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 .
Election/Restrictions
Applicant’s election without traverse of claims 51-68 in the reply filed on 17 February 2026 is acknowledged.
Claims 69-70 are withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected invention, there being no allowable generic or linking claim. Election was made without traverse in the reply filed on 17 February 2026.
Information Disclosure Statement
The information disclosure statement (IDS) filed 27 March 2023 is considered, initialed, and attached hereto.
Specification
The disclosure is objected to because it contains an embedded hyperlink and/or other form of browser-executable code on page 106 line 19, page 115 line 30, and page 135 line 10. Applicant is required to delete the embedded hyperlink and/or other form of browser-executable code; references to websites should be limited to the top-level domain name without any prefix such as http:// or other browser-executable code. See MPEP § 608.01.
The use of the term IDT, Integrated DNA Technologies, PyroPhage, Lucigen, Bioron, NEB, New England Biolabs, Invitrogen, NEBNext, Oxford Nanopore Technologies, AMPure, Beckman Coulter, MinION, MinKNOW, and USER, which is a trade name or a mark used in commerce, has been noted in this application. The term should be accompanied by the generic terminology; furthermore the term should be capitalized wherever it appears or, where appropriate, include a proper symbol indicating use in commerce such as ™, SM , or ® following the term.
Although the use of trade names and marks used in commerce (i.e., trademarks, service marks, certification marks, and collective marks) are permissible in patent applications, the proprietary nature of the marks should be respected and every effort made to prevent their use in any manner which might adversely affect their validity as commercial marks.
Claim Rejections - 35 USC § 112(b) - Indefiniteness
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.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claims 51-68 rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
Claim 51 recites the limitation "moves freely" in line 3. The metes and bounds of free movement are unclear. The initial broadest reasonable interpretation (BRI) in view of the claim would be that the polynucleotide is free in solution and is not restricted in any way that would prevent or decrease its ability to move with respect to the detector. However, the specification provides that “controlling the speed of the translocation of the first part of the polynucleotide through or across the nanopore using an ultra-fast polynucleotide-handling enzyme, a slider and/or a brake is an example of freely translocating the first part of the polynucleotide in the sample through or across the nanopore” (Specification, page 41 lines 5-9). The specification additionally provides that “Ultra-fast polynucleotide-handling enzymes suitable for controlling the movement of the polynucleotide with respect to the detector (e.g. for controlling the translocation of a first part of a polynucleotide through or across a nanopore) […] include translocases […] packaging motors […] and helicases and helicase-nucleases” (Specification, page 42 line 33 - page 43 line 4). The specification therefore broadens the BRI of this phrase in claim 51 to include polynucleotides that are bound by various substances that restrict the movement of the polynucleotide but in broadening the BRI it fails to make clear where the metes and bounds of the phrase “moves freely” lie (e.g. what substances can the polynucleotide interact with and still be considered to move freely and what substances would interaction with prevent free movement). For the purpose of examination, a polynucleotide bound by any enzyme of the classes given as examples of ultra-fast polynucleotide-handling enzyme (translocases, packaging motors, helicases, and helicase-nucleases) is considered to fall within the BRI of a polynucleotide moving freely with respect to a detector. Claims 52-68 are rejected due to their dependency on claim 51.
Every instance of “the polynucleotide” in claims 51-68 lack antecedent basis because it is unclear whether they refer to the recited “a polynucleotide” in claim 51 line 1 or the recited “a polynucleotide” in claim 51 line 2.
Regarding claim 53, the phrase "preferably" renders the claim indefinite because it is unclear whether the limitations following the phrase are part of the claimed invention. See MPEP § 2173.05(d). The phrase “preferably […] nanopore” in lines 4-9 is considered an optional limitation.
Claim 53 recites the limitation "the nanopore" in line 7. There is insufficient antecedent basis for this limitation in the claim.
Regarding claim 54, the phrase "preferably" renders the claim indefinite because it is unclear whether the limitations following the phrase are part of the claimed invention. See MPEP § 2173.05(d). The phrase “preferably […] adapter” in lines 3-4 is considered an optional limitation.
Claim 56 recites the limitation "move freely" in lines 4-5. This limitation has unclear metes and bounds for the same reasons described above for the phrase "moves freely" in claim 51.
Claim 56 recites “A method according to claim 51, comprising:” followed by 3 steps denoted (i), (ii a), and (ii b). It is unclear whether these are additional steps or are intended to further limit the respective steps (i), (ii a), and (ii b) in claim 51.
Claim 56 recites the limitation "the polynucleotide" in lines 3, 4, and 6. There is insufficient antecedent basis for this limitation in the claim. It is unclear whether these instances refer to the polynucleotide of claim 56 line 2 or claim 51 line 2.
