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 .
Response to Arguments
Applicant’s arguments, see Remarks page 5, filed 22 July 2025, with respect to the rejections under 35 U.S.C 112(b) have been fully considered and are persuasive in light of the amendments. The rejections of claims 14 and 15 have been withdrawn.
Applicant’s arguments, see Remarks page 5, filed 22 July 2025, with respect to the rejections of claims 1-16 under 35 U.S.C. 103 have been fully considered and are persuasive in light of the amendments. Therefore, the rejection has been withdrawn. However, upon further consideration, a new grounds of rejection is made in view of Bayley et al in view of He et al in view of Natale et al and further in view of Jovanovich et al.
Status of Claims
Applicant’s amendments to the claims, filed 22 July 2025, have been entered. Applicant’s remarks filed 22 July 2025 are acknowledged.
Claims 1, 2, 6, 12, and 14 are in status “Currently Amended.” Claims 4, 5, 7-11, 13, 15, and 16 are in status “Original.” Claim 3 is in status “Cancelled.”
Claim Rejections - 35 USC § 103
The text of those sections of Title 35, U.S. Code not included in this action can be found in a prior Office action.
Claims 1, 2, 4-7, 9-14, and 16 are rejected under 35 U.S.C. 103 as being unpatentable over Bayley et al (US 20080311582 A1) in view of He et al (US 20110312529 A1) in view of Natale et al (WO 2014111723 A1, priority claimed to 18 January 2013, cited on the IDS submitted 22 February 2022) and further in view of Jovanovich et al (US 20080014576 A1).
With regards to claim 1, Bayley et al teaches;
The claimed “a fluid impermeable barrier having a first side and second side separating a first fluid reservoir from a second fluid reservoir” has been read on the taught ([0075], "…the apparatus comprises a chamber comprising an aqueous solution and a barrier that separates the chamber into two sections."; [0072], “The nucleotide may be contacted with the pore on either side of the membrane.”; The barrier separating the chamber reads on a fluid impermeable barrier. The two sections created by the barrier reads on the first and second fluid reservoir. Either side of the membrane reads on a first side and second side.);
The claimed “a nanopore positioned in the fluid impermeable barrier to form a passage through which a nucleotide triphosphate can pass from the first fluid reservoir to the second fluid reservoir” has been read on the taught ([0005], “Stochastic sensing involves placing a nanometer sized pore in an insulating lipid bilayer membrane and measuring the ionic transport through the pore.”; [0072], “The nucleotide may be contacted with the pore on either side of the membrane. The nucleotide may be introduced to the pore on either side of the membrane… The nucleotide is preferably contacted with a side of the membrane that allows the nucleotide to pass through the pore to the other side of the membrane.");
The claimed “a reaction mix in the first fluid reservoir” has been read on the taught ([0075], "…the apparatus comprises a chamber comprising an aqueous solution and a barrier that separates the chamber into two sections." The aqueous solution reads on a reaction mix.);
The claimed “an electrical device that generates and electrical circuit between the first fluid reservoir and second fluid reservoir” has been read on the taught ([0076], “Therefore the apparatus also comprises an electrical circuit capable of applying and measuring an electrical signal across the membrane and pore.”);
The claimed “a polymerase attached to the first side of the fluid impermeable barrier at the nanopore in the first fluid reservoir” has been read on the taught ([0097], “The processive exonuclease is preferably covalently attached to the transmembrane protein pore.”).
Bayley et al additionally teaches that other means of progressively digesting the nucleic acid may be used in sequencing methods ([0096], “The method […] involves contacting the nucleic acid sequence with the processive exonuclease so that the nucleotides are digested... Methods for doing this are well known in the art… A homologous method may be used in the present invention.”).
Bayley et al further teaches that the target nucleic acid is typically amplified prior to sequencing, as read on the taught ([0093], “The nucleic acid sequence is typically amplified prior to being sequenced using the method of the second embodiment.”).
