DETAILED ACTION
Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Effective Filing Date
The present application, filed on March 11, 2022 is a continuation of 15/513,472, filed on March 22, 2017, which is a 371 of PCT/US2015/052729, filed on September 28, 2015, which claims the benefit of 62/056,378, filed on September 26, 2014.
Election/Restrictions
Applicant’s election without traverse of Group I, claims 1-30, drawn to methods of detecting polynucleotides in the reply filed on March 4, 2025 is acknowledged.
Claim Status and Action Summary
Claims 1-5, 9-30, and 40-43 are pending in the present application. Claims 6-8 and 31-39 were canceled by applicant and claims 40-43 were newly added in the amendment filed on September 22, 2025.
Claims 1-5, 9-30, and 40-43 are under examination.
Any objections and rejections not reiterated below are hereby withdrawn.
The rejections of record under 35 U.S.C. 102(a)(1) have been withdrawn in view of the amendments to the claims.
Claim Rejections - 35 USC § 112
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
Claims 9 and 43 are rejected under 35 U.S.C. 112(b) as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor regards as the invention.
This is a new rejection necessitated by the amendments to the claims.
Claim 9 recites “The method of claim 8, wherein…”, however, claim 8 was canceled. Claim 43 depends upon claim 9. It is unclear which claim the present claim 9 is intended to reference.
For the purposes of compact prosecution, the examiner has interpreted claim 9 as being dependent from claim 1, upon which the canceled claim 8 depended in previous versions of the claims.
Claim Rejections - 35 USC § 103
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.
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.
Claims 1-3, 9 and 14-18 are rejected under 35 U.S.C. 103 as being unpatentable over Meller et al., US 2012/0276530A1 (published November 11, 2012) in view of Korlach et al., US 2013/0109577 A1 (Published May 2, 2013).
This rejection has been updated as necessitated by the amendments to the claims.
Regarding claim 1, Meller et al. teach methods of detecting polynucleotides comprising multiple target sequences wherein targets bound by bis-PNA probes are detected as they pass through a nanopore device (Meller et al., paragraph 0011) from a first chamber, through a nanopore through which the first and second chambers are in fluidic and electrical communication, and into a second chamber (Meller et al., figure 1). Meller et al. teach the probe comprises a peptide nucleic acid (Meller et al., paragraph 0011). Meller et al. teach that the probe comprises a PNA bound to a secondary molecule configured to facilitate detection of the complex (Meller et al., paragraphs 0058-0060). Meller et al. teach that the secondary molecule comprises polyethylene glycol (PEG) (Meller et al., paragraph 0059).
Meller et al. further teaches said PNA probes may be bound to a polyethylene glycol (PEG) (i.e. a secondary molecule) configured to facilitate detection of the complex by enhancing the bulk of the dsDNA-PNA complex… to enhance the signal amplitude… and ease of detection (Meller et al., paragraphs 0058-0060).
Meller does not teach the molecular weight of said bulk- and signal- enhancing PEG secondary molecule.
However, Korlach et al. teach a method of detecting polynucleotides using DNA probes coupled to detectable tags (i.e. a secondary molecule) comprising a polyethylene glycol (PEG) linker (Korlach et al., paragraph 0141). Korlach et al. further teach PEG linkers that comprise 77 ethylene glycol monomers (Korlach et al., paragraph 0184). PEG (CH2CH2O)n has an approximate molecular weight of 44n Da, where n=the number of monomers in the polymer. Therefore, a PEG linker comprising 77 monomers has a molecular weight ≈ 3.388 kDa (i.e. at least 1 kDa…).
The teachings of Meller et al. and Korlach et al. are in the same very narrow field of scientific inquiry, namely detection of nucleic acids labeled with tags comprising polyethylene glycol coupled to a nucleic acid configured to facilitate detection of the nucleic acid.
Therefore, it would have been prima facie obvious prior to the effective filing date of the claimed invention for one of ordinary skill in the art to modify the method of nanopore detection of polynucleotides containing specific target sequences through binding of sequence-specific PNA-probes associated to bulky-second molecules comprising PEG, taught by Meller et al. by substituting the bulk-and signal- enhancing PEG secondary molecule with the 77-mer PEG detectable labels taught by Korlach et al. The ordinary artisan would have been motivated to substitute the 77-mer PEG labels into the method taught by Meller et al. because Korlach et al. and Meller et al. teach bulky PEG linkers are useful for increasing nanopore detection of nucleic acids. The ordinary artisan would have been reasonably confident that the 77-mer PEG molecules taught by Korlach et al. would have produced predictable bulk- and signal- enhancement in the method taught by Meller et al. because the teachings of Korlach et al. predictably result in useful linkers for nanopore sequencing of tagged nucleic acid molecules.
Regarding claim 2, Meller et al. teach the polynucleotide is DNA or RNA (Meller et al., paragraph 0021)
Regarding claim 3, Meller et al. teach electrical detection of the polynucleotide (Meller et al., paragraph 0011).