Claim 56 recites the limitation "the " in line 6. There is insufficient antecedent basis for this limitation in the claim. It is unclear whether this refers to the first part of the polynucleotide as recited in claim 56 line 4 or claim 51 line 3.
Regarding claim 57, the phrase "preferably" renders the claim indefinite because it is unclear whether the limitations following the phrase are part of the claimed invention. See MPEP § 2173.05(d). The phrase “preferably […] polynucleotide” in lines 2-5 is considered an optional limitation.
Claim 59 recites the limitation "the polynucleotide binding protein" in line 3. There is insufficient antecedent basis for this limitation in the claim.
Claim 60 recites the limitation "the polynucleotide binding protein" in line 3. There is insufficient antecedent basis for this limitation in the claim.
Claim 61 recites the limitation "the first end of the polynucleotide" in lines 2-3. There is insufficient antecedent basis for this limitation in the claim.
Claim 61 recites the limitation "the polynucleotide binding protein" in line 3. There is insufficient antecedent basis for this limitation in the claim.
Regarding claim 62, the phrase "preferably" renders the claim indefinite because it is unclear whether the limitations following the phrase are part of the claimed invention. See MPEP § 2173.05(d). The phrase “preferably […] polynucleotide” in lines 2-8 is considered an optional limitation.
Claim 62 recites the limitation "the first end of the polynucleotide" in line 5. There is insufficient antecedent basis for this limitation in the claim.
Claim 62 recites the limitation "the polynucleotide binding protein" in line 7. There is insufficient antecedent basis for this limitation in the claim.
Claim 62 recites the limitation "the polynucleotide is single-stranded" in line 3 and “the double-stranded polynucleotide” in line 10. These conflicting limitations render unclear the metes and bounds of the claim.
Claim 62 recites the limitation "the first strand" in line 10. There is insufficient antecedent basis for this limitation in the claim.
Claim 62 recites the limitation "the nanopore" in line 15. There is insufficient antecedent basis for this limitation in the claim.
Claim 63 recites the limitation "the second end of the first strand" in line 6. There is insufficient antecedent basis for this limitation in the claim.
Claim 63 recites the limitation "the polynucleotide binding protein" in line 8. There is insufficient antecedent basis for this limitation in the claim.
Claim 63 recites the limitation "the nanopore" in line 17. There is insufficient antecedent basis for this limitation in the claim.
Regarding claim 64, the phrase "preferably" renders the claim indefinite because it is unclear whether the limitations following the phrase are part of the claimed invention. See MPEP § 2173.05(d). The phrase “preferably […] detector” in lines 2-3 is considered an optional limitation.
Claim 65 recites the phrase “wherein the polynucleotide is double stranded and the portion which is complementary to a tag sequence is a portion of the first strand of the polynucleotide and/or the portion having an oligonucleotide hybridized thereto is a portion of the first strand” (emphasis added). The option of “or” given in the second conjunction makes it unclear whether the claim includes instances where only the 3rd clause occurs ((clause 1 AND clause 2) OR clause 3) or whether it is limited to instances where the 1st clause occurs and at least one of the 2nd and 3rd clauses occur (clause 1 AND (clause 2 AND/OR clause 3)).
Claim 65 recites the limitation "the first strand" in line 3. There is insufficient antecedent basis for this limitation in the claim.
Claim 65 recites the limitation "the portion having an oligonucleotide hybridised thereto" in line 4. There is insufficient antecedent basis for this limitation in the claim.
Claim 65 recites the limitation "the second strand" in line 5. There is insufficient antecedent basis for this limitation in the claim.
Claim 67 recites the limitation "the read force" in line 6. There is insufficient antecedent basis for this limitation in the claim.
Claim 68 recites the limitation "the free movement of the polynucleotide" in lines 1-2. This limitation has unclear metes and bounds for the same reasons described above for the phrase "move freely" in claim 51.
Claim 68 recites the limitation “an ultra-fast polynucleotide-handling enzyme” in lines 2-3. This limitation has unclear metes and bounds because the specification does not limit the term (see discussion of the phrase "the polynucleotide moves freely with respect to the detector" for claim 51) and one of ordinary skill in the art would not know what enzymes fall within the limitation. For the purpose of examination, this limitation is interpreted to include all examples of ultra-fast polynucleotide-handling enzyme given in the specification: “Suitable examples include translocases (e.g. DNA translocases) such as those in the FtsK and SpoIIIE families; packaging motors such as the phi29 packaging motor; and helicases and helicase-nucleases such as AddAB and RecBCD” (Specification, page 43 lines 1-4) (i.e. any translocase, packaging motor, helicase, or helicase-nuclease).
Claim Rejections - 35 USC § 112(d)
The following is a quotation of 35 U.S.C. 112(d):
(d) REFERENCE IN DEPENDENT FORMS.—Subject to subsection (e), a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.