However, Bayley et al does not explicitly disclose at least one pump fluidly connected to the first fluid reservoir for adding and/or removing a component of the reaction mix; the reaction mix including a pyrophosphorolytic concentration of phosphate; and a plurality of single stranded DNA template strand attached to the first side of the fluid impermeable barrier in the first fluid reservoir.
In the analogous art of nucleotide sequencing, He et al teaches;
The claimed “a plurality of single stranded DNA template strand attached to the first side of the fluid impermeable barrier in the first fluid reservoir” has been read on the taught ([0086], “…For example, a nucleic acid, nucleic acid enzyme or nucleotide can be in or on a solid support during the course of a solid-phase reaction. A nucleic acid that is attached to the solid support can be a template nucleic acid such as one that is copied by a polymerase… or a double stranded nucleic acid…”; [0087], “Any of a variety of solid-support materials can be used in a method or composition set forth herein… Useful materials include […] those that form a continuous material such as a flow cell, microchip or other chip, microscope slide or other planar surface, or the like.”; A template nucleic acid that is copied by a polymerase reads on a single stranded DNA, as supported by the later recitation of double-stranded nucleic acid. A planar surface reads on an impermeable barrier).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify the sequencing device including a fluid impermeable barrier as taught by Bayley et al with the single stranded DNA templates attached to a solid support as taught by He et al, for the benefit of controlling which components in solution can interact with the single stranded DNA template, as taught by He et al ([0089], “A reaction component can be attached to a support in a way that provides detection at a single molecule level or at an ensemble level. For example, a population of nucleic acids can be attached to a solid support in a way that conformationally labeled polymerases that interact with individual nucleic acid molecules in the population can be distinguished from conformationally labeled polymerases that interact with other nucleic acid molecules on the support.”).
He et al does not explicitly disclose that the template nucleic acid is attached to the first side of the fluid impermeable barrier. However, as Bayley et al teaches that the nucleic acid sequence is typically amplified prior to sequencing, one of ordinary skill in the art would understand that placing a template strand on the first side of the barrier would allow the resulting amplified nucleic acids to pass from the first reservoir to the second reservoir as taught by Bayley et al.
Neither Bayley et al nor He et al explicitly disclose at least one pump fluidly connected to the first fluid reservoir for adding and/or removing a component of the reaction mix; or the reaction mix including a pyrophosphorolytic concentration of phosphate.
In the analogous art of nucleic acid sequencing, Natale et al teaches;
A method of sequencing nucleic acids, as read on the taught (Abstract, “Disclosed is a method for determining the sequence of nucleotide bases in a polynucleotide analyte.”);
Wherein the method involves digesting nucleic acids in pyrophosphorolytic concentrations of pyrophosphate, as read on the taught (Page 4, lines 17-20, "…the single nucleotide bases can be generated from the polynucleotide analyte by pyrophosphorolysis in the presence of high levels of pyrophosphate anion...");
And wherein the method involves a polymerase, as read on the taught (Page 4, line 27, "The enzyme used in this second embodiment is suitably one which can cause progressive 3'-5' pyrophosphorolytic degradation of the analyte to yield deoxyribonucleotide triphosphates with high selectivity and at a reasonable reaction rate…. The enzyme is suitably selected from the group consisting of those polymerases which show essentially neither exo- nor endonuclease activity under the reaction conditions.”).
