Office Action 2: Notice of Allowance (2025-03-17)

CLAIMS:
IN THE CLAIMS

1-18. (Cancelled)217

19\. (Currently Amended) A method for enriching a double-stranded target polynucleotide in a sample to obtain an amplification product, the method comprising the steps of:(a) preparing a reaction mixture comprising:
(i) the sample, wherein the sample comprises a double-stranded reference polynucleotide and the double-stranded target polynucleotide, wherein the target polynucleotide comprises a genetic alteration and the reference polynucleotide lacks the genetic alteration, wherein the genetic alteration is a substitution of one or more nucleotides, a deletion of one or more nucleotides, and/or an insertion of one or more nucleotides;
(ii) a DNA polymerase that lacks 5'->3' nuclease activity and comprises 3'45' nuclease activity;
(iii) a blocking oligonucleotide that has a length of 8-20 nucleotides, consists of locked nucleic acids (LNAs) comprising a sugar moiety having a 2'-O conjugated to 4'-C via a methylene group, and is complementary to the reference polynucleotide and not complementary to the target polynucleotide;
(iv) a pair of oligonucleotide primers that flank the genetic alteration, wherein a forward primer of the primer pair is capable of hybridizing to a first strand of the double-stranded reference polynucleotide and to a first strand of the double-stranded target polynucleotide, andwherein a reverse primer of the primer pair is capable of hybridizing to a second strand of the double-stranded reference polynucleotide and to a second strand of the double-stranded target polynucleotide,
wherein the forward and reverse primers of the primer pair flank the genetic alteration in the target polynucleotide, and

wherein the forward and reverse primers of the primer pair do not overlap with the sequence complementary to the blocking oligonucleotide;
   ;
(b) heating the reaction mixture above a temperature to melt the double stranded reference polynucleotide and the double stranded target polynucleotide;
(c) cooling the reaction mixture to a temperature wherein the blocking oligonucleotide hybridizes to the reference polynucleotide to form a blocking duplex,but wherein the blocking oligonucleotide does not hybridize to the target polynucleotide, and wherein the forward and reverse primers of the primer pair do not hybridize to the reference polynucleotide or to the target polynucleotide ;
(d) cooling the reaction mixture, wherein the forward primer hybridizes to the reference polynucleotide and the reverse primer hybridizes with the reference polynucleotide, and wherein the forward primer hybridizes with the target polynucleotide and the reverse primer hybridizes with the target polynucleotide;
(e) extending the primers hybridized to the target polynucleotide or to the reference polynucleotide; and
(f) repeating steps (b) through (e) for two or more cycles to form the amplification product containing target polynucleotides, thereby enriching thequantity of target polynucleotides relative to the reference polynucleotides in the amplification product as compared to the sample.

20\. (Cancelled)

21\. (Previously Presented) The method of claim 19, wherein the DNA polymerase has an error rate of less than 10-5.

22-23. (Cancelled)

24\. (Previously Presented) The method of claim 19, wherein the blocking oligonucleotide comprises one or more modified nucleotides that enhance the selectivity of the blocking oligonucleotide for hybridizing to the reference polynucleotide relative to the target polynucleotide.

25-26. (Cancelled)

27\. (Previously Presented) The method of claim 19, wherein a DNA binding fluorophore is added to the mixture.

28\. (Cancelled)

29\. (Previously Presented) The method of claim 19, wherein an oligonucleotide probe is added to the mixture.

30\. (Cancelled)

31\. (Previously Presented) The method of claim 19, further comprising (g) sequencing amplification product containing target polynucleotides.

32\. (New) A method for enriching a double-stranded target polynucleotide in a sample to obtain an amplification product, the method consisting of the steps of:(a) preparing a reaction mixture comprising:
(i) the sample, wherein the sample comprises a double-stranded reference polynucleotide and the double-stranded target polynucleotide, wherein the

target polynucleotide comprises a genetic alteration and the reference polynucleotide lacks the genetic alteration, wherein the genetic alteration is a substitution of one or more nucleotides, a deletion of one or more nucleotides, and/or an insertion of one or more nucleotides;
(ii) a DNA polymerase that lacks 5'-43' nuclease activity and comprises 3'-45' nuclease activity;
(iii) a blocking oligonucleotide that has a length of 8-20 nucleotides, consists of locked nucleic acids (LNAs) comprising a sugar moiety having a 2'-O conjugated to 4'-C via a methylene group, and is complementary to the reference polynucleotide and not complementary to the target polynucleotide;
(iv) a pair of oligonucleotide primers that flank the genetic alteration, wherein a forward primer of the primer pair is capable of hybridizing to a first strand of the double-stranded reference polynucleotide and to a first strand of the double-stranded target polynucleotide, and
wherein a reverse primer of the primer pair is capable of hybridizing to a second strand of the double-stranded reference polynucleotide and to a second strand of the double-stranded target polynucleotide,
wherein the forward and reverse primers of the primer pair flank the genetic alteration in the target polynucleotide, and wherein the forward and reverse primers of the primer pair do not overlap with the sequence complementary to the blocking oligonucleotide;
(b) heating the reaction mixture above a temperature to melt the double stranded reference polynucleotide and the double stranded target polynucleotide;
(c) cooling the reaction mixture to a temperature wherein the blocking oligonucleotide hybridizes to the reference polynucleotide to form a blocking duplex,
but wherein the blocking oligonucleotide does not hybridize to the target polynucleotide, and wherein the forward and reverse primers of the primer pair do not hybridize to the reference polynucleotide or to the target polynucleotide

(d) cooling the reaction mixture, wherein the forward primer hybridizes to the reference polynucleotide and the reverse primer hybridizes with the reference polynucleotide, and wherein the forward primer hybridizes with the target polynucleotide and the reverse primer hybridizes with the target polynucleotide;
(e) extending the primers hybridized to the target polynucleotide or to the reference polynucleotide;
(f) repeating steps (b) through (e) for two or more cycles to form the amplification product containing target polynucleotides, thereby enriching the quantity of target polynucleotides relative to the reference polynucleotides in the amplification product as compared to the sample; and optionally (g) sequencing amplification product containing target polynucleotides.