The following is a quotation of pre-AIA 35 U.S.C. 112, fourth paragraph:
Subject to the following paragraph [i.e., the fifth paragraph of pre-AIA 35 U.S.C. 112], a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.
Claim 62 is rejected under 35 U.S.C. 112(d) or pre-AIA 35 U.S.C. 112, 4th paragraph, as being of improper dependent form for failing to further limit the subject matter of the claim upon which it depends, or for failing to include all the limitations of the claim upon which it depends. Claim 62 recites "A method according to claim 51, wherein the polynucleotide is single stranded or double stranded, followed by a clause "preferably [...] polynucleotide" in lines 2-8 and a clause "optionally [...] force" in lines 9-15. Because the clauses starting with preferably and optionally are both interpreted as being optional, they do not limit the scope of the claim. Claim scope is not limited by claim language that suggests or makes optional but does not require steps to be performed (MPEP §2111.04). The instant specification defines a polynucleotide as a nucleic acid ("nucleic acids, also referred to herein as "polynucleotides"" page 25 line 5) and nucleic acids as being single or double stranded ("The term "nucleic acid" as used herein, is a single or double stranded covalently-linked sequence of nucleotides" page 24 lines 28-29). Based on these definitions, the polynucleotide of claim 51 is interpreted as being single or double stranded. Therefore, claim 62's required limitation, that the polynucleotide is single stranded or double stranded, does not further limit the polynucleotide and claim 62 as a whole fails to further limit claim 51 on which it depends. Applicant may cancel the claim(s), amend the claim(s) to place the claim(s) in proper dependent form, rewrite the claim(s) in independent form, or present a sufficient showing that the dependent claim(s) complies with the statutory requirements.
Claim Rejections - 35 USC § 102
The following is a quotation of the appropriate paragraphs of 35 U.S.C. 102 that form the basis for the rejections under this section made in this Office action:
(a)(1) the claimed invention was patented, described in a printed publication, or in public use, on sale, or otherwise available to the public before the effective filing date of the claimed invention.
(a)(2) the claimed invention was described in a patent issued under section 151, or in an application for patent published or deemed published under section 122(b), in which the patent or application, as the case may be, names another inventor and was effectively filed before the effective filing date of the claimed invention.
Claims 51-53, 55, 59, 62, and 68 are rejected under 35 U.S.C. 102(a)(1) and (a)(2) as being anticipated by Reid et al. (US 2017/0233804, published 17 August 2017), herein Reid.
The applied reference appears to have a common assignee with the instant application. Based upon the earlier effectively filed date of the reference, it constitutes prior art under 35 U.S.C. 102(a)(2). This rejection under 35 U.S.C. 102(a)(2) might be overcome by: (1) a showing under 37 CFR 1.130(a) that the subject matter disclosed in the reference was obtained directly or indirectly from the inventor or a joint inventor of this application and is thus not prior art in accordance with 35 U.S.C. 102(b)(2)(A); (2) a showing under 37 CFR 1.130(b) of a prior public disclosure under 35 U.S.C. 102(b)(2)(B) if the same invention is not being claimed; or (3) a statement pursuant to 35 U.S.C. 102(b)(2)(C) establishing that, not later than the effective filing date of the claimed invention, the subject matter disclosed in the reference and the claimed invention were either owned by the same person or subject to an obligation of assignment to the same person or subject to a joint research agreement. However, should Reid be disqualified as 102(a)(2) prior art, Reid is still eligible as 102(a)(1) prior art due to being publicly available before the effective filing date of the claimed invention.
Regarding claim 51, Reid teaches a method of characterizing a polynucleotide in a sample (“method shown in FIG. 7 of controlling the biochemical analysis system 1 to analyse polymers” [0179], “the polymer may be a polynucleotide” [0100]), the method comprising: (i) contacting a detector with a polynucleotide (“In step C1, the biochemical analysis system 1 is operated by controlling the bias control circuit 30 to apply a bias voltage across the pore 32 of the sensor element 30 that is sufficient to enable translocation of polymer” [0180]); (ii a) taking measurements as a first part of the polynucleotide moves freely with respect to the detector under an applied force (“translocation is detected and a measurements start to be taken” [0180]; “Step C2 is performed when a polymer has partially translocated through the nanopore, i.e. during the translocation. At this time, the series of measurements taken from the polymer during the partial translocation is collected for analysis” [0195]; “to allow measurements to be taken as the polymer translocates through the pore 32, the rate of translocation can be controlled […] Suitable enzymes for controlling the rate of translocation of polynucleotides include […] helicases” [0134] as discussed in the 35 U.S.C. 112(b) rejection of claim 51 for indefiniteness of the limitation “moves freely” above, based on examples given in the specification a polynucleotide bound by a helicase is interpreted as moving freely with respect to a detector; “bias voltage across the pore” [0180] is the applied force); (ii b) assessing one or more properties of the first part of the polynucleotide (“In step C3, the chunk of measurements collected in step C2 is analysed. This analysis uses reference data 50 […] The analysis performed in step C3 provides a measure of similarity between (a) the sequence of polymer units of the partially translocated polymer from which measurements have been taken and (b) the one reference sequence” [0196]); (iii) (a) where the first part of the polynucleotide has one or more desired properties, controlling the movement of a second part of the polynucleotide with respect to the detector and taking measurements as the second part of the polynucleotide moves with respect to the detector to determine one or more characteristics of the polynucleotide, thereby characterizing the polynucleotide; or (b) where the first part of the polynucleotide does not have one or more desired properties, rejecting the polynucleotide (“In step C4, a decision is made responsive to the measure of similarity determined in step C3 either (a) to reject the polymer being measured, (b) that further measurements are needed to make a decision, or (c) to continue taking measurements until the end of the polymer” [0199]).
