e., a finger prick [0009]) wherein the biological sample is 25 µL ([0042]). Yeh teaches that the cfDNA is modified (i.e., tagged) by adding an exogenous nucleic acid sequence to both the 5' and 3' ends of the cfDNA ([0031]). Yeh also teaches that the cfDNAs are end repaired to produce blunt ends using a suitable polymerase (i.e., in a first reaction), and that the polymerase has exonuclease activity (i.e., one or more exonucleases [0163]). Yeh teaches that exogenous nucleic acid sequences (i.e., adapters) are added to the 3' and 5' ends of the cfDNA using a ligase (i.e., in a second reaction; [0163] and [0168]) with a reaction buffer comprising DTT (i.e., a small molecule enhancer [0268]), and that the tagged cfDNA is analyzed by next generation sequencing (NGS [0084]).
It is noted that Yeh teaches the contacting of the cfDNA with a polymerase and a ligase as described above, and each of these enzymes performs a distinct reaction on the cfDNA. There is no language in amended claim 1 that requires that the first and second reaction do not occur at the same time.
Yeh teaches that BSA, a molecular crowding reagent known in the art, is added to the reaction buffers taught in this disclosure ([0268] and [0288]). Yeh does not teach that BSA is present at a concentration sufficient to enhance the first reaction or the second reaction, nor does Yeh teach that the blunt ends of the cfDNA are dephosphorylated.
However, Raymond teaches that cfDNA fragments are dephosphorylated as part of the end repair process ([0076] and [0255]). These dephosphorylated DNAs are inherently ligation-competent when ligated to an adapter comprising a 5' phosphate as taught by Raymond ([0076]).
It would have been obvious to one having ordinary skill in the art to have modified the end polishing method taught by Yeh with the dephosphorylation step taught by Raymond, and one having ordinary skill in the art would have been motivated to make this modification in order to reduce the likelihood of self-ligation between ends of the blunted cfDNA molecules. In addition, it would have been obvious to the ordinary artisan that the known techniques in the cited references could have been combined with predictable results because the known techniques of the cited references result in techniques useful for the processing of cfDNA samples for library generation.
Additionally, Raghunath teaches that DNA polymerases and ligases benefit from the use of macromolecular crowding agents (columns 9 – 12) at concentrations sufficient to enhance these reactions (columns 28-29).
It would have been obvious to one having ordinary skill in the art to have modified the first and second reactions taught by Yeh and Raymond to include a macromolecular crowding agent as taught by Raghunath to arrive at the instantly claimed invention with a reasonable expectation of success. The ordinary artisan would have been motivated to make this modification because Raghunath specifically teaches that macromolecular crowding enhances enzyme processivity (column 8, ¶ 3, column 9 ¶ 7, and column 10 ¶ 2). In addition, the ordinary artisan would have recognized that the known techniques in the cited references could have been combined with predictable results because the known techniques in the cited references predictably result in the polymerization and ligation of nucleic acids.
Regarding claims 2-5, Yeh teaches a sample volume of 25 µL is obtained by finger stick (i.e., a capillary blood sample [0042]).
Regarding claim 7, Yeh teaches the determining of characteristics of cfDNA molecules including copy number variations (i.e., the representation of a sequences in a cfDNA sample [0085]).
Regarding claims 8 and 9, Raymond teaches the detection of genetic variants in fetal cfDNA ([0056]). If fetal cfDNA is being detected from a subject, it is inherent that the subject is pregnant with a fetus.
Regarding claim 10, Yeh teaches that cfDNA isolated from a subject has an original concentration of .0083 ng/µL (8.3 pg/µL; [0410] and associated table). A complete human genome weighs approximately 6 pg, and a patient sample of 25 µL at a concentration of 8.3 pg/µL contains ~207.5 pg of cfDNA. This represents approximately 34.5 genome equivalents of cfDNA (i.e., about 10 genome equivalents).
Regarding claim 11, Raymond teaches illustrative examples of alleles suitable for paternity testing that are detected using cfDNA ([0226] and [0236]).
Regarding claim 12, Yeh teaches that a pool of cell free nucleic acids is amplified for analysis ([0026]) and that analysis of cell free nucleic acids is performed by NGS ([0084]).
Regarding claim 13, Yeh teaches that the characteristics of cfDNA are related to tumor evolution ([0245]), a pathogen ([0228]), or transplantation rejection ([0251]).
Regarding claim 14, Yeh teaches that cfDNA characteristics included the determination of the presence of an agent such as a virus, bacteria or fungi ([0028]).
5. Claim 6 is rejected under 35 U.S.C. 103 as being unpatentable over Yeh (US Patent Application No. US 20190024127 A1, published 01-24-2019) in view of Raymond et al (US Patent Application No. US 20180245072 A1, published 08-30-2018) and Raghunath et al (United States Patent No. US8568982, issued 29 October 2013) as applied to claim 1 above, and further in view of Dhingra et al (WHO Guidelines on Drawing Blood: Best Practices in Phlebotomy. Geneve: World Health Organization, 7, 2010).
Regarding claim 6, Yeh teaches a drawing a biological sample from a finger stick (i.e., a first transdermal puncture to produce a first fraction of a biological sample [0042]).
Yeh does not teach the steps of discarding the first fraction of the biological sample and collecting a second fraction of the biological sample thereby reducing or eliminating contamination due to white blood cell lysis.
However, Dhingra teaches a method of capillary sampling where the first drop of blood is wiped away because it may be contaminated with tissue debris (i.e., cellular debris, section 7.2.2). It would have been obvious to one having ordinary skill in the art to have modified the finger stick technique taught by Yeh with the collection technique taught by Dhingra to arrive at the instantly claimed invention with a reasonable expectation of success. One having ordinary skill in the art would have been motivated to make this combination in order to reduce potential contamination from cellular debris as taught by Dhingra (Section 7.2.2). In addition, it would have been obvious to the ordinary artisan that the known techniques in the cited references could have been combined with predictable results because the known techniques in the cited references predictably result in the drawing of a blood sample from a capillary finger stick.