Regarding claim 9, Korlach et al. teach Korlach et al. teach a detectable nucleic acid tag comprising PEG having a molecular weight greater than 3 kDa.
Regarding claim 14, Korlach et al. teach detectably labeled nucleic acids (i.e. probes) comprising a 77-mer PEG molecule coupled to a nucleic acid that may be cleaved. (Korlach et al., paragraph 0039).
Regarding claim 15, Meller et al. teach that the probe interacts with the target sequence by forming Watson-Crick and/or Hoogsteen base-pairs (i.e. a hydrogen bond) (Meller et al., paragraph 0021 and paragraph 0058).
Regarding claim 16, Meller et al. teach small particles, molecules, proteins, or peptides can be conjugated to the pseudopeptide backbone to enhance the bulk of the dsDNA-PNA complex… to enhance the signal amplitude… and ease of detection (Meller et al., paragraph 0059) (i.e. detectable labels capable of binding to the probe or to the complex).
Regarding claim 17, Meller et al. teach polynucleotides comprising at least two target sequences (Meller et al., Figure 1).
Regarding claim 18, Meller et al. teach the nanopore is between 3-10 nm in diameter (Meller et al., paragraph 0085), is fabricated in a thin (about 20 nm) membrane (i.e. the nanopore is less than 20 nm in length, and that each of the chambers comprises an electrode (Meller et al., paragraph 0178).
Response to arguments
The response argues that, as amended, the requirement that the PEG label has a molecular weight of at least 1 kDa is distinct from the art because Meller et al. discloses PEG among a list of bulk- and signal- enhancing PNA modifications that enhance nanopore signal detection without disclosing the molecular weight of the PEG molecule required to accomplish said signal enhancement. The response asserts that the ordinary artisan would not know what size PEG molecule would be sufficient to accomplish the differential detection or signal enhancement taught by Meller et al. The response further asserts that the PEG labels taught by Korlach et al. (comprising PEG labels greater than 3kDa) are used for an “entirely different purpose” because Korlach et al. does not detect nucleic acids with a nanopore.
This argument has been thoroughly reviewed but is not persuasive because, as described in the 103 rejection above, the PEG-labels taught by Korlach et al. are clearly used in the same field of research as the PEG-labels on the probes taught by Meller et al. As to the assertion that the ordinary artisan would not know what size PEG labels would accomplish differential detection, it is well known in the art that mass tags (comprising PEG) added to nucleotides in nanopore sequencing allow for discrimination between nucleic acid species AND allow for enhanced detection relative to unlabeled nucleic acid probes based on the known, predictable properties of increased bulk and dwell time within a nanopore. For example, Kumar et al., “PEG-Labeled Nucleotides and Nanopore Detection for Single Molecule DNA Sequencing by Synthesis” Scientific Reports 2:684, published September 21, 2012 (cited here as an evidentiary reference for the purposes of argument) demonstrate that as early as 2012, incorporation of individual nucleotides comprising cleavable mass tags comprising PEG molecules differing in mass for each of the four nucleotides were readily distinguishable by a nanopore (Kumar et al., figures 3 and 7, reproduced below for applicant’s convenience). Furthermore, Kumar et al., as early as 2012, describe that further increasing the molecular weight of the tag would have been expected to “further enhance nanopore discrimination” (Kumar et al., page 5, column 1, paragraph 2).
PNG
media_image1.png
499
808
media_image1.png
Greyscale
PNG
media_image2.png
654
1186
media_image2.png
Greyscale
Therefore, the argument that the ordinary artisan would not have known what size PEG tags would accomplish the claimed effect is not persuasive, as the cited references, evidenced by Kumar et al. clearly demonstrate that PEG mass tags comprising the claimed mass range (i.e. at least 1kDa) were widely known and used in the art to discriminate between different nucleic acid species using nanopore detection methods. Furthermore, it is clear that the ordinary artisan would have expected that using multiple different size mass tags together (which is an unclaimed embodiment argued in the response) would have been easily discriminated by a nanopore (as evidenced by Kumar et al.) and selection of particular mass tags distinguishable by a nanopore would have a constituted routine optimization step well within the skill of the ordinary artisan.
Claim 43 is rejected under 35 U.S.C. 103 as being unpatentable over Meller et al. and Korlach et al., as applied to claims 1-3, 9 and 14-18 above, and further in view of Kumar et al., “PEG-Labeled Nucleotides and Nanopore Detection for Single Molecule DNA Sequencing by Synthesis” Scientific Reports 2:684, published September 21, 2012.
This is a new grounds of rejection necessitated by newly added claim 43.
As described in the 103 rejection above, Meller et al. in view of Korlach et al. teach methods of detecting a polynucleotide comprising hybridizing a PEG-labeled PNA probe with the target nucleic acid, loading the resulting complex into a nanopore device, and determining the presence of the target nucleic acid-PEG-label-PNA complex during translocation through the nanopore, wherein the PEG-label has a molecular weight of at least 1 kDa.