Regarding claim 52, Reid teaches a method according to claim 51 as discussed above wherein, when the first part of the polynucleotide does not have one or more desired properties, step (iii) comprises repeating steps (i), (ii a) and (ii b) with further polynucleotides from the sample until a polynucleotide having a first part having one or more desired properties is identified (“If the decision made in step C4 is (a) to reject the polymer being measured, then the method proceeds to step C5 wherein the biochemical analysis system 1 is controlled to reject the polymer, so that measurements can be taken from a further polymer” [0200]; “After ejection such ejection in step C5, the method returns to step C1” [0202]).
Regarding claim 53, Reid teaches a method according to claim 51 as discussed above, wherein said one or more desired properties are selected from the approximate length of the first part of the polynucleotide, the structure of the first part of the polynucleotide, and the composition of the first part of the polynucleotide (“The analysis performed in step C3 provides a measure of similarity between (a) the sequence of polymer units of the partially translocated polymer from which measurements have been taken and (b) the one reference sequence” [0196] the sequence is equivalent to the composition in the claim, this interpretation of “composition” in the claim is supported by the instant specification reciting “information on their composition such as their nucleotide sequence” on page 1 lines 27-28). The remaining limitations in claim 53 (“preferably […] nanopore” claim 53 lines 4-9) are within a “preferably” clause and therefore are interpreted as optional.
Regarding claim 55, Reid teaches a method according to claim 51 as discussed above, wherein determining one or more characteristics of the polynucleotide comprises determining the sequence of the polynucleotide (“(c) to continue taking measurements until the end of the polymer” [0199]; “Examples of various applications of the method shown in FIG. 7, and more generally in accordance with the first aspect of the present invention, will now be described” [0323]; “retained in the nanopore and sequenced in its entirety” [0326] describing the measurement taken in an example when a polymer is retained instead of rejected).
Regarding claim 59, Reid teaches a method according to claim 51 as discussed above, wherein in step (ii a) the first part of the polynucleotide moves freely with respect to the detector in a first direction relative to the applied force, and in step (iii) the polynucleotide binding protein controls the movement of the second part of the polynucleotide with respect to the detector in a second direction relative to the applied force (“In order to allow measurements to be taken as the polymer translocates through the pore 32, the rate of translocation can be controlled by a polymer binding moiety […] Where the polymer is a polynucleotide there are a number of methods proposed for controlling the rate of translocation including use of polynucleotide binding enzymes” [0134] teaches a polynucleotide binding protein controlling the movement; the additional limitations in step (ii a) of a first direction and step (iii) of a second direction do not further limit claim 51 as these directions are not defined in relation to each other and therefore could be the same or any different directions).
Regarding claim 62, Reid teaches a method according to claim 51 as discussed above, wherein the polynucleotide is single stranded or double stranded (“The polynucleotide may be single-stranded, be double-stranded” [0101]). The remaining limitations in claim 62 (“preferably […] force” claim 62 lines 2-15) are within “preferably” and “optionally” clauses and therefore are interpreted as optional.
Regarding claim 68, Reid teaches a method according to claim 51 as discussed above, wherein in step (ii a) the free movement of the polynucleotide with respect to the detector is governed by an ultra-fast polynucleotide-handling enzyme (“polynucleotide binding enzymes. Suitable enzymes for controlling the rate of translocation of polynucleotides include […] helicases” [0134] helicases are interpreted as ultra-fast polynucleotide-handling enzymes in view of the instant specification giving helicases as an example of ultra-fast polynucleotide-handling enzymes, see 35 U.S.C. 112(b) rejection of claim 68 for the limitation “an ultra-fast polynucleotide-handling enzyme”).