OFFICE 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 .
REASONS FOR ALLOWANCE
The following is an examiner’s statement of reasons for allowance:
A. The terminal disclaimer filed on 12 February 2025 disclaiming the terminal portion of any patent granted on this application which would extend beyond the expiration date of any patent granted on Application Number 18/691,325 has been reviewed and is accepted.  The terminal disclaimer has been recorded.
B. The previous rejections of the claims under 35 U.S.C. 112(b) have been obviated by the amendments to the claims.
C. The previous rejections of the claims under 35 U.S.C. 103 as unpatentable over Makrigiorgos in view of Huang, as evidenced by Cline and over Makrigiorgos in view of Huang, as evidenced by Cline, and further in view of Albitar have been obviated by the amendments to the claims and in view of Applicant’s arguments.
In particular, the teachings of Makrigiorgos are discussed in detail in the Office action of 18 November 2024. As noted in Applicant’s response, Makrigiorgos teaches a method of “COLD-PCR” in which four distinct temperatures are cycled. Mauger (Mol Diagn Ther. 2017. 21: 269-283) provides a review of full-COLD-PCR, fast-COLD-PCR, ice-COLD-PCR, E-ice-COLD-PCR and TT-COLD-PCR). See Figure 1a-d of Mauger below:
PNG
media_image1.png
798
728
media_image1.png
Greyscale
In these methods of PCR, when the temperature is reduced following the denaturing step, the blocking oligonucleotide hybridizes to both the wildtype reference nucleic acid and the target nucleic acid comprising a mutation.
Further, Makrigiorgos teaches blocking oligonucleotides that include LNAs (e.g., para [0018]). The cited prior art of Huang also teaches blocking oligonucleotides that include LNAs. Huang exemplifies only LNA-DNA chimera blocking oligonucleotides in which only 6 of  the 13 nucleotides are locked nucleotides. Further, Nelson et al (WO 2021050100) discloses blocking oligonucleotides in which each of the nucleotides is a locked nucleotide (e.g., para [0418] – i.e., “The blocking oligonucleotide is a locked nucleic acid (LNA) and all nucleotides of the blocking oligonucleotide are locked nucleotides”).
The advantage of the higher melting temperature of a blocking oligonucleotide consisting of only locked nucleotides, and the resulting greater melting temperature differential of blocking oligonucleotides consisting of only locked nucleotides, as it relates to the claimed amplification methods,  is discussed in detail in Applicant’s response of 12 February 2025 (p. 13-15).
The prior art does not teach or suggest the presently claimed method which requires, in part, a blocking oligonucleotide that consists of locked nucleic acids (LNAs) comprising a sugar moiety having a 2'-O conjugated to 4'-C via a methylene group, and is complementary to the reference polynucleotide and not complementary to the target polynucleotide, which method has 3 functional temperature cycles, including, following a step of heating the reaction mixture to melt / denature double-stranded reference polynucleotides and double-stranded target polynucleotides, a step of cooling the reaction mixture to a temperature wherein the blocking oligonucleotide hybridizes to the reference polynucleotide to form a blocking duplex, but wherein the blocking oligonucleotide does not hybridize to the target polynucleotide, and wherein the forward and reverse primers of the primer pair do not hybridize to the reference polynucleotide or to the target polynucleotide.
Any comments considered necessary by applicant must be submitted no later than the payment of the issue fee and, to avoid processing delays, should preferably accompany the issue fee.  Such submissions should be clearly labeled “Comments on Statement of Reasons for Allowance.”