Natale et al additionally teaches that digestion by phosphorolysis is suitable for nucleotide detection as taught by Bayley et al. The apparatus as taught by Bayley et al involves removal of digested nucleotides through the pore, which meets Natale et al’s requirement that liberated nucleotide bases are continuously removed from the reaction zone (Page 4, lines 17-20, "Alternatively, the single nucleotide bases can be generated from the polynucleotide analyte by pyrophosphorolysis in the presence of high levels of pyrophosphate anion under conditions where the liberated nucleotide bases are continuously removed from the reaction zone to avoid establishment of the reverse conventional polymerase chain reaction."). Natale et al teaches that digestion by phosphorolysis allows the use of an enzyme which yields deoxyribonucleotide triphosphates with high selectivity and at a reasonable reaction rate, which would be desirable for sequential detection based on a nucleotide’s electric charge (Natale et al, Page 4, line 27, "The enzyme used in this second embodiment is suitably one which can cause progressive 3'-5' pyrophosphorolytic degradation of the analyte to yield deoxyribonucleotide triphosphates with high selectivity and at a reasonable reaction rate."; Bayley et al, [0006], “It has been surprisingly demonstrated that individual nucleotides can be identified at the single molecule level from their current amplitude when they interact with a transmembrane pore.”).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify the apparatus including reagents for progressive digestion of nucleic acid as taught by Bayley et al in view of Turner et al with the method for progressive digestion of a nucleic acid using a polymerase and pyrophosphorolytic concentrations of pyrophosphate as taught by Natale et al. MPEP 2143(I)(A) teaches that “combining prior art elements according to known methods to yield predictable results” supports a case of prima facie obviousness. Combining the prior art elements taught by Bayley et al with the known nucleic acid digestion methods of Natale et al yields the predictable result of a processive digestion of a target nucleic acid with a high selectivity for deoxyribonucleotide triphosphates. As Bayley et al teaches that homologous methods may be used, the combination would have been within the ordinary skill of the art.
However, Bayley et al in view of He et al and further in view of Natale et al fails to teach wherein the apparatus further comprises at least one pump fluidly connected to the first fluid reservoir for adding and/or removing a component of the reaction mix.
In the analogous art of DNA sequencing apparatuses, Jovanovich et al teaches;
A DNA sequencing apparatus, as read on the taught (Abstract, "The technology disclosed can be used as sample preparation and analysis systems for various applications, such as DNA sequencing…");
Wherein the DNA sequencing apparatus includes “at least one pump fluidly connected to the first fluid reservoir for adding and/or removing a component of the reaction mix”, as read on the taught ([0061], "The microchips disclosed herein can be manufactured by microfabrication techniques known in the art and can comprise valves, pumps, chambers, channels, reservoirs etc. and can be suitable for processing or analyzing one or more target analytes.").
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify the DNA sequencing apparatus as taught by Bayley et al in view of He et al in view of Natale et al with the sequencing apparatus including a pump as taught by Jovanovich et al, for the benefit of controlling reaction times and reaction sequences as taught by Jovanovich et al ([0109], "In some embodiments, the various types of chips can process samples in individual bioprocessor modules using MOV valves, pumps, and routers as system control elements to thereby control reaction times and sequences.").
With regards to claim 2, the apparatus of claim 1 is obvious over Bayley et al in view of He et al in view of Natale et al and further in view of Jovanovich et al.
Bayley et al additionally teaches;
Wherein the apparatus of claim 1 further comprises electrodes positioned to create difference in potential for the first fluid reservoir compared to the second fluid reservoir ([0043], "The pore preferably permits the nucleotide to flow from one side of the membrane to the other along an electrochemical gradient."; [0108], "The cis side of the chamber was at ground, and the trans side of the chamber was connected to the head stage. The potential refers to the potential value of the trans side electrode."; The head stage applying a potential, which permits the nucleotide to flow from one side of the membrane to the other along an electrochemical gradient reads on electrodes positioned to create a difference in potential for the first fluid reservoir compared to the second fluid reservoir.).
With regards to claim 4, the apparatus of claim 1 is obvious over Bayley et al in view of He et al in view of Natale et al and further in view of Jovanovich et al.
Bayley et al additionally teaches;
The claimed “wherein the fluid impermeable barrier comprises a membrane” has been read on the taught ([0041], “The membrane forms a barrier to the flow of ions and nucleotides. The membrane is preferably a lipid bilayer.”)
With regards to claim 5, the apparatus of claim 4 is obvious over Bayley et al in view of He et al in view of Natale et al and further in view of Jovanovich et al.