Therefore, claims 51-53, 55, 59, 62, and 68 are anticipated by Reid.
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.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
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 54, 57-58, 60, and 64-67 are rejected under 35 U.S.C. 103 as being unpatentable over Reid et al. (US 2017/0233804, published 17 August 2017), herein Reid, as applied to claims 51-53, 55, 59, 62, and 68, in view of Clarke et al. (in IDS filed 27 March 2023)(WO 2018/100370, published 7 June 2018), herein Clarke.
Regarding claim 54, Reid teaches a method according to claim 51 as discussed in the 35 U.S.C. 102 rejection above, wherein the polynucleotide is a double-stranded polynucleotide (“The polynucleotide may be […] double-stranded” [0101]). Additionally, Reid teaches their method possesses the advantage of improved speed and efficiency by reducing the amount of wasted data acquisition spent on polymers/polynucleotides that are not of interest (“The method potentially provides a significantly faster time to result wherein continued measurement is carried out only on those polymers determined to be of interest and those determined to be not of interest are rejected. This advantage of reducing the amount of wasted data acquisition is particularly significant in applications where a large amount of data acquisition is required” [0011]). However, Reid does not teach that the polynucleotide comprises a first strand connected to a second strand by a hairpin or hairpin adapter. The remaining limitations of claim 54 (“preferably […] adapter” claim 54 lines 3-4) are in a “preferably” clause and therefore are interpreted as optional.
Regarding claim 54, Clarke teaches a method of characterizing a double stranded polynucleotide comprising a first strand connected to a second strand by a hairpin by translocating the polynucleotide through a nanopore detector (“Figures 1A-1B illustrate a prior art method of sequencing a double stranded polynucleotide (e.g., DNA) construct, in which the template and complement strands are attached via a hairpin loop and the template strand comprises a 5' leader sequence, using a transmembrane pore. Figure 1A is a schematic representation of the polynucleotide (e.g., DNA) construct translocating through a nanopore under the control of an enzyme” page 11 lines 12-16; Figure 1A). Additionally, Clarke teaches that the method they teach provide the advantage of higher confidence measurements by measuring both complementary strands (“Measurement of both strands in this way is advantageous as information from the two complementary linked strands can be combined and used to provide higher confidence observations than may be achieved from measurement of template strands only” page 2 lines 6-8).
Regarding claim 57, Clarke teaches a method of characterizing a polynucleotide wherein an adapter is attached to one or both ends of the polynucleotide prior to contacting a detector with the polynucleotide and wherein the polynucleotide is characterized by translocating the polynucleotide through a nanopore detector (“Figures 2A-B illustrate a method of "follow-on" sequencing a double stranded polynucleotide (e.g., DNA) construct without the use of a hairpin according to one embodiment described herein. Both the template and complement polynucleotide (e.g., DNA) strands comprise an adaptor at each end, which adaptor comprises a leader sequence. Figure 2A is a schematic representation of a double stranded polynucleotide (e.g., DNA) construct translocating through the nanopore under enzyme control” page 11 lines 24-29; Figure 2A). The remaining limitations of claim 57 (“preferably […] polynucleotide” claim 57 lines 2-5) are in a “preferably” clause and therefore are interpreted as optional.
Regarding claim 58, Clarke teaches a method of characterizing a polynucleotide wherein prior to contacting a detector with the polynucleotide a polynucleotide binding protein capable of controlling the movement of the polynucleotide is stalled on the polynucleotide or on an adapter attached to the polynucleotide (“In some embodiments, the adaptor described herein can comprise one or more spacers to prevent pre-bound polynucleotide binding protein (e.g., a polynucleotide unwinding enzyme) from moving along and unwinding a double stranded polynucleotide. These spacers prevent further movement of the polynucleotide binding protein (e.g., a polynucleotide unwinding enzyme) until the polynucleotide binding protein (e.g., a polynucleotide unwinding enzyme) is located at the pore and a potential difference is applied across the pore. The additional force provided by the potential difference pushes the polynucleotide binding protein (e.g., a polynucleotide unwinding enzyme) over the spacers and allows it to unwind and control movement of the polynucleotide through the nanopore” page 47 line 30 - page 48 line 3).
Regarding claim 60, Clarke teaches a method of characterizing a polynucleotide wherein a detector is contacted with a first end of the polynucleotide or an adapter attached to the first end of the polynucleotide and a polynucleotide binding protein is bound to a second end of the polynucleotide or to an adapter attached to the second end of the polynucleotide (“In some embodiments, an adaptor is attached to one or both of the two ends of the double-stranded polynucleotide […] In some embodiments, a polynucleotide unwinding enzyme is prebound to one or each of both adaptors” page 5 lines 17-18 and 27-28; Figure 2A, in the leftmost frame polynucleotide binding proteins are attached to each end of the polynucleotide and in the second frame one of the ends is contacted with and translocating through a nanopore detector).