APPLICANT REBUTTAL: REMARKS
Applicant respectfully requests entry of the amendments and consideration of the remarks submitted herein. Support for the amendments to claim 19 and new claim 31 can be found throughout the specification as originally filed.
For example, support for the blocking oligonucleotide consisting of locked nucleic acids (LNAs) comprising a sugar moiety having a 2'-O conjugated to 4'-C via a methylene group can be found in original claims 25 and 26.
For example, support for the blocking oligonucleotide having a length of 8-20 nucleotides can be found in original claim 9.
For example, support for the target polynucleotide comprising a genetic alteration and the reference polynucleotide lacking the genetic alteration can be found in  0027,  0043 and  0060 of the published PCT application WO 2023/039228. As discussed in the specification at  0027, a "reference oligonucleotide" is also called a "non-target oligonucleotide," which may include a "wild-type oligonucleotide." A "target oligonucleotide" is an oligonucleotide that contains one or more genetic alterations at one or more positions as compared to the reference oligonucleotide (i.e., is a "mutant" oligonucleotide). A mutant (i.e., "target") oligonucleotide is distinguishable from the reference (i.e., "non-target") oligonucleotide based on this genetic alteration. Support for the feature that the genetic alteration is a substitution of one or more nucleotides, a deletion of one or more nucleotides, and/or an insertion of one or more nucleotides can be found in original claims 15-17 and in the specification at  0035.
For example, support for the blocking oligonucleotide selectively forming a duplex with the reference polynucleotide as compared to the target polynucleotide (i.e., at a higher temperature) can be found at  0054-0057.
For example, support for the elements that the primers are used to amplify the target and reference polynucleotides, and that the primer oligonucleotides flank the position where the blocking oligonucleotide hybridizes can be found in the specification at  0056. Support for the presence of forward and reverse primers in a primer pair can be found in the specification at
0082-0084 and 0087-0088.
For example, support for the phrase that the blocking oligonucleotide is complementary to the reference polynucleotide and not complementary to the target polynucleotide can be found at  0039.
In claim 19, steps (b) and (c) have been amended to have proper antecedent basis.
No new matter is added by these amendments or new claims.
Rej
ection under 35 U.S.C.
§
112(b) or 35 U.S.C.
§
p
re-AIA)
,
Second Para
graph
Claims 1, 3, 6-9, 11-15, 19, 21, 24-27 and 29-31 are rejected under 35 U.S.C. §112(b) or 35 U.S.C. § 112 (pre-AIA), second paragraph, as allegedly 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. Applicant respectfully traverses for at least the following reasons.
A.
Claims 1, 3, 6-9, 11-15, 19, 21, 24-27 and 29-31 allegedly are indefinite over the
recitation of "the reference polynucleotide" at (ii) of claims 1 and 19 because this phrase lacks proper antecedent basis. The Office Action indicates that claims 1 and 19 should be amended at
(ii) to recite "a reference polynucleotide." Applicant presumes that the Office Action intended to refer to "(iii)" in claims 1 and 19 rather than "(ii)," because "(iii)" and not "(ii)" recites "the reference polynucleotide." If Applicant's understanding of the rejection is incorrect, Applicant respectfully requests clarification in a further non-final Office Action.
Claim 1 has been cancelled, thereby rendering this rejection moot with respect to this claim.
Claim 19 at (i) has been amended to recite that the sample "a double-stranded reference polynucleotide and the double-stranded target polynucleotide" thereby providing antecedent basis for recitations of "the reference polynucleotide" later in the claim.
B.
Claims 1, 3, 6-9, 11-15, 19, 21, 24-27 and 29-31 are indefinite over the recitation
of "the genetic alteration" at (iv) of claims 1 and 19 because this phrase lacks proper antecedent basis.
As indicated above, claims 1 has been cancelled. Claim 19 at (i) has been amended to recite that "the target polynucleotide comprises a genetic alteration" thereby providing antecedent basis for the recitation of "the genetic alteration" later in the claim.
The Office Action further indicates that it is not clear as to whether the genetic alteration that the pair of primers flanks is the same as or different from the genetic alteration in the target
polynucleotide. Claim 19 has been amended to clarify that the "genetic alternation" that the pair of primers flank, is the same "genetic alteration" in the target polynucleotide.
The Office Action indicates that the claims are also vague and indefinite over the
recitation that the primers do not interfere with the hybridization of the blocking oligonucleotide per se. As suggested by the Office Action, claim 19 has been amended at (iv) to clarify that the pair of primers flanks the genetic alteration in the target polynucleotide and the pair of primers does not overlap with the sequence complementary to the blocking oligonucleotide.
The Office Action indicates that claims 30 and 31 are indefinite over the recitations of "sequencing the amplification product containing target polynucleotides" (claim 30) and "sequencing amplification product containing target polynucleotides" (claim 31). The Office Action states that these phrases lack proper antecedent basis because, while the claims previously refer to target polynucleotides, the claims do not previously refer to amplification products and particularly amplification products containing (any) target polynucleotides. Claim 19 has been amended to clarify that the amplification product contains target polynucleotides, thereby providing antecedent basis for the recitations of "sequencing the amplification product containing target polynucleotides" in claim 31. Claim 30 has been cancelled.
Claim 1 has been cancelled, as have claims 3, 6-9, 11-15, 25-26 and 30, which depended from claim 1. Applicant respectfully asserts that the amendments to claim 19 discussed above address these indefiniteness rejections. Claims 21, 24, 27, 29 and 31 depend from claim 19. Applicant respectfully asserts that these amendments also address the indefiniteness rejections as applied to the dependent claims, and requests that these rejections be withdrawn.
Double Patenting
Claims 1, 3, 6-9, 11-15, 19, 21, 24-26 and 30-31 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1-18 and 20-28 of copending Application No. 18/691,325 (reference application).
Claims 27 and 29 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1-18 and 20-28 of copending Application No. 18/691,325 (reference application) in view of Makrigiorgos, G. (U.S. 20110217714).