Meller et al. in view of Korlach et al. do not expressly teach that the molecular weight of the PEG is at least 5 kDa or at least 10 kDa. However, Kumar et al. clearly demonstrate that as early as 2012, incorporation of individual nucleotides comprising cleavable mass tags comprising PEG molecules differing in mass for each of the four nucleotides were readily distinguishable by a nanopore (Kumar et al., figures 3 and 7, reproduced below for applicant’s convenience). Furthermore, Kumar et al., as early as 2012, describe that further increasing the molecular weight of the tag would have been expected to “further enhance nanopore discrimination” (Kumar et al., page 5, column 1, paragraph 2).
PNG
media_image1.png
499
808
media_image1.png
Greyscale
PNG
media_image2.png
654
1186
media_image2.png
Greyscale
Therefore, it would have been prima facie obvious prior to the effective filing date of the claimed invention for one of ordinary skill in the art to have modified the PEG mass tags in the methods taught by Meller et al. in view of Korlach et al. to comprise increased molecular weight to enhance nanopore discrimination between nucleic acid species of interest because of the express teachings of Kumar et al. that species as small and closely related in mass/structure/charge as single nucleotides are distinguishable with low molecular weight PEG tags. Furthermore, figure 7 of Kumar et al. clearly demonstrates increasing resolution between different species with increasing size of the PEG tag coupled thereto. Therefore, the selection and/or optimization of PEG tag(s) for the methods taught by Meller et al. in view of Korlach et al. were clearly within the skill of the ordinary artisan and the ordinary artisan would have been motivated to use larger PEG tags than those disclosed by Kumar et al. because Kumar et al. teaches that increasing the molecular weight of said tags increases the resolution of target discrimination in nanopore detection (see above).
Claims 4, 5, 10-13, 15, 16, and 18 are rejected under 35 U.S.C. 103 as being unpatentable over Meller et al. and Korlach et al., as applied to claims 1-3, 9 and 14-18 above, and further in view of Drndic et al., US 20130092541 A1 (published April 18, 2013).
This rejection is modified from the previous 103 rejection over Meller et al. in view of Korlach et al. further incorporating the cited teachings of Drndic et al. in the 102(a)(1) rejection of record, as necessitated by the amendments to the claims.
As described in the 103 rejection above, Meller et al. in view of Korlach et al. teach methods of detecting a polynucleotide comprising hybridizing a PEG-labeled PNA probe with the target nucleic acid, loading the resulting complex into a nanopore device, and determining the presence of the target nucleic acid-PEG-label-PNA complex during translocation through the nanopore, wherein the PEG-label has a molecular weight of at least 1 kDa.
Meller et al. in view of Korlach et al. do not teach that the detectable signal is an optical signal or that the probe further comprises a molecule selected from the list recited by claim 5.
However, Drndic et al. teaches a method of detecting a miRNA (i.e. a polynucleotide) comprising hybridizing a probe that specifically binds to the miRNA to form a miRNA-probe complex and detecting the miRNA-probe complex using a nanopore device. (Drndic et al., Figure 21a, and paragraph 0043) Drndic further teaches a nanopore device comprises at least one nanopore, at least two chambers in electrical and fluidic communication through the nanopore, and wherein each of the chambers comprises an electrode (i.e. the voltage across the nanopore is controllable) (Drndic et al., figure 18 and paragraph 0038). Drndic et al. teaches that the polynucleotide is miRNA (i.e. RNA). (Drndic et al., Figure 21a, and paragraph 0043) Drndic et al. further teaches that the detectable signal detected by the nanopore device can be an electrical signal or an optical signal. (Drndic et al., paragraph 0144) Drndic et al. further teaches that the probe is a nucleic acid (paragraph 0043).
Further regarding claims 5 and 10-11, Drndic et al. teach binding the probe:miRNA duplex to beads coupled to a p19 (Drndic et al., paragraph 0043) (a protein that preferentially binds the duplex; Drndic et al., paragraph 0163) followed by thorough washing to remove other RNAs from the mixture. (Drndic et al., paragraph 0043) (i.e. applying a condition (changing an initial pH, salt, or temperature condition) to alter the binding interaction between the probe and the target sequence).
Therefore, it would have been prima facie obvious prior to the effective filing date of the claimed invention for one of ordinary skill in the art to have modified the methods for nanopore detection of a target nucleic acid with a PEG-labeled PNA probe, having a PEG label with a molecular weight of at least 1 kDa comprising observing an electrical signal, taught by Meller et al. in view of Korlach et al., to detect the translocation event using an optical signal and an RNA-binding protein. The ordinary artisan would have been motivated to modify the method of Meller et al. in view of Korlach et al. with the teachings of Drndic et al. because of the teaching of Drndic et al. that detection of miRNAs with miRNA-specific nucleic acid probes and RNA-binding proteins using a nanopore accomplishes miRNA-specific detection from complex lysates comprising total cellular RNA (Drndic et al., paragraph 0043). The ordinary artisan would have been reasonably confident that the detection system taught by Meller et al. in view of Korlach et al. comprising PEG-labeled PNA probes would have functioned well in the assay taught by Drndic et al. for specific detection of miRNAs in a complex mixture comprising capture of the miRNA of interest on a complementary nucleic acid probe and RNA binding protein followed by detection of the complex using a nanopore.