Regarding claim 64, Clarke teaches a method of characterizing a polynucleotide wherein the polynucleotide comprises a portion which is complementary to a tag sequence (“binding the capture sequence of the adaptor that is attached to the second strand to the tag (e.g., a capture polynucleotide)” page 13 lines 6-7, Figures 9 and 10).
Regarding claim 65, Clarke teaches a method of characterizing a polynucleotide wherein the polynucleotide comprises a portion which is complementary to a tag sequence and wherein the polynucleotide is double stranded and the portion which is complementary to a tag sequence is a portion of the first strand of the polynucleotide and wherein the second strand hybridizes to the tag sequence as the first strand moves with respect to the detector (“A system for characterizing a polynucleotide […] at least one nucleic acid having sequence complementarity with a portion of the adaptor. […] An adaptor may be attached at each of two ends of the polynucleotide, wherein each adaptor comprises a duplex stem and a first single strand extending from the duplex stem. The first single strand of one adaptor may be contiguous with the template strand and the first single strand of the other adaptor may be contiguous with the complement strand” page 7 line 3 - page 8 line 5, the at least one nucleic acid having sequence complementarity with a portion of the adapter is the tag sequence; Figure 10).
Regarding claim 66, Clarke teaches a method of characterizing a polynucleotide wherein prior to contacting a detector with the polynucleotide a polynucleotide binding protein is stalled on the polynucleotide and wherein there is a step comprising the destalling of the polynucleotide binding protein (“In some embodiments, the adaptor described herein can comprise one or more spacers to prevent pre-bound polynucleotide binding protein (e.g., a polynucleotide unwinding enzyme) from moving along and unwinding a double stranded polynucleotide. These spacers prevent further movement of the polynucleotide binding protein (e.g., a polynucleotide unwinding enzyme) until the polynucleotide binding protein (e.g., a polynucleotide unwinding enzyme) is located at the pore and a potential difference is applied across the pore. The additional force provided by the potential difference pushes the polynucleotide binding protein (e.g., a polynucleotide unwinding enzyme) over the spacers and allows it to unwind and control movement of the polynucleotide through the nanopore” page 47 line 30 - page 48 line 3).
Regarding claim 67, Clarke teaches a method of characterizing a polynucleotide wherein prior to contacting a detector with the polynucleotide a polynucleotide binding protein is stalled on the polynucleotide, wherein there is a step comprising the destalling of the polynucleotide binding protein, and wherein destalling the polynucleotide binding protein comprises applying a destalling force to the polynucleotide wherein said destalling force is of opposite direction to the force applied in the step of taking measurements as a first part of the polynucleotide is translocated through a nanopore under an applied force (“In some embodiments, the adaptor described herein can comprise one or more spacers to prevent pre-bound polynucleotide binding protein (e.g., a polynucleotide unwinding enzyme) from moving along and unwinding a double stranded polynucleotide. These spacers prevent further movement of the polynucleotide binding protein (e.g., a polynucleotide unwinding enzyme) until the polynucleotide binding protein (e.g., a polynucleotide unwinding enzyme) is located at the pore and a potential difference is applied across the pore. The additional force provided by the potential difference pushes the polynucleotide binding protein (e.g., a polynucleotide unwinding enzyme) over the spacers and allows it to unwind and control movement of the polynucleotide through the nanopore” page 47 line 30 - page 48 line 3; Figure 2A, in the second panel to the left the leftmost binding protein is stalled on the second strand/part while the first strand/part is translocated through the nanopore under an applied force indicated by the downward arrow, in the fourth panel to the left the binding protein has been destalled, which the quoted text describes as involving the binding protein being pushed over the spacers, which in the figure is the upward direction).
It would have been obvious to one of ordinary skill in the art prior to the effective filing date of the claimed invention to combine the method of characterizing a polynucleotide taught by Reid with the method of characterizing a polynucleotide taught by Clarke in view of the advantages taught Reid and Clarke for each method. One of ordinary skill in the art would recognize that Clarke’s method, while providing the advantage of higher confidence observations, requires a longer time to characterize both strands instead of just one, so they would be motivated to combine it with methods that reduce the time taken, which Reid provides by rejecting undesired polynucleotides before characterizing their full length. One of ordinary skill in the art would have a reasonable expectation of success because both methods are modifications of the same general method of nanopore sequencing and because the sequencing of a template strand and then its complement as taught by Clarke would not impair the ability for the polynucleotide to be rejected as taught by Reid or vice versa. Therefore, the invention as a whole of claims 54, 57-58, 60, and 64-67 would have been prima facie obvious to one of ordinary skill in the art prior to the effective filing date of the claimed invention.