Terminal disclaimers are submitted herewith. Applicant, therefore, respectfully requests withdrawal of these nonstatutory double patenting rejections.
Rej
ection under 35 U.S.C.
&
Claims 1, 3, 6, 7, 9, 11-15, 19, 21, 24, 25, 27 and 29-31 are rejected under 35 U.S.C. § 103 as allegedly being unpatentable over Makrigiorgos, G. (U.S. 20110217714, hereinafter "Makrigiorgos") in view of Huang et al. (Molecular and Cellular Probes. 2010. 24: 376-380, hereinafter "Huang"), as evidenced by Cline et al. (Nucleic Acids Research. 1996. 24(18): 3546- 3551, "Cline"). Claims 1, 3, 6, 7, 9, 11-15, 25 and 30 have been cancelled. Claim 19 has been amended. Claims 21, 24, 27, 29 and 31 depend from claim 19. Applicant respectfully traverses for at least the following reasons.
A rejection of obviousness under 35 U.S.C. § 103 requires that the Examiner establish a prima facie case of obviousness. To establish a prima facie case of obviousness, the Examiner has the burden to establish three basic elements. First, the Examiner must establish that there is some suggestion or motivation, either in the cited documents themselves or in the knowledge generally available to an art worker, to modify the documents or to combine document teachings so as to arrive at the claimed invention. Second, the Examiner must establish that there is a reasonable expectation of success. Finally, the Examiner must establish that the cited documents teach or suggests all the claim limitations. M.P.E.P. § 2143. Applicant respectfully submits that the Examiner has not demonstrated that the claims are prima facie obvious in view of the cited documents, for example, because the cited documents do not teach or suggest the motivation to modify the documents or to combine the document teachings so as to arrive at the claimed invention, or to teach all the elements of the claimed invention.
Makrigiorgos teaches a different method of enriching nucleic acids as compared to the presently claimed invention. Makrigiorgos teaches a variation on the method called "co- amplification at Lower denaturation" temperature ("COLD PCR"). Makrigiorgos at  [0010]. Their method is depicted in FIGS. 1 and 2, and is described at   [0011]-[0018] and in claim 1. COLD PCR has
four
functional temperatures, which are cycled. Each temperature has a defined and distinct function. The present invention is performing a distinct thermal cycling process using only
t
hr
ee
functional temperatures. Makrigiorgos, therefore, does not teach all the elements of the claimed invention.
Briefly, in the Makrigiorgos method, a reaction mixture is subjected to the four functional temperatures as follows:
Subjecting the reaction mixture suspected of having said target sequence to a first
denaturing temperature that is above the melting temperature (Tm) of the reference
sequence so as to form denatured reference strands and denatured target strands;
Reducing the temperature of the reaction mixture so as to permit the formation of
heteroduplexes of the reference blocking sequencing and the complementary
reference strand and heteroduplexes of the reference blocking sequence and target
strands;
Subjecting said reaction mixture to a critical temperature (Tc) sufficient to permit
preferential denaturation of said heteroduplexes of the reference blocking
sequence and target strands, yet insufficient to denature heteroduplexes of the
reference blocking sequence and the complementary refence strand; and
Reducing the temperature of the reaction mixture so as to permit said primer pair
to anneal to free target strands and any free reference strands in the reaction
mixture.
The first step in Makrigiorgos is similar to that of the present invention, namely the sample mixture is heated so that the double-stranded polynucleotides melt (denature).
The two methods, however, diverge at the second step. Makrigiorgos teaches that in the second step of their method, the functional temperature needs to be such that heteroduplexes are created between the blocker and
reference,
and also for heteroduplexes to form between the blocker and the
t
arget.
The present invention does not perform this step of forming heteroduplexes between the blocker and the target polynucleotides. In the present invention, claim 19, step (c) recites that the temperature is reduced to a temperature where the blocking oligonucleotide is in a duplex with the
reference
polynucleotide, but
n
ot
the
t
arget
polynucleotide (or the primers). The present invention has no requirement, condition, or need to create a heteroduplex between the blocker and the target strand.
Creating a duplex between the blocker and the target polynucleotide, as described by Makrigiorgos, can be very detrimental to the goal of selective amplification of the target polynucleotide compared to amplification of the reference polynucleotide. Polymerase chain reaction requires both strands of DNA to be replicated for exponential amplification. Blocking oligonucleotides hybridize to one of the strands, creating linear replication for only one strand. For example, duplex between the blocker (top-strand) and the target polynucleotide (bottom- strand) is similar to a duplex between the target polynucleotide (top-strand) and the reference
polynucleotide (bottom-strand). Forming a duplex between the target polynucleotide (top- strand) and the reference polynucleotide (bottom-strand) reduces the efficiency of the enrichment, by either preventing the replication of the target polynucleotide (top-strand) or facilitating the replication of the reference polynucleotide (bottom-strand).
The results in the present invention are a non-obvious improvement over the
Makrigiorgos method. The Makrigiorgos method demonstrates a sample containing 3% target polynucleotide enriching to between 45 - 47% (Figures 5 - 7). A separate publication refers to the Makrigiorgos method taught in U.S. 20110217714 as Ice-COLD-PCR (Milbury et al., 2011, of record). In Figure 4b, a sample containing 3% target polynucleotide is enriched to 37.5%, a 0.3% sample is enriched to 14.6% and a 0% sample has a signal of 3.4% (Milbury et al., 2011). This represents a 12.5x and a 49x enrichment of the 3% and 0.3% samples respectively. In contrast, the results in the present invention show a sample containing 0.25% target polynucleotide being enriched to 99.37%, which is a
397x
enrichment. Thus, the present invention represents a major improvement in the enrichment process, which impacts the accuracy (both the sensitivity and the specificity) of the method. This has commercial implications because higher sensitivity can detect cancer at an earlier stage, which is known to improve patient outcomes. High specificity is also required to prevent the wrong treatment for cancer patients, which can have severe negative side effects.