Bayley et al additionally teaches;
The claimed “wherein the nanopore comprises a protein nanopore that is embedded in the membrane” has been read on the taught ([0043], “The method of the invention is carried out using a transmembrane protein pore.”).
With regards to claim 6, the apparatus of claim 4 is obvious over Bayley et al in view of He et al in view of Natale et al and further in view of Jovanovich et al.
Bayley et al teaches that the target nucleic acid is typically amplified prior to sequencing, as read on the taught ([0093], “The nucleic acid sequence is typically amplified prior to being sequenced using the method of the second embodiment.”).
Neither Natale et al nor Jovanovich et al explicitly disclose wherein a strand of target nucleic acid is complementary to a DNA template strand of the plurality of single stranded DNA template strands attached to the membrane.
He et al additionally teaches;
The claimed “wherein a strand of target nucleic acid is complementary to a DNA template strand of the plurality of single stranded DNA template strands attached to the membrane” has been read on the taught ([0028], “For example, a polymerase can use a first nucleic acid strand as a template to sequentially build a second, complementary nucleic acid strand by sequential addition of nucleotides to the second strand.”).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify the device as taught by Bayley et al with the target nucleic acid being complementary to a template strand as taught by He et al, in order to analyze the amplified target nucleic acid strand, as taught by Bayley et al ([0093], “The nucleic acid sequence is typically amplified prior to being sequenced using the method of the second embodiment.”).
With regards to claim 7, the apparatus of claim 6 is obvious over Bayley et al in view of He et al in view of Natale et al and further in view of Jovanovich et al.
Bayley et al additionally teaches;
The claimed “wherein the target nucleic acid includes at least one base moiety that is non-naturally occurring in DNA or RNA” has been read on the taught ([0081], "The method of the invention may be used to identify any nucleotide. The nucleotide can be naturally-occurring or artificial.").
With regards to claim 9, the apparatus of claim 1 is obvious over Bayley et al in view of He et al in view of Natale et al and further in view of Jovanovich et al.
Natale et al additionally teaches;
The claimed “wherein the pyrophosphorolytic concentration comprises at least 100 pM pyrophosphate” has been read on the taught (Page 5, lines 5-7, "Suitably, the pyrophosphorolytic degradation is carried out in the presence of a medium which further comprises pyrophosphate anion and magnesium cations in preferably millimolar concentrations."; millimolar concentrations of pyrophosphate reads on a concentration of at least 100 pM of pyrophosphate.).
MPEP 2143(I)(A) teaches that “combining prior art elements according to known methods to yield predictable results” supports a case of prima facie obviousness. Accordingly, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to combine the prior art elements of an apparatus including a pyrophosphorolytic concentration of pyrophosphate as taught by Bayley et al in view of Natale et al with the known method of using a pyrophosphorolytic concentration comprising at least 100 pM pyrophosphate as taught by Natale et al, to yield the predictable result of pyrophosphorolytic digestion of nucleic acids as taught by Natale et al.
With regards to claim 10, the apparatus of claim 1 is obvious over Bayley et al in view of He et al in view of Natale et al and further in view of Jovanovich et al.
Bayley et al additionally teaches;
wherein the polymerase lacks 3' to 5' exo nuclease activity ([0094], "The processive exonuclease can digest the nucleic acid in the 5' to 3' direction or 3' to 5' direction. The end of the nucleic acid to which the processive exonuclease binds is typically determined through the choice of enzyme used…").
With regards to claim 11, the apparatus of claim 1 is obvious over Bayley et al in view of He et al in view of Natale et al and further in view of Jovanovich et al.