Claim 61 is rejected under 35 U.S.C. 103 as being unpatentable over Reid et al. (US 2017/0233804, published 17 August 2017), herein Reid, as applied to claims 51-53, 55, 59, 62, and 68, in view of Rand (US 2018/0030525, published 1 February 2018).
Regarding claim 61, Reid teaches a method according to claim 51 as discussed in the 35 U.S.C. 102 rejection above. However, Reid does not teach that step (i) comprises contacting the detector with a leader sequence at the first end of the polynucleotide and the polynucleotide binding protein is stalled at a second end of the polynucleotide or on an adapter attached to the second end of the polynucleotide; and b) the first part of the polynucleotide is the part between the leader sequence and the polynucleotide binding protein and the second part of the polynucleotide is the same as the first part of the polynucleotide; and c) the polynucleotide binding protein is orientated on the polynucleotide such that the polynucleotide binding protein controls the movement of the second part of the polynucleotide with respect to the detector against the applied force. This deficiency is made up for in the teachings of Rand.
Regarding claim 61, Rand teaches a method of characterizing a polynucleotide using a nanopore detector (“method for analyzing properties of a target portion polynucleotide using a nanopore in a membrane separating a cis fluid area from a trans fluid area” [0018]) comprising contacting a detector with the polynucleotide (“adding to the cis fluid side a polynucleotide to be analyzed” [0018]), wherein the polynucleotide comprises a leader sequence at one end of the polynucleotide (“FIG.3 […] 310 on the other side of the folded sequence, showing an enzyme displacement sequence; and adjacent to that is a thread threading sequence 314” [0059]; FIG. 3, 310 and 314 together are equivalent to the claimed leader sequence) and a polynucleotide binding protein is stalled at another end of the polynucleotide (“FIG. 3 […] 304 showing a folded sequence (e.g. GQ) adjacent to the strand of interest; 306 showing an enzyme binding sequence”; FIG. 3; “if the folded portion 304 is in place, the enzyme will bind to the strand, but will not move forward past the folded portion, when it is folded” [0062]), the part of the polynucleotide that is characterized first is the part between the leader sequence and the polynucleotide binding protein and the part of the polynucleotide that is characterized second is the same as the part of the polynucleotide that is characterized first, and the polynucleotide binding protein is orientated on the polynucleotide such that the polynucleotide binding protein controls the movement of the second part of the polynucleotide with respect to the detector against the applied force (“The enzyme ratchets the DNA against the voltage, allowing sequencing of the strand as it passes through the nanopore, and the GQ refolds behind the helicase […] the electric potential pulls the strand to the starting position at the GQ and the read starts over” [0058], by having the read start over, the same part is characterized again, so the first and second parts characterized are the same; see FIG. 1 and [0058] in its entirety for full cycle and its ability to repeat).
It would have been obvious to one of ordinary skill in the art prior to the effective filing date of the claimed invention to combine the method of characterizing a polynucleotide taught by Reid with the method of characterizing a polynucleotide taught by Rand in view of the advantages taught Reid and Rand for each method. One of ordinary skill in the art would recognize that Rand’s method, while providing the advantage “the advantage of removing stochastic undesired enzyme behavior by comparing reads of the same molecule” (Rand [0049]), requires a longer time to repeatedly characterize a molecule instead of characterizing it just once, so they would be motivated to combine it with methods that reduce the time taken, which Reid provides by rejecting undesired polynucleotides before characterizing their full length. One of ordinary skill in the art would have a reasonable expectation of success because both methods are modifications of the same general method of nanopore sequencing and because the sequencing of a target strand multiple times as taught by Rand would not impair the ability for the polynucleotide to be rejected as taught by Reid or vice versa. The first cycle of sequencing in Rand would constitute the initial measurement of a sequence to compare with a reference sequence as taught by Reid. Therefore, the invention as a whole of claim 61 would have been prima facie obvious to one of ordinary skill in the art prior to the effective filing date of the claimed invention.
Claims 56 and 63 are rejected under 35 U.S.C. 103 as being unpatentable over Reid et al. (US 2017/0233804, published 17 August 2017), herein Reid, in view of Rand (US 2018/0030525, published 1 February 2018) as applied to claim 61 above and further in view of Maglia et al. (US 2018/0364214, published 20 December 2018), herein Maglia.
Regarding claim 56, Reid teaches a method according to claim 51, as discussed in the 35 U.S.C. 102 rejection above, and Rand teaches contacting a detector with a polynucleotide having a polynucleotide binding protein capable of controlling the movement of the polynucleotide stalled thereon, as discussed in the 35 U.S.C. 103 rejection of claim 61 above. However, neither Reid nor Rand teach determining the time taken for a first part of the polynucleotide to move freely with respect to the detector under an applied force or determining the approximate length of the first part of the polynucleotide. This deficiency is made up for in the teachings of Maglia.