The present invention is also distinguishable from the Makrigiorgos method because it does not require a duplex to be form between the blocking polynucleotide and the target polynucleotide. The requirement for the target polynucleotide duplex means that a different method must be designed for each target polynucleotide. For example, certain cancer patients lack a mutation in the KRAS 12 or 13 codon. There are at least 7 different common mutations between those two codons, which would require 7 different tests. The present invention, however, is a significant improvement, and it allows for the use of a single test because it does not require the formation of the target polynucleotide with the blocking polynucleotide.
Also, because the blocking oligonucleotide does not hybridize to the target
polynucleotide, step 3 of the Makrigiorgos method is not needed in the method of the present invention, which allows for a more efficient method.
The Office Action at pages 10-11 discusses the teachings of Makrigiorgos in view of Huang and Cline. The Office Action acknowledges that Makrigiorgos does not exemplify as a single embodiment a method where a DNA polymerase that lacks 5'-3' exonuclease activity is
specifically mentioned. The Office Action, however, notes that Makrigiorgos teaches the use of Pyrococcusfuriosus (Pfu) DNA polymerase, which lacks 5'-3' exonuclease activity. The Office Action at page 11 states that Cline is only cited to establish inherent features of Pfu DNA polymerase, i.e., to show that it was known that a property of Pfu DNA polymerase is that it has an error rate of less than 10-5.
The Office Action then cites to Huang as teaching method of perform PCR using wild- type blocking (WTB) locked nucleic acid (LNA) oligonucleotide blockers (Huang, at Figure 1 and p. 377) using Pfu DNA polymerase. It should be noted that the WTB LNA oligonucleotides taught by Huang are
chimeras
composed of both LNA and DNA. (Huang, Abstract and p. 377). In Huang's WTB seven of the 13 nucleotides are DNA, and six of the 13 are LNA. (Huang at p. 377). Thus, Huang's WTB does not consist of LNA, as recited by the current claims. Like Huang, Makrigiorgos also lacks any teaching of a blocking oligonucleotide consisting of LNAs. Makrigiorgos at  [0018] suggests that their reference blocking sequence can take the form of "a chimera between single stranded DNA RNA, peptide nucleic acid (PNA) or locked nucleic acid (LNA), or another modified nucleotide. The PNA or LNA position on the chimera sequence can be selected to match positions where mutations are likely, so as to maximize the effect of potential mismatches at those positions." Id.
Claim 19 as amended recites that the blocker
consists
of locked nucleic acids (LNAs). This is relevant because the reference polynucleotide and target polynucleotide in the present invention are DNA. Within a blocking oligonucleotide, a DNA duplex forms a B-form double helix (see, Hardison, 2024, "Working with Molecular Genetics," Chapter 2.5,) but a LNA duplex forms a A-form helix (see, Pande & Nilsson, 2008, "Insights into structure, dynamics and hydration of locked nucleic acid (LNA) strand-based duplexes from molecular dynamics simulations," Nucl. Acids Res., Vol. 36, no. 5, 1508-1516). A-form and B-form helices are distinct helical structures. "In B-form, the base-pairs are almost centered over the helical axis (Figure 2.5.4), but in A-form, they are displaced away from the central axis and closer to the major groove. The result is a ribbon-like helix with a more open cylindrical core in A-form" (Hardison, 2024). Figure 2.5.5 from Hardison provides a depiction of different helical structures:
Figure 2.5.5: B-form (left), A-form (middle) and Z-DNA (right). (CC BY-SA 4.0; Mauroesguerroto)
Helical structures represent the most stable state of polynucleotide molecules, and
therefore require the most energy to denature. In a chimeric oligonucleotide (e.g., a DNA/RNA molecule or a DNA/LNA molecule), at each transition point within the chimera between DNA and LNA a "helical axis misalignment" occurs (Cofsky et al., 2022, "Crystal structure of an RNA/DNA strand exchange junction," PLOS ONE, 17(4): e0263547). Each time a blocker transitions from an A-form to a B-form a misalignment is created, the effectiveness of the blocker is reduced. In the present invention, the goal is to have the blocker bind to the reference sequence and not to the target sequence, where the difference in the preferential binding of the blocker to the reference polynucleotide rather than to the target polynucleotide is because of the misalignment between the blocker and the target sequences. If, however, the blocker and the reference sequence already have misalignments because the blocker is a chimera (i.e., have misalignments due to helicalforms), the differential of misalignments between the blocker and the target sequence is less. Thus, in the present invention, having a blocker consisting of LNAs will have a higher melting temperature (and a greater melting temperature differential) as
compared to a chimeric LNA/DNA blocker, because the non-chimeric LNA blocker will not have competing LNA and DNA helical structures. This higher melting temperature of a blocking consisting of LNA results in more efficient enrichment, thereby reducing the number of false positives.
Applicant respectfully asserts that even when combined, Makrigiorgos, Huang and Cline do not teach or suggest all the elements of the presently claimed invention, namely, a three-step method where the blocking oligonucleotide is in a duplex with the
reference
polynucleotide, but
not
the
t
arget
polynucleotide (or the primers). The present invention has no requirement, condition, or need to create a heteroduplex between the blocker and the target strand. Further, none of the cited references teach the use of a blocking oligonucleotide that consists of LNA. Thus, even when combined, the cited references do not teach or suggest all the features of the claimed invention. Since the combination of references do not teach or suggest all the features of the claimed invention, the claims are non-obvious in view of the cited references. Accordingly, Applicant respectfully submits that this rejection is overcome and respectfully requests that it be withdrawn.
Claims 8 and 26 are rejected under 35 U.S.C. § 103 as allegedly as being unpatentable
over Makrigiorgos in view of Huang, as evidenced by Cline, and further in view of Albitar et al. (J. Visualized Experiments. 2017. 121: e55130, 8 pages, hereinafter "Albitar").
Claims 8 and 26 have been cancelled. Therefore, this rejection is rendered moot.
Conclusion