Regarding claims 12-13, Drndic et al. teaches that the polynucleotide comprises cytosine methylation (Drndic et al., figure 84 and paragraph 0108).
Regarding claim 15, Drndic et al. teaches that the probe interacts with the target sequence by hybridization (i.e. hydrogen bonds) (Drndic et al., Figure 21a, and paragraph 0043).
Regarding claim 16, Drndic et al. teaches contacting the sample with the DNA intercalating dye SYBR Green I (i.e. a detectable label capable of binding to the polynucleotide probe complex) (Drndic et al., paragraph 0475).
Regarding claim 18, Drndic et al. teaches at least a nanopore 4 nm in diameter and with a range of lengths between 8 and 60 nm (i.e. about 1 nm-about 100 nm in diameter and length) (Drndic et al., figure 12A).
Claims 1-3, 5, 10-11, and 15-22 are rejected under 35 U.S.C. 103 as being unpatentable over Meller et al. and Korlach et al., as applied to claims 1-3, 9 and 14-18 above, and further in view of Dunbar et al., WO 2013/012881 A2 (published January 24, 2013)
This rejection is modified from the previous 103 rejection over Meller et al. in view of Korlach et al. further incorporating the cited teachings of Dunbar et al. in the 102(a)(1) rejection of record, as necessitated by the amendments to the claims.
As described in the 103 rejection above, Meller et al. in view of Korlach et al. teach methods of detecting a polynucleotide comprising hybridizing a PEG-labeled PNA probe with the target nucleic acid, loading the resulting complex into a nanopore device, and determining the presence of the target nucleic acid-PEG-label-PNA complex during translocation through the nanopore, wherein the PEG-label has a molecular weight of at least 1 kDa.
Meller et al. in view of Korlach et al. do not teach that the probe further comprises a molecule listed in claim 5.
However, Dunbar et al. teaches a method of detecting a polynucleotide comprising a target sequence comprising binding a DNA-binding protein (i.e. a probe) such as RecA, NF-kB, and p53 to a DNA molecule to form a polynucleotide-probe complex (Dunbar et al., paragraph 0084). Dunbar et al. further teaches loading said sample into a first chamber of a nanopore device, wherein the nanopore device comprises at least two chambers, at least one nanopore through which the at least two chambers are in electrical and fluidic communication, and wherein the voltages across each of the nanopores are independently-controlled and the nanopores are associated with a sensor configured to identify objects passing through the nanopore (Dunbar et al., paragraph 0086 and Figure 1).
Therefore, it would have been prima facie obvious prior to the effective filing date of the claimed invention for one of ordinary skill in the art to have modified the methods for nanopore detection of a target nucleic acid with a PEG-labeled PNA probe, having a PEG label with a molecular weight of at least 1 kDa comprising observing an electrical signal, taught by Meller et al. in view of Korlach et al., to further comprise binding a DNA binding protein to the molecule of interest for detection on a nanopore device. The ordinary artisan would have been motivated to modify the methods of Meller et al. in view of Korlach et al. to further bind the complex with a DNA binding protein because of the teachings of Dunbar et al. that binding of said proteins to a target nucleic acid is useful for detection of modifications to the nucleic acid (Dunbar et al., paragraph 0084-0085) because protein binding alters the ionic current observed in the nanopore by virtue of the size of the resulting complex relative to the non-protein bound substrate. (Dunbar et al., paragraph 0084).
Regarding claim 2, Dunbar et al. teaches that the polynucleotide is DNA (Dunbar et al., paragraph 0084)
Regarding claim 3, Dunbar et al. teaches that the signal is an electrical signal. (Dunbar et al., paragraph 0086)
Regarding claim 5-6, Dunbar et al. teaches the probe comprises polypeptides/proteins such as: RecA, NF-kB, and p53 (Dunbar et al., paragraph 0084).
Regarding claims 10-11, Dunbar et al. teaches mapping RecA on DNA by assembling complexes at physiological salt and increasing to high salt using a poorly hydrolysable ATP analogue ATP (gamma)S or a non-hydrolyzable ADP-AlF4 that causes RecA to bind more tightly to the DNA (i.e. applying a condition to the sample suspected to alter the binding interaction between the probe and target sequence) (Dunbar et al., paragraph 0103).
Regarding claim 15, Dunbar et al. teaches binding sequence-specific DNA binding proteins (such as lac repressor and phage lambda repressor) (i.e. probes) to DNA (i.e. by hydrogen bonding) (Dunbar et al., paragraph 0110).