6. Claims 17 and 18 are rejected under 35 U.S.C. 103 as being unpatentable over Yeh (US Patent Application No. US 20190024127 A1, published 01-24-2019) in view of Raymond et al (US Patent Application No. US 20180245072 A1, published 08-30-2018) and Raghunath et al (United States Patent No. US8568982, issued 29 October 2013) as applied to claim 12 above, and further in view of Babiarz et al (US Patent Application No. 20150147815 A1, published 05-28-2015).
Regarding claims 17 and 18, the method of claim 12 is discussed fully above and incorporated here.
Yeh does not teach that the cfDNA sample is amplified by a massively multiplexed amplification assay.
However, Babiarz teach that cfDNA samples are collected and amplified by massively multiplex PCR (mmPCR [0066]). It would have been obvious to one of ordinary skill in the art to have substituted the amplification method taught by Yeh with the mmPCR method taught by Babiarz to arrive at the instantly claimed method with a reasonable expectation of success, in order to increase the efficiency of sample preparation by multiplexing the amplification step. In addition, it would have been obvious to the ordinary artisan that the known techniques in the cited references could have been combined with predictable results because the known techniques in the cited references predictably result in techniques useful for the amplification of cfDNAs.
7. Claim 20 is rejected under 35 U.S.C. 103 as being unpatentable over Yeh (US Patent Application No. US 20190024127 A1, published 01-24-2019) in view of Raymond et al (US Patent Application No. US 20180245072 A1, published 08-30-2018) and Raghunath et al (United States Patent No. US8568982, issued 29 October 2013) as applied to claim 1 above, and further in view of Osborne (US Patent Application No. US 20180245148 A1, published 08-30-2018).
Regarding claim 20, Yeh teaches that multiple analyzable pools (i.e., two or more) of cfDNA from the same sample can be combined prior to sampling ([0220]).
Neither Yeh, Raymond, no Raghunath teach pooling two or more biological samples obtained from different subjects.
However, Osborne teaches that nucleotides are modified with identifier sequences by ligation, and that nucleotides from different individuals (i.e., different subjects) are tagged with different identifier sequences and pooled together for sequencing analysis ([0060]). Osborne teaches that these nucleotides are cfDNA ([0097]). It would have been obvious to one having ordinary skill in the art to have substituted the pooled cfDNAs taught by Yeh with the pooled and tagged cfDNAs taught by Osborne to arrive at the instantly claimed invention with a reasonable expectation of success. An ordinary artisan would have recognized the advantages of pooling samples from different individuals prior to sequencing, as this has the known benefit of generating more data from a single sequencing run while only using a single batch of sequencing reagents. Additionally, one having ordinary skill in the art would have recognized that these two techniques could be substituted with predictable results because both techniques predictably result in the pooling of cfDNA samples.
8. Claim 21 is rejected under 35 U.S.C. 103 as being unpatentable over Yeh (US Patent Application No. US 20190024127 A1, published 01-24-2019) in view of Raymond et al (US Patent Application No. US 20180245072 A1, published 08-30-2018) and Raghunath et al (United States Patent No. US8568982, issued 29 October 2013) as applied to claim 1 above, and further in view of Grolz (US Patent Application No. US 20160374330 A1, published 12-29-2016).
Regarding claim 21, the method of claim 1 is discussed fully above and incorporated here.
Neither Yeh, Raymond, nor Raghunath teach contact a biological sample with a white blood cell stabilizer.
However, Grolz teaches a method for reducing lysis of white blood cells (i.e., stabilization) in a blood sample by contacting said sample with a stabilizer ([0101]). It would have been obvious to one having ordinary skill in the art that the method taught by Yeh and Raymond could have been modified with the stabilization step taught by Grolz in order to arrive at the instantly claimed invention with a reasonable expectation of success. One having ordinary skill in the art would have been motivated to make this modification in order to reduce DNA contamination from nucleated cell in the blood sample as taught by Grolz ([0101]). In addition, it would have been obvious to the ordinary artisan that the known techniques in the cited references could have been combined with predictable results because the known techniques of the cited references predictably result in techniques for the collection and processing of blood samples.
Response to Arguments
9. Any issue not repeated in this Office Action was overcome by amendment to the claims.
Applicant’s arguments, see pg. 6 ¶ 6 of applicant’s remarks, filed 02 July 2025, with respect to the rejections of claims 1-5 and 7-14 under U.S.C. § 103 have been fully considered and are persuasive. Therefore, the rejection has been withdrawn. However, upon further consideration, a new grounds of rejection is made in view of Yeh, Raymond, and prior art reference Raghunath et al (United States Patent No. US8568982, issued 29 October 2013). As discussed fully above and incorporated here, Raghunath teaches that molecular crowding agents are used to enhance the processivity of enzymes including polymerases and ligases.
10. Applicant's amendment necessitated the new ground(s) of rejection presented in this Office action. Accordingly, THIS ACTION IS MADE FINAL. See MPEP § 706.07(a). Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any nonprovisional extension fee (37 CFR 1.17(a)) pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
Conclusion
11. No claims are allowed.
12. Any inquiry concerning this communication or earlier communications from the examiner should be directed to BRIAN ELLIS YOUNG whose telephone number is (703)756-5397. The examiner can normally be reached M-F 0730 - 1700.
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/BRIAN ELLIS YOUNG/Examiner, Art Unit 1684
/JULIET C SWITZER/Primary Examiner, Art Unit 1682