Jovanovich et al additionally teaches;
The claimed “wherein the at least one pump provides pyrophosphate to the first fluid reservoir” has been read on the taught ([0119], “The chip comprises MOV valves, pumps, and reaction chambers, sample ports for the injection of reagents…”; [0245], “The nucleic acid, DNA or RNA, is moved […] using MOV pumps 251 into a chamber and then the single reaction mix added from one of the reagent channels 252.”; The injection of reagents and the addition of a single reaction mix reads on providing pyrophosphate to the first fluid reservoir.).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify the DNA sequencing apparatus as taught by Bayley et al in view of Natale et al in view of Jovanovich et al with the sequencing apparatus including a pump as taught by Jovanovich et al, for the benefit of controlling reaction times and reaction sequences as taught by Jovanovich et al ([0109], "In some embodiments, the various types of chips can process samples in individual bioprocessor modules using MOV valves, pumps, and routers as system control elements to thereby control reaction times and sequences.").
With regards to claim 12, the apparatus of claim 1 is obvious over Bayley et al in view of He et al in view of Natale et al and further in view of Jovanovich et al.
Bayley et al additionally teaches;
The claimed “wherein the target nucleic acid is DNA” has been read on the taught ([0083], “The nucleotide may be derived from the digestion of a nucleic acid sequence such as […] deoxyribonucleic acid.”).
With regards to claim 13, the apparatus of claim 12 is obvious over Bayley et al in view of He et al in view of Natale et al and further in view of Jovanovich et al.
Bayley et al additionally teaches;
The claimed “wherein the target nucleic acid comprises at least one base moiety that is non-naturally occurring in DNA” has been read on the taught ([0081], "The method of the invention may be used to identify any nucleotide. The nucleotide can be naturally-occurring or artificial.").
With regards to claim 14, the apparatus of claim 12 is obvious over Bayley et al in view of He et al in view of Natale et al and further in view of Jovanovich et al.
Bayley et al additionally teaches;
The claimed “wherein the target nucleic acid comprises at least one base moiety and wherein at least one base moiety comprises 5-methyl cytosine or 5-hydroxymethylcytosine” has been read on the taught ([0081], "Suitable nucleobases include purines and pyrimidines and more specifically […] cytosine."; [0084], "The nucleotide is typically methylated."; The cytosine which is typically methylated reads on 5-methyl cytosine.).
With regards to claim 16, the apparatus of claim 14 is obvious over Bayley et al in view of He et al in view of Natale et al and further in view of Jovanovich et al.
Bayley et al additionally teaches;
The claimed “wherein enzyme activity is limited as a result of pyrophosphate concentrations” has been read on the taught ([0100], "Another way of limiting the rate of the enzyme is to carry out the method of the second embodiment at a salt concentration that reduces the rate of the activity of the enzyme without adversely affecting its activity.”; The salt concentration reads on the concentration of the pyrophosphate anion.).
Neither He at al nor Natale et al explicitly disclose wherein the pump cycles pyrophosphate into and out of the first fluid reservoir under conditions to pause pyrophosphorolytic cleavage.
Jovanovich et al additionally teaches;
The claimed “wherein the pump cycles pyrophosphate into and out of the first fluid reservoir under conditions to pause pyrophosphorolytic cleavage” has been read on the taught ([0109], "In some embodiments, the various types of chips can process samples in individual bioprocessor modules using MOV valves, pumps, and routers as system control elements to thereby control reaction times and sequences."; Controlling reaction times reads on pausing a pyrophosphorolytic cleavage reaction.).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify the DNA sequencing apparatus as taught by Bayley et al in view of He et al in view of Natale et al in view of Jovanovich et al with the sequencing apparatus including a pump as taught by Jovanovich et al, for the benefit of controlling reaction times and reaction sequences as taught by Jovanovich et al ([0109], "In some embodiments, the various types of chips can process samples in individual bioprocessor modules using MOV valves, pumps, and routers as system control elements to thereby control reaction times and sequences.").
Claim 8 is rejected under 35 U.S.C. 103 as being unpatentable over Bayley et al (US 20080311582 A1) in view of He et al (US 20110312529 A1) in view of Natale et al (WO 2014111723 A1, priority claimed to 18 January 2013, cited on the IDS submitted 22 February 2022) in view of Jovanovich et al (US 20080014576 A1), and further in view of Clarke et al (WO 2012164270 A1).