Regarding claim 16-17, Dunbar et al. teaches binding additional proteins (i.e. detectable labels) to the RecA-DNA (i.e. probe-DNA) complex including LexA and phage lambda repressors “which has multiple operator sites on lambda-DNA” (Dunbar et al., paragraph 0110).
Regarding claim 18, Dunbar et al. teaches the nanopores are about 1 to about 100 nm in diameter, each chamber comprises an electrode, and the nanopore length is 0.3-50 nm (i.e. about 1 to about 100 nm) (Dunbar et al., figure 1).
Regarding claim 19, Dunbar et al. teaches the nanopore device comprises two nanopores (Dunbar et al., figure 1) configured to control the movement of the polynucleotide through both nanopores simultaneously (Dunbar et al., figure 1).
Regarding claim 20, Dunbar et al. teaches reversing the independently controlled voltages after initial detection of the complex and identifying the presence of the complex again (Dunbar et al., paragraph 0069-0070).
Regarding claim 21, Dunbar et al. teaches that the polynucleotide is simultaneously located within both of the two nanopores (Dunbar et al., figure 1).
Regarding claim 22, Dunbar et al. teaches adjusting the magnitude or direction of the voltages in each nanopore to generate an opposing force and control the rate of translocation through the nanopores (Dunbar et al., paragraph 0069-0070).
Claims 23-30 are rejected under 35 U.S.C. 103 as being unpatentable over Drmanac, US 2009/0111115 A1 (published April 30, 2009) in view of Meller et al., US 2012/0276530A1 (published November 11, 2012) and Korlach et al., US 2013/0109577 A1 (Published May 2, 2013).
This rejection has been updated as necessitated by the amendments to the claims.
Regarding claim 23, Drmanac teaches a method of analyzing sequence information (i.e. detecting a polynucleotide in a sample) (Drmanac, Abstract) comprising binding a first and second probe to target sequences in the polynucleotide (Drmanac, figures 3B and 4D). Drmanac further teaches contacting the sample with additional probes that hybridize to the first and second probes and generate “junction structures” (i.e. a third molecule that binds to the first and second probes when the probes are in sufficient proximity, thereby forming a fusion complex) (Drmanac, figure 5, paragraph 0106, and paragraphs 0120-0126). Finally, Drmanac teaches loading the sample into a nanopore device and detecting the fusion complex using a nanopore device (Drmanac, paragraphs 0057 and 0081).
Drmanac does not expressly teach that the nanopore device comprises at least one nanopore through which at least a first and second chamber are in electrical and fluidic communication, and wherein the device comprises a controlled voltage potential across the nanopore and a sensor.
However, Meller et al. teach methods of detecting polynucleotides comprising multiple target sequences wherein targets bound by multiple nucleic acid probes are detected as they pass through a nanopore device (Meller et al., paragraph 0011) from a first chamber, through a nanopore through which the first and second chambers are in fluidic and electrical communication, and into a second chamber (Meller et al., figure 1).
Therefore, it would have been prima facie obvious prior to the effective filing date of the claimed invention for one of ordinary skill in the art to combine the method of detecting polynucleotides comprising simultaneously binding at least two target-specific polynucleotide probes and a third molecule to which the two target-specific polynucleotide probes bind comprising detection with a nanopore device, taught by Drmanac, with the method of detecting multiple target-specific polynucleotide probes bound to a target nucleic acid sequence with nanopore device comprising the elements recited by claim 23, taught by Meller et al. The ordinary artisan would have been motivated to combine the method of Drmanac with the method of Meller et al. because of the teaching of Meller et al. that secondary molecules associated with bound probes comprising bulk-increasing modifications (analogous to the third molecules taught by Drmanac) facilitate detection of the complex by enhancing the bulk of the dsDNA-PNA complex… to enhance the signal amplitude… and ease of detection (Meller et al., paragraphs 0058-0060). The ordinary artisan would have been reasonably confident that the combination of the methods of Drmanac with the methods taught by Meller et al. would have successfully enhanced detection of a specific polynucleotide because both Drmanac and Meller et al. teach that probe-secondary molecule fusion complexes increase the ease of detection of said complexes in nanopore devices relative to single probe-target binding events.
Drmanac et al. and Meller et al. does not teach the molecular weight of said bulk- and signal- enhancing PEG secondary molecule.
However, Korlach et al. teach a method of detecting polynucleotides using DNA probes coupled to detectable tags (i.e. a secondary molecule) comprising a polyethylene glycol (PEG) linker (Korlach et al., paragraph 0141). Korlach et al. further teach PEG linkers that comprise 77 ethylene glycol monomers (Korlach et al., paragraph 0184). PEG (CH2CH2O)n has an approximate molecular weight of 44n Da, where n=the number of monomers in the polymer. Therefore, a PEG linker comprising 77 monomers has a molecular weight ≈ 3.388 kDa (i.e. at least 1 kDa…).