Regarding claim 56, Maglia teaches a method of characterizing a polynucleotide wherein the length of the polynucleotide is determined (“a method of characterizing a target polynucleotide […] examples of the characteristics of the target polynucleotides that can be determined using the methods described herein include (i) the length of the target polynucleotide” [0017]) by determining the time taken for the polynucleotide to move freely with respect to the detector under an applied force (“the length of the polynucleotide may be measured for example by determining […] the duration of interaction between the polynucleotide and the pore” [0132]; “measuring, during application of a potential across the nanopore” [0017]; “the rate of translocation of the double stranded DNA analyte may be controlled by a double stranded DNA translocase such as FtsK” [0074] as FtsK is one of the example ultra-fast polynucleotide-handling enzymes on page 43 of the instant specification, this indicates that the polynucleotide was capable of moving freely as discussed in the 35 U.S.C. 112(b) rejection of claim 56). Therefore, in the combination of Reid, Rand, and Maglia, step B in Figure 1 of Rand represents the movement of the polynucleotide as in step (ii a) of the claim, which Maglia teaches could be used to assess the length of the polynucleotide based on the amount of time taken, wherein the method of Reid would decide whether to reject the polynucleotide or continue with the characterization of the sequence as in step C and onward in Figure 1 of Rand.
Regarding claim 63, Reid teaches a method according to claim 51, as discussed in the 35 U.S.C. 102 rejection above, wherein the polynucleotide is double stranded (“The polynucleotide may […] be double-stranded” [0101]).
Regarding claim 63, Maglia teaches a method of characterizing a polynucleotide wherein the polynucleotide is double stranded (“the target polynucleotide can be […] double-stranded” [0018]), comprises a leader sequence (“attaching to the polynucleotide a leader sequence which preferentially threads into the pore” [0164]) and a hairpin adapter connecting the two strands (“a target double stranded polynucleotide is provided with a hairpin loop adaptor at one end and the method comprises contacting the polynucleotide with any one of the modified ClyA nanopores described herein such that both strands of the polynucleotide move through the pore” [0163]). As the leader sequence is taught to thread into the pore and the hairpin adapter to link the translocation of one strand to the following translocation of the other strand, the relationship of the leader to the hairpin adapter is that the leader is at a first end of a first strand and the hairpin adapter connects a second end of the first strand to a first end of a second strand.
Regarding claim 63, Rand teaches a method of characterizing a polynucleotide wherein a polynucleotide binding protein is stalled at the end of a strand of polynucleotide that will translocate through a nanopore wherein it is on the opposite end of the strand as a leader/threading sequence (“FIG. 3 […] 304 showing a folded sequence (e.g. GQ) adjacent to the strand of interest; 306 showing an enzyme binding sequence”; FIG. 3; “if the folded portion 304 is in place, the enzyme will bind to the strand, but will not move forward past the folded portion, when it is folded” [0062]; FIG. 3, threading sequence 314 and enzyme binding sequence 306 are opposite, portion of interest to be sequenced 302 between them). Therefore, in combination with the teachings of Reid and Maglia discussed in the two paragraphs above, Rand teaches the polynucleotide binding protein stalled at the second end of the second strand, since that is the end that is opposite to the end with the leader sequence due to the hairpin adapter connecting the two strands.
It would have been obvious to one of ordinary skill in the art prior to the effective filing date of the claimed invention to perform the simple substitution of the length of a polynucleotide taught by Maglia for the sequence taught by Reid as the measure of similarity assessed in the combination of Reid and Rand to decide whether to reject or further characterize a polynucleotide (see 35 U.S.C. 103 rejection of claims 61 above for reasons the combination of Reid and Rand would have been obvious). One of ordinary skill in the art would be able to substitute one analysis (the analysis of duration of interaction taught by Maglia) for another analysis (the analysis of sequence taught by Reid) that both are calculated from fundamentally the same measurements (measurements of ion flow through the nanopore: “measuring, during application of a potential across the nanopore, ion flow through the modified ClyA nanopore” Maglia [0017]; “measurements of the ion current flowing through the pore” Reid [0133]). One of ordinary skill in the art would have a reasonable expectation of success because both methods are applied to the characterization of polynucleotides translocating through a nanopore and both methods are based on similar measurements of ion flow through the nanopore. Therefore, the invention as a whole of claims 56 and 63 would have been prima facie obvious to one of ordinary skill in the art prior to the effective filing date of the claimed invention.
Conclusion
Claims 51-68 are rejected. Claims 69-70 are withdrawn.
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/JEFFREY BELLAH/Examiner, Art Unit 1683
/ANNE M. GUSSOW/Supervisory Patent Examiner, Art Unit 1683