CITED REFERENCE SUMMARIES: Ref WO2021050100: Claims:
What is claimed is: 1. A method of detecting the presence of a first genetic variant in a target nucleic acid comprising: (a) contacting the target nucleic acid with (i) a first primer, (ii) a second primer comprising a first member of a binding pair, (iii) a polymerase and (iv) a blocking oligonucleotide, wherein the blocking oligonucleotide comprises a sequence complementary to a second genetic variant of the target nucleic acid, and the first and second primers are configured for amplification of the target nucleic acid; (b) amplifying the target nucleic acid thereby providing amplicons of the target nucleic acid; (c) contacting the amplicons with a capture nucleic acid, wherein the capture nucleic acid comprises a sequence complementary to the first genetic variant of the target sequence, thereby providing captured amplicons comprising the first member of the binding pair; (d) contacting the captured amplicons with a first detectable label comprising a second member of the binding pair; and (e) detecting a presence, absence, amount, or change thereof, of the first detectable label. 2. The method of claim 1, wherein the capture nucleic acid is attached to a surface of a sensor. 3. The method of claim 2, wherein the detecting of (e) comprises detecting the presence, absence, amount, or change thereof, of the first detectable label at the surface of the sensor. 4. The method of any one of claims 1 to 3, wherein the detecting of (e) comprises a dynamic detection process. 5. The method of claim 4, wherein the dynamic detection process comprises increasing a temperature at or near the sensor, or at the surface of the sensor, during the detecting of (e). 6. The method of claim 4 or 5, wherein the dynamic detection process comprises changing a salt or cation concentration at or near the sensor, or at the surface of the sensor, during the detecting of (e). 7. The method of any one of claims 4 to 6, wherein the dynamic detection process comprises flowing a fluid across the surface of the sensor during the detecting of (e). 8. The method of any one of claims 1 to 7, wherein the detecting of (e) comprises detecting binding of one or more amplicons that bind to the capture nucleic acid. 9. The method of any one of claims 2 to 8, wherein the detecting of (e) comprises detecting a change in an amount of amplicons that are bound to the surface of the sensor. 10. The method of any one of claims 2 to 9, wherein the sensor comprises a magnetic sensor, the first detectable label comprises a magnetic particle, and the detecting of (e) comprises detecting a presence, absence, amount, or change of magnetoresistance at or near the surface of the magnetic sensor. 11. The method of claim 10, wherein the detecting of (e) comprises detecting a change in magnetoresistance at the surface of the sensor. 12. The method of any one of claims 1 to 11, wherein the blocking oligonucleotide comprises one or more locked nucleotides. 13. The method of any one of claims 1 to 12, wherein the first primer comprises a 5’- phosphorylated nucleotide. 14. The method of any one of claims 1 to 13, wherein the capture nucleic acid comprises one or more locked nucleotides. 15. The method of any one of claims 11 to 14, wherein the detecting the change in magnetoresistance comprises increasing the temperature of the surface by at least 5 °C while detecting the magnetoresistance at the surface of the sensor prior to, during and/or after increasing the temperature. 16. The method of any one of claims 1 to 15, wherein the blocking oligonucleotide, when hybridized to the second genetic variant, substantially prevents amplification of the target nucleic acid. 17. The method of any one of claims 1 to 16, wherein the blocking oligonucleotide comprises a melting temperature of at least 75 °C, at least 80 °C or at least 85 °C. 18. The method of any one of claims 1 to 17, wherein the blocking oligonucleotide has length in a range from 9 to 20 oligonucleotides. 19. The method of any one of claims 1 to 18, wherein the blocking oligonucleotide comprises at least 3 locked nucleotides. 20. The method of any one of claims 1 to 19, wherein after (b), the amplicons are contacted with a 5’ -3’ exonuclease. 21. The method of any one of claims 1 to 20, wherein the capture nucleic acid has a length in a range from 9 to 30 oligonucleotides. 22. The method of any one of claims 1 to 21, wherein the capture nucleic acid comprises a melting temperature of at least 50 °C, at least 55 °C or at least 65 °C. 23. The method of any one of claims 1 to 22, wherein the capture nucleic acid comprises at least 3 locked nucleotides. 24. The method of any one of claims 1 to 23, wherein the presence of the first genetic variant in the target nucleic acid is determined according to a change of magnetoresistance detected in (e). 25. The method of any one of claims 2 to 24, wherein the detecting of (e) comprises distinguishing the presence, absence, or amount of the first genetic variant at the surface of the sensor compared to a presence, absence, or amount of the second genetic variant at the surface of the sensor. 26. The method of any one of claims 2 to 25, wherein the detecting of (e) comprises distinguishing the presence, absence, or amount of the first genetic variant at the surface of the sensor compared to a presence, absence or amount of another nucleic acid at the surface of the sensor. 27. The method of any one of claims 1 to 26, wherein the first member of the binding pair comprises biotin and the second member of the binding pair comprises streptavidin. 28. The method of any one of claims 1 to 27, wherein the amplifying of (b) comprises a polymerase chain reaction. 29. The method of claim 28, wherein the amplifying of (b) comprises at least 40, or at least 50 cycles a polymerase chain reaction. 30. The method of any one of claims 1 to 29, wherein the method is conducted on a sample obtained from a subject, wherein the sample comprises the target nucleic acid. 31. The method of claim 30, wherein the sample comprises cell free DNA. 32. The method of claim 30 or 31, wherein the sample is obtained from a pregnant female. 33. The method of any one of claims 1 to 32, wherein prior to (a) the sample is contacted with a microfluid channel, wherein the microfluidic channel is operably and/or fluidically connected to the sensor. 34. The method of any one of claims 1 to 33, wherein prior to (a) the sample is contacted with a membrane configured to reversibly and/or non-specifically bind to nucleic acids in the sample, thereby providing bound nucleic acids, wherein the membrane is operably and/or fluidically connected to the microfluidic channel and to the sensor. 