The teachings of Meller et al., Drmanac et al., and Korlach et al. are in the same very narrow field of scientific inquiry, namely detection of nucleic acids labeled with tags comprising polyethylene glycol coupled to a nucleic acid configured to facilitate detection of the nucleic acid.
Therefore, it would have been prima facie obvious prior to the effective filing date of the claimed invention for one of ordinary skill in the art to modify the method of nanopore detection of polynucleotides containing specific target sequences through binding of sequence-specific PNA-probes associated to bulky-second molecules comprising PEG, taught by Meller et al. by substituting the bulk-and signal- enhancing PEG secondary molecule with the 77-mer PEG detectable labels taught by Korlach et al. The ordinary artisan would have been motivated to substitute the 77-mer PEG labels into the method taught by Meller et al. because Korlach et al. and Meller et al. teach bulky PEG linkers are useful for increasing nanopore detection of nucleic acids. The ordinary artisan would have been reasonably confident that the 77-mer PEG molecules taught by Korlach et al. would have produced predictable bulk- and signal- enhancement in the method taught by Meller et al. because the teachings of Korlach et al. predictably result in useful linkers for nanopore sequencing of tagged nucleic acid molecules.
Regarding claim 24, Drmanac teaches that the polynucleotide is DNA (Drmanac, paragraph 003).
Regarding claim 25, Meller et al. teaches that the detectable signal is an electric signal (Meller et al., paragraph 0011).
Regarding claim 26, Drmanac teaches that the detectable signal is an optical signal (Drmanac, paragraph 007).
Regarding claim 27, Meller et al. teaches the proximity between the two target-specific probes are at least 100 bp … at least 150 bp… at least 1000 bp apart, including all the whole integers between 20-1000 bp (Meller et al., paragraph 0087).
Regarding claim 28, Meller et al. and Drmanac teach nucleic acid probes (i.e. a third molecule) may comprise a PEG (Meller et al., paragraph 0059 and Drmanac, paragraph 0113). Drmanac specifically teaches third molecules (Drmanac, figure 5, paragraph 0106, and paragraphs 0120-0126) may comprise linkers including PEG groups and PNA (Drmanac, paragraph 0113).
Regarding claim 29, Drmanac teaches third molecules comprise polynucleotide sequences that are complementary to portions of the first and second probes, wherein the first and second probes hybridize to ssDNA targets (Drmanac, figure 5, paragraph 0106, and paragraphs 0120-0126)
Regarding claim 30, Drmanac teaches that the third molecule may be further comprise secondary labels that may be bound by a suitable binding partner. Drmanac teaches that such binding partner:label pairs include antigens and antibodies, biotin and streptavidin, nucleic acids and nucleic acid binding proteins… (Drmanac, paragraph 0105-0106).
Response to arguments
The response argues that Drmanac teaches PEG coupled to probes acts only as a linker to connect a dye molecule to a probe, which is asserted to be an “entirely different purpose” and that Drmanac does not teach that the increased molecular weight of the PEG molecules improves nanopore current blockade signals.
These arguments have been reviewed and are not persuasive because these limitations, incorporated from claim 9 (dependent upon claim 1), were not present in independent claim 23 in the previous version of the claims. The newly added requirement to claim 23 that the PEG label has a particular molecular weight has been addressed in the updated 103 rejection above.
Newly added claims 40-42 are rejected under 35 U.S.C. 103 as being unpatentable over Drmanac, Meller et al., and Korlach et al., as applied to claims 23-30 above, and further in view of Kumar et al., “PEG-Labeled Nucleotides and Nanopore Detection for Single Molecule DNA Sequencing by Synthesis” Scientific Reports 2:684, published September 21, 2012.
This is a new grounds of rejection necessitated by new claims 40-42.
Claims 40-42 require that: the PEG coupled to the first PNA probe has a molecular weight of at least 1 kDa (claim 40) and that the PEG coupled to the second PNA probe has a molecular weight of at least 2 kDa (claim 41) or the PEG coupled to the third PNA probe has a molecular weight of at least 1 kDa. It is noted that the claim language “at least 1 kDa” and “at least 2 kDa” does not require that the probes comprise PNAs having different molecular weights, as an embodiment wherein both the first and second probe comprise PEG molecules having molecular weights of 3 kDa is encompassed by the claims.
Regarding claim 40, Drmanac in view of Meller et al. and Korlach et al. teach methods of nanopore detection of polynucleotides containing specific target sequences through binding of sequence-specific PNA-probes associated to bulky-second molecules comprising PEG, taught by Meller et al. by substituting the bulk-and signal- enhancing PEG secondary molecule with the 77-mer PEG (i.e. at least 3 kDa) detectable labels taught by Korlach et al.