With regards to claim 8, the apparatus of claim 1 is obvious over Bayley et al in view of He et al in view of Natale et al and further in view of Jovanovich et al.
However, that combination fails to teach wherein the nanopore comprises a solid state nanopore.
In the analogous art of nanopore devices for nucleic acid sequencing, Clarke et al teaches;
The claimed “wherein the nanopore comprises a solid state nanopore” has been read on the taught (Page 6, line 3, "Fig. 17 shows analyte tethering schemes for solid state nanopores."; Page 17, line 15, "In another preferred embodiment, the membrane is a solid state layer.").
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify the nucleopore sequencing apparatus as taught by Bayley et al in view of He et al in view of Natale et al and further in view of Jovanovich et al with the solid-state nanopore as taught by Clarke et al, for the benefit of creating an apparatus with a membrane that can be formed in a factory and which has an extended shelf life, as taught by Clarke et al (Page 64, lines 22-25, "Nanopores in solid state materials, such as silicon nitride offer advantages over the biological channels as the pores. Solid state materials are far less fragile than lipid membranes. Nanopores in solid state material can be formed in a factory and have a long shelf life, unlike biological membranes which are often formed in situ.").
Claim 15 is rejected under 35 U.S.C. 103 as being unpatentable over Bayley et al (US 20080311582 A1) in view of Natale et al (WO 2014111723 A1, priority claimed to 18 January 2013, cited on the IDS submitted 22 February 2022) in view of Jovanovich et al (US 20080014576 A1), and further in view of Clarke et al (Clarke J, Wu HC, Jayasinghe L. et al. "Continuous base identification for single-molecule nanopore DNA sequencing." Nature Nanotech 2009; 4: 265–270).
With regards to claim 15, the apparatus of claim 14 is obvious over Bayley et al in view of Natale et al and further in view of Jovanovich et al.
However, this combination fails to teach wherein the 5-methyl cytosine or 5- hydroxymethylcytosine is distinguished from cytosine, thereby facilitating an epigenetic analysis.
In the analogous art of nanopore sequencing devices for nucleic acids, Clarke et al teaches;
The claimed “wherein the 5-methyl cytosine or 5- hydroxymethylcytosine is distinguished from cytosine, thereby facilitating an epigenetic analysis” has been read on the taught (Conclusion, paragraph 1, "The pore can also discriminate between other nucleotides, including methylated dCMP."; methylated dCMP reads on 5-methylcytosine. See Nucleotide discrimination and translocation, paragraph 6, "DNA methylation, manifested as 5-methylcytosine (m5C), is a critical factor in epigenetics.").
It would have been obvious to one of ordinary skill in the art before the effective fining date of the claimed invention to modify the nanopore sequencing device for nucleic acids as taught by Bayley et al in view of Natale et al and further in view of Jovanovich et al with the method of detecting methylated bases in a nanopore as taught by Clarke et al, in order to directly observe modified nucleotides for gathering epigenetic information, using a method which removes the need for amplifying target DNA, lowers the cost of sample preparation, and is scalable using established technology (Clarke et al, Conclusion, paragraph 3, “…a nanopore DNA sequencing system would offer many advantages. The sequencing of single molecules removes the need for amplification of the target DNA, lowering the time and cost of sample preparation and avoiding amplification errors. Single-molecule detection also allows the direct observation of modified nucleotides such as methylated dCMP, which would significantly benefit the field of epigenetics… Additionally, the electrical nature of the signal means that the approach is scaleable using established technology and cheap fabrication techniques.”).
Conclusion
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 ALISON CLAIRE GERHARD whose telephone number is (571)270-0945. The examiner can normally be reached M-F, 9:00 - 5:30pm EST.
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/ALISON CLAIRE GERHARD/Examiner, Art Unit 1797 /LYLE ALEXANDER/Supervisory Patent Examiner, Art Unit 1797