35. The method of any one of claims 1 to 34, wherein the amplifying of (b) is performed inside of an amplification chamber that is operably and/or fluidically connected to a microfluidic channel, to the sensor and optionally to a membrane. 36. The method of any one of claims 1 to 35, wherein prior to (a), the method comprises (i) contacting the sample with (i) a cell lysis solution, (ii) a membrane, (iii) optionally a wash solution, and (iv) an elution buffer, wherein after the contacting of (iv) bound nucleic acids are released from the membrane. 37. The method of any one of claims 33 to 36, wherein the nucleic acids in the sample are transported through the microfluidic channel to the membrane, transported through the microfluidic channel from the membrane to the amplification chamber, and transported through the microfluidic channel from the amplification chamber to the surface of the sensor. 38. The method of any one of claims 1 to 37, wherein the sensor comprises a giant magnetomagnetoresistance (GMR) sensor. 39. The method of any one of claims 1 to 38, wherein the first genetic variant comprises at least one single nucleotide polymorphism (SNP). 40. The method of any one of claims 1 to 38, wherein the first genetic variant comprises at least one single nucleotide mutation. 41. The method of any one of claims 1 to 40, wherein the first genetic variant comprises at least one single nucleotide deletion or insertion. 42. The method of any one of claims 1 to 41, wherein the captured amplicons are in fluid contact with a buffer and prior to, or during, the detecting of (e), a concentration of positively charged cations in the buffer is decreased by at least 50%. 43. The method of claim 42, wherein the positively charged cations comprise sodium, potassium, calcium or magnesium. 44. The method of any one of claims 1 to 43, wherein a sensitivity of detection of the first genetic variant is less than 15 copies per mL of the sample. 45. The method of any one of claims 1 to 43, wherein the method detects the presence of the first genetic variant at a concentration as low as 1% of the target sequence in the sample. 46. The method of any one of claims 1 to 45, wherein the method is performed in a mircrofluidic device. 47. A mircrofluidic device for carrying out the method of any one of claims 1 to 46, the device comprising: (a) a microfluidic channel; (b) a first chamber comprising a membrane; (c) an amplification chamber; (d) 3 or more miniature solenoid valves; and (d) a sensor comprising a surface comprising a plurality of capture nucleic acids; wherein the microfluidic channel is operably connected and/or fluidically connected with the first chamber, the amplification chamber, the 3 or more valves and the sensor. 48. The microfluidic device of claim 47, wherein the sensor is a magnetic sensor. 49. The mircrofluidic device of claim 46 or 47, further comprising a sample port, and one or more wash chambers comprising a wash buffer, wherein the sample port and one or more wash chambers are operably and/or fluidically connected to the microfluidic channel and the first chamber. 50. The mircrofluidic device of any one of claims 47 to 49, further comprising a second chamber comprising magnetic particles, wherein the second chamber is operably and/or fluidically connected to the microfluidic channel and to the magnetic sensor. 51. The mircrofluidic device of any one of claims 47 to 50, wherein the magnetic sensor is housed in a third chamber. 52. The mircrofluidic device of any one of claims 47 or 51, further comprising one or more waste collection chambers, wherein the one or more waste collection chambers are operably and/or fluidically connected to the microfluidic channel. 53. The mircrofluidic device of any one of claims 47 or 52, further comprising a first heat source operably connected to the amplification chamber. 54. The mircrofluidic device of any one of claims 47 or 53, further comprising a cooling source operably connected to the amplification chamber. 55. The mircrofluidic device of any one of claims 471 or 54, further comprising a second heat source operably connected to the magnetic magnetoresistance sensor and/or to the third chamber. 56. The mircrofluidic device of any one of claims 47 or 55, wherein the microfluidics channel is operably connected to one or more diaphragm pumps or vacuum pumps. 57. The mircrofluidic device of any one of claims 47 or 56, wherein the microfluidic device comprises one or more electrical contact pads that are operably connected to the three or more valves. 58. The mircrofluidic device of any one of claims 47 or 57, wherein the microfluidic device comprises a memory chip. 59. The mircrofluidic device of any one of claims 47 or 58, wherein the microfluidic device has a length of 3 to 10 cm long, a width of 1 to 5 cm, and a thickness of 0.1 to 0.5 cm. 60. The mircrofluidic device of any one of claims 47 or 59, wherein the microfluidic device comprises or consists of a self contained cartridge or card comprising lyophilized amplification reagents, and lyophilized magnetic beads. 61. A method of any detecting at least one genetic variant comprising at least one target nucleic acid in a sample comprising, or suspected of comprising, the at least least one genetic variant, the method comprising: (a) providing the sample; (b) contacting the sample with (i) a first primer or a plurality of different first primers and (ii) a second primer or a plurality of different second primers, wherein each second primer comprises a first member of a binding pair, and (iii) a polymerase; (c) amplifying the at least one genetic variant thereby providing amplicons of the at least one genetic variant; (c) contacting the amplicons with a plurality of different capture nucleic acids, wherein each different capture nucleic acid comprises a sequence complementary to at a different genetic variant of a class of genetic variants, thereby providing distinguishable captured amplicons comprising the first member of the binding pair; (d) contacting the distinguishable captured amplicons with a first detectable label comprising a second member of the binding pair; and (e) detecting a presence, absence, amount, or change thereof, of the first detectable label. 62. The method of claim 61, wherein the detecting of (e) comprises detecting the presence, absence, amount, or change thereof, of the first detectable label at the surface of the sensor. 63. The method of any one of claims 61 to 2, wherein the detecting of (e) comprises a dynamic detection process. 