Regarding claims 41 and 42, Drmanac, Meller et al., and Korlach et al. do not teach that the probes comprise PEG labels having different molecular weights. However, Kumar et al. clearly demonstrate that as early as 2012, incorporation of individual nucleotides comprising cleavable mass tags comprising PEG molecules differing in mass for each of the four nucleotides were readily distinguishable by a nanopore (Kumar et al., figures 3 and 7, reproduced below for applicant’s convenience). Furthermore, Kumar et al., as early as 2012, describe that further increasing the molecular weight of the tag would have been expected to “further enhance nanopore discrimination” (Kumar et al., page 5, column 1, paragraph 2).
PNG
media_image1.png
499
808
media_image1.png
Greyscale
PNG
media_image2.png
654
1186
media_image2.png
Greyscale
Therefore, it is the position of the examiner that it is clear that the ordinary artisan would have expected that using multiple different size mass tags together would have been easily discriminated by a nanopore (as clearly taught by Kumar et al.) and selection of particular mass tags distinguishable by a nanopore would have a constituted routine optimization step well within the skill of the ordinary artisan.
Response to arguments
The response asserts that the ability of the claimed method to distinguish between probes labeled with PEG molecules having different molecular weights constitute unexpected results. However, as evidenced by Kumar et al., increasing the molecular weight (i.e. bulk) of mass tags comprising PEG has been a known strategy for increasing resolution between different detectable species using a nanopore. Therefore, this argument/assertion is not persuasive.
Double Patenting
The nonstatutory double patenting rejection is based on a judicially created doctrine grounded in public policy (a policy reflected in the statute) so as to prevent the unjustified or improper timewise extension of the “right to exclude” granted by a patent and to prevent possible harassment by multiple assignees. A nonstatutory double patenting rejection is appropriate where the conflicting claims are not identical, but at least one examined application claim is not patentably distinct from the reference claim(s) because the examined application claim is either anticipated by, or would have been obvious over, the reference claim(s). See, e.g., In re Berg, 140 F.3d 1428, 46 USPQ2d 1226 (Fed. Cir. 1998); In re Goodman, 11 F.3d 1046, 29 USPQ2d 2010 (Fed. Cir. 1993); In re Longi, 759 F.2d 887, 225 USPQ 645 (Fed. Cir. 1985); In re Van Ornum, 686 F.2d 937, 214 USPQ 761 (CCPA 1982); In re Vogel, 422 F.2d 438, 164 USPQ 619 (CCPA 1970); In re Thorington, 418 F.2d 528, 163 USPQ 644 (CCPA 1969).
A timely filed terminal disclaimer in compliance with 37 CFR 1.321(c) or 1.321(d) may be used to overcome an actual or provisional rejection based on nonstatutory double patenting provided the reference application or patent either is shown to be commonly owned with the examined application, or claims an invention made as a result of activities undertaken within the scope of a joint research agreement. See MPEP § 717.02 for applications subject to examination under the first inventor to file provisions of the AIA as explained in MPEP § 2159. See MPEP § 2146 et seq. for applications not subject to examination under the first inventor to file provisions of the AIA . A terminal disclaimer must be signed in compliance with 37 CFR 1.321(b).
The filing of a terminal disclaimer by itself is not a complete reply to a nonstatutory double patenting (NSDP) rejection. A complete reply requires that the terminal disclaimer be accompanied by a reply requesting reconsideration of the prior Office action. Even where the NSDP rejection is provisional the reply must be complete. See MPEP § 804, subsection I.B.1. For a reply to a non-final Office action, see 37 CFR 1.111(a). For a reply to final Office action, see 37 CFR 1.113(c). A request for reconsideration while not provided for in 37 CFR 1.113(c) may be filed after final for consideration. See MPEP §§ 706.07(e) and 714.13.
The USPTO Internet website contains terminal disclaimer forms which may be used. Please visit www.uspto.gov/patent/patents-forms. The actual filing date of the application in which the form is filed determines what form (e.g., PTO/SB/25, PTO/SB/26, PTO/AIA /25, or PTO/AIA /26) should be used. A web-based eTerminal Disclaimer may be filled out completely online using web-screens. An eTerminal Disclaimer that meets all requirements is auto-processed and approved immediately upon submission. For more information about eTerminal Disclaimers, refer to www.uspto.gov/patents/apply/applying-online/eterminal-disclaimer.
Claim 1 is rejected on the ground of nonstatutory double patenting as being unpatentable over claim 1 of U.S. Patent No. 10,670,590 B2 (herein referred to as ‘590). Although the claims at issue are not identical, they are not patentably distinct from each other in view of Meller et al. and Korlach et al.
This rejection has been updated in view of the amendments to the pending claims.
Claim 1 of ‘590 recites a method of detecting the presence or absence of a target molecule in a sample (i.e. a polynucleotide) comprising contacting the sample with a complex comprising a fusion molecule bound to a polymer scaffold wherein the fusion molecule comprises a target binding domain (i.e. a probe), loading the sample into a nanopore device, and determining whether the complex is bound to the target molecule.
The claims of ‘590 do not recite that the probe comprises a PEG having a molecular weight of at least 1 kDa. However, Meller et al. and Korlach et al. teach methods of detecting a polynucleotide comprising hybridizing a PEG-labeled PNA probe with the target nucleic acid, loading the resulting complex into a nanopore device, and determining the presence of the target nucleic acid-PEG-label-PNA complex during translocation through the nanopore, wherein the PEG-label has a molecular weight of at least 1 kDa.
Therefore, it would have been prima facie obvious prior to the effective filing date of the claimed invention for one of ordinary skill in the art to have modified the claims of ‘590 with the teaching of Meller et al. and Korlach et al. comprising detection of nucleic acid sequences of interest with nanopore-detectable PEG-labeled PNA probes wherein the PEG molecules have a molecular weight of at least 1 kDa. The ordinary artisan would have been motivated to modify the probes recited by the claims of ‘590 in this way because of the teaching of Meller et al. that secondary molecules associated with bound probes comprising bulk-increasing modifications facilitate detection of the complex by enhancing the bulk of the dsDNA-PNA complex… to enhance the signal amplitude… and ease of detection (Meller et al., paragraphs 0058-0060).
Claim 1 is rejected on the ground of nonstatutory double patenting as being unpatentable over claim 1 of U.S. Patent No. 9,983,191 B2 (herein referred to as ‘191). Although the claims at issue are not identical, they are not patentably distinct from each other in view of Meller et al. and Korlach et al.
This rejection has been updated in view of the amendments to the pending claims.
Claim 1 of ‘191 recites a method for detecting the presence or absence of a target molecule (i.e. a polynucleotide) in a sample comprising contacting the sample with a fusion molecule comprising a target molecule binding domain (i.e. a probe), loading the sample into a nanopore device, and detecting the presence or absence of the target molecule in the sample.
The claims of ‘191 do not recite that the probe comprises a PEG having a molecular weight of at least 1 kDa. However, Meller et al. and Korlach et al. teach methods of detecting a polynucleotide comprising hybridizing a PEG-labeled PNA probe with the target nucleic acid, loading the resulting complex into a nanopore device, and determining the presence of the target nucleic acid-PEG-label-PNA complex during translocation through the nanopore, wherein the PEG-label has a molecular weight of at least 1 kDa.
Therefore, it would have been prima facie obvious prior to the effective filing date of the claimed invention for one of ordinary skill in the art to have modified the claims of ‘191 with the teaching of Meller et al. and Korlach et al. comprising detection of nucleic acid sequences of interest with nanopore-detectable PEG-labeled PNA probes wherein the PEG molecules have a molecular weight of at least 1 kDa. The ordinary artisan would have been motivated to modify the probes recited by the claims of ‘191 in this way because of the teaching of Meller et al. that secondary molecules associated with bound probes comprising bulk-increasing modifications facilitate detection of the complex by enhancing the bulk of the dsDNA-PNA complex… to enhance the signal amplitude… and ease of detection (Meller et al., paragraphs 0058-0060).
Response to arguments
The response acknowledges the double patenting rejections of record, but does not present any arguments responsive to the rejections. Rather the response states, “If the rejection is maintained and the claims are deemed otherwise allowable, Applicant will consider filing a Terminal Disclaimer as needed.”
This has been interpreted as a request to hold the double patenting rejections in abeyance rather than an incomplete response.
This request has been noted and is denied.
See MPEP 804(I)(b)(1) and 37 C.F.R. 1.111(b), which allows that some objections may be held in abeyance, but includes no provision for holding rejections in abeyance. Thus, for the reasons above and those already of record, the rejection is maintained.
Conclusion
No claim is allowed.
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.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to ZACHARY MARK TURPIN whose telephone number is (703)756-5917. The examiner can normally be reached Monday-Friday 8:00 am - 5:00 pm.
Examiner interviews are available via telephone, in-person, and video conferencing using a USPTO supplied web-based collaboration tool. To schedule an interview, applicant is encouraged to use the USPTO Automated Interview Request (AIR) at http://www.uspto.gov/interviewpractice.
If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, Winston Shen can be reached at 5712723157. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
Information regarding the status of published or unpublished applications may be obtained from Patent Center. Unpublished application information in Patent Center is available to registered users. To file and manage patent submissions in Patent Center, visit: https://patentcenter.uspto.gov. Visit https://www.uspto.gov/patents/apply/patent-center for more information about Patent Center and https://www.uspto.gov/patents/docx for information about filing in DOCX format. For additional questions, contact the Electronic Business Center (EBC) at 866-217-9197 (toll-free). If you would like assistance from a USPTO Customer Service Representative, call 800-786-9199 (IN USA OR CANADA) or 571-272-1000.
/Z.M.T./Examiner, Art Unit 1682
/WU CHENG W SHEN/Supervisory Patent Examiner, Art Unit 1682