64. The method of claim 63, wherein the dynamic detection process comprises increasing a temperature at or near the sensor, or at the surface of the sensor, during the detecting of (e). 65. The method of claim 63 or 64, wherein the dynamic detection process comprises changing a salt or cation concentration at or near the sensor, or at the surface of the sensor, during the detecting of (e). 66. The method of any one of claims 63 to 65, wherein the dynamic detection process comprises flowing a fluid across the surface of the sensor during the detecting of (e). 67. The method of any one of claims 61 to 66, wherein the detecting of (e) comprises detecting binding of one or more of the distinguishable amplicons that bind to each different capture nucleic acid, thereby distinguishing one genetic variant from another genetic variant. 68. The method of any one of claims 62 to 67, wherein the detecting of (e) comprises detecting a change in an amount of distinguishable amplicons that are bound to the surface of the sensor. 69. The method of any one of claims 62 to 68, wherein the sensor comprises a magnetic sensor, the first detectable label comprises a magnetic particle, and the detecting of (e) comprises detecting a presence, absence, amount, or change of magnetoresistance at or near the surface of the magnetic sensor. 70. The method of claim 69, wherein the detecting of (e) comprises detecting a change in magnetoresistance at the surface of the sensor. 71. The method of any one of claims 61 to 70, wherein the each of the different first primers and second primers comprises a 5’-phosphorylated nucleotide. 72. The method of any one of claims 61 to 71, wherein the capture nucleic acids comprise one or more locked nucleotides. 73. The method of any one of claims 70 to 72, wherein the detecting the change in magnetoresistance comprises increasing the temperature of the surface by at least 5 °C while detecting the magnetoresistance at the surface of the sensor prior to, during and/or after increasing the temperature. 74. The method of any one of claims 61 to 73, wherein after (b), the distinguishable amplicons are contacted with a 5’-3’ exonuclease. 75. The method of any one of claims 61 to 74, wherein the capture nucleic acids have a length in a range from 9 to 30 oligonucleotides. 76. The method of any one of claims 61 to 75, wherein the capture nucleic acids comprise a melting temperature of at least 50 °C, at least 55 °C or at least 65 °C. 77. The method of any one of claims 61 to 76, wherein the capture nucleic acids comprise at least 3 locked nucleotides. 78. The method of any one of claims 61 to 77, wherein the presence of the at least one genetic variant in the target nucleic acid is determined according to a change of magnetoresistance detected in (e). 79. The method of any one of claims 62 to 78, wherein the detecting of (e) comprises distinguishing the presence, absence, or amount of the at least one genetic variant at the surface of the sensor compared to a presence, absence, or amount of another genetic variant at the surface of the sensor. 80. The method of any one of claims 62 to 79, wherein the detecting of (e) comprises distinguishing the presence, absence, or amount of at least one genetic variant at the surface of the sensor compared to a presence, absence or amount of another nucleic acid at the surface of the sensor. 81. The method of any one of claims 61 to 80, wherein the first member of the binding pair comprises biotin and the second member of the binding pair comprises streptavidin. 82. The method of any one of claims 61 to 81, wherein the amplifying of (b) comprises a polymerase chain reaction. 83. The method of claim 82, wherein the amplifying of (b) comprises at least 20, or at least 50 cycles a polymerase chain reaction. 84. The method of any one of claims 61 to 83, wherein the method is conducted on a sample obtained from a subject, wherein the sample comprises, or is suspected of comprising, the least one genetic variant. 85. The method of claim 84, wherein the sample comprises cell free DNA. 86. The method of any one of claims 61 to 85, wherein prior to (a) the sample is contacted with a microfluid channel, wherein the microfluidic channel is operably and/or fluidically connected to the sensor. 87. The method of any one of claims 61 to 86, wherein prior to (a) the sample is contacted with a membrane configured to reversibly and/or non-specifically bind to nucleic acids in the sample, thereby providing bound nucleic acids, wherein the membrane is operably and/or fluidically connected to the microfluidic channel and to the sensor. 88. The method of any one of claims 61 to 87, wherein the amplifying of (b) is performed inside of an amplification chamber that is operably and/or fluidically connected to a microfluidic channel, to the sensor and optionally to a membrane. 89. The method of any one of claims 61 to 88, wherein prior to (a), the method comprises (i) contacting the sample with (i) a cell lysis solution, (ii) a membrane, (iii) optionally a wash solution, and (iv) an elution buffer, wherein after the contacting of (iv) bound nucleic acids are released from the membrane. 90. The method of any one of claims 87 to 89, wherein the nucleic acids in the sample are transported through the microfluidic channel to the membrane, transported through the microfluidic channel from the membrane to the amplification chamber, and transported through the microfluidic channel from the amplification chamber to the surface of the sensor. 91. The method of any one of claims 61 to 90, wherein the sensor comprises a giant magnetomagnetoresistance (GMR) sensor. 92. The method of any one of claims 61 to 91, wherein the at least one genetic variant comprises at least one single nucleotide polymorphism (SNP). 93. The method of any one of claims 61 to 92, wherein the at least one genetic variant comprises at least one single nucleotide mutation. 94. The method of any one of claims 61 to 93, wherein the at least one genetic variant comprises at least one single nucleotide deletion or insertion. 95. The method of any one of claims 61 to 94, wherein the captured amplicons are in fluid contact with a buffer and prior to, or during, the detecting of (e), a concentration of positively charged cations in the buffer is decreased by at least 50%. 96. The method of claim 95, wherein the positively charged cations comprise sodium, potassium, calcium or magnesium. 97. The method of any one of claims 61 to 96, wherein a sensitivity of detection of the at least one genetic variant is less than 15 copies per mL of the sample. 98. The method of any one of claims 61 to 96, wherein the meth
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