Office Action 1: Non-Final Rejection (2024-11-18)
Rejection types: 103 - Obviousness, 112 - Written Description/Enablement, Double Patenting

CLAIMS:
CLAIMS

We claim:

1\. A method for detecting a target polynucleotide comprising a genetic alteration relative to a reference polynucleotide lacking the genetic alteration in a sample mprising a mixture of the target polynucleotide and the reference polynucleotide, the m hod comprising.
(a) performing a polymerase chain reaction (PCR) amplification to obtain an amplification product in a reaction mixture comprising:(i) the sample,
(ii) a high fidelity DNA polymerase that lacks 5'43' nuclease activity and comprises 3'45' nuclease activity;
(iii) a pair of primers that flank the genetic alteration; and
(iv) a blocking oligonucleotide that hybridizes selectively to the reference polynucleotide lacking the genetic alteration to form a blocking duplex; and
(b) sequencing the amplification product and detecting the genetic alteration in the amplification product, thereby detecting the target polynucleotide comprising the genetic alteration in the sample

2\. The method of claim 1,wherein the target polynucleotide represents no ore than about 3%,2%, 1%, 0.5%,0.25%. or 0.125% of the mixture.

3\. The method of claim 1 or 2,wherein the target polynucleotide is present in e sample at a copy number less than about 30, 20, or 10.

4\. The method of any of the foregoing claims, wherein the PCR amplification is performed for no more than 60, 50, 40, or 30 cycles.5 The method of any of the foregoing claims, wherein the DNA polymerase as an error rate of less than 10', 10k, or 10'.

6\. The method of any of the foregoing claims, the amplification product is sequenced to obtain multiple reads, and the genetic alteration is present in at least about 90%, 95%, 96%, 97%, 98%, or 99o of the multiple reads.7 The method of any of the foregoing claims, wherein the genetic alteration is present in at least about 90%, 95%, 96%, 97%, 98%, or 99% of the amplification product.

8\. The method of any of the foregoing claims, the method comprising removing the blocking oligonucleotide from the amplification product prior to sequencing the amplification product.

9\. The method of any of the foregoing claims, further comprising determining the minimum number of PCR cycles necessary for detecting a target polynucleotide that is present in the mixture at a percentage of no more than 0.1%, 0.05%, 0.02%, or 0.01%.
10\. The method of any of the foregoing claims, wherein the blocking oligonucleotide comprises one or more modified nucleotides that enhance the stability of the duplex formed by the blocking oligonucleotide and the reference polynucleotide.SVG {EMBED HERE} 0.1130.033
I1. The method of any of the foregoing claims, 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.

12\. The method of any of the foregoing claims, wherein the blocking oligonucleotide comprises one or more locked nucleic acids (LNAs) comprising a sugar moiety having a 2'-O conjugated to 4'-C via a methylene group.

13\. The method of claim 12, wherein all of the nucleotides of the blocking oligonucleotide are LNAs.
14\. The method of any of the foregoing claims, wherein the blocking oligonucleotide has a length that is no more than about 20, 19, 18, 17, 16, 15, 14, 13, 1 SVG {EMBED HERE} 0.1370.11I1SVG {EMBED HERE} 0.140.087 or 10 nucleotides.

15\. The method of any of the foregoing claims, wherein the genetic alteration comprises a substitution.

16\. The method of any of the foregoing claims, wherein the genetic alteration comprises a deletion.
17\. The methSVG {EMBED HERE} 0.0870.04od of any of the foregoing claims, wherein the genetic alteration comprises an insertion.
18 The method of any of the foregoing claims, wherein the genetic alteration is present in a genetic loci selected from PIK3(A,KRAS, APCFAT4, KMT21),K7T2(, and BRAF.

19\. A method for detecting two or more target polynucleotides each comprising a different genetic alteration relative to a reference polynucleotide lacking the genetic alterations in a sample comprising a mixture of the two or more target polynucleotides and the reference polynucleotide, the method comprising- (a) performing a polymerase chain reaction (PCR) amplification to obtain an amplification product in a reaction mixture comprising:(i) the sample;
(ii) a DNA polymerase that lacks 5'43' nuclease activity and comprises 3'45' nuclease activity;
(iii) two or more pairs of primers, wherein each pair of primers flank one of the different genetic alterations; and
(iv) two or more blocking oligonucleotides that hybridize selectively to the reference polynucleotides lacking the different genetic alterations to form two or more blocking duplexes;
(b) sequencing the amplification product and detecting the different genetic alterations in the amplification product, thereby detecting the two or more target polynucleotides comprising the different genetic alterations in the sample.

20\. The method of any of the foregoing claims, wherein the sample is a biological sample from a subject.

21\. The method of any of the foregoing claims, wherein the sample is a blood sample from a subject

22\. The method of claim 21, wherein the sample is a cell-free blood sample and/or the blood sample is treated to remove cells prior to performing PCR amplification.
23 The method of any of the foregoing claims, wherein prior to the method being performed the sample was treated with a reagent that selectively modifies non- methylated cytosine residues to produce detectable modified residues but which does not modify methylated cytosine residues.

24\. The method of any of the foregoing claims, wherein the genetic alteration is associated with cancer in a subject.

25\. The method of claim 24, wherein prior to performing step (a), the method comprises sequencing a cancer sample from the subject and detecting the genetic alteration in the cancer sample.

26\. The method of any of the foregoing claims, wherein the reference polynucleotide comprises a wild-type sequence with respect to the genetic alteration and the genetic alteration is a genetic mutation present in the target polynucleotide.

27\. The method of any of the foregoing claims, wherein the sample is obtained from a subject who has cancer or is at risk for developing cancer.

28\. The method of any of claims 1-27, wherein the subject has been diagnosed with cancer and the subject currently is in remission.
29 A kit comprising one or more components for performing any of the foregoing methods.
30 The kit of claim 29, wherein the kit comprises- (i) a DNA polymerase that lacks 5'43' nuclease activity and comprises 3'+5' nuclease activity; (ii) a pair of primers
that flank the genetic alteration, and (iii) a blocking oligonucleotide that hybridizes selectively to the reference polynucleotide lacking the genetic alteration to form a blocking duplex and obtaining an amplified fragment sample.31 The kit of claim 29, wherein the kit comprises: (i) a DNA polymerase that lacks 5'->3' nuclease activity and comprises 3'->5' nuclease activity; (ii) a pair of primers that flank a genetic alteration in a genetic loci selected from P/K3CA,KRAS. APCSVG {EMBED HERE} 0.1370.13PAT4,KM72),KM72C. and HRAF; and (iii) a blocking oligonucleotide that hybridizes selectively to the reference polynucleotide lacking the genetic alteration to form a blocking duplex and obtaining an amplified fragment sample.

32\. The kit of any of claims 29-31, wherein the blocking oligonucleotide consists of LNAs and the blocking oligonucleotide is no longer than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, or 10 nucleotides.

OFFICE ACTION:
DETAILED ACTION
Notice of Pre-AIA  or AIA  Status
1. The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
2. Claims 1, 3, 6-9, 11-15, 19, 21, 24-27 and 29-31 are pending and have been examined herein.
Claim Rejections - 35 USC § 112(b)
3. 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 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 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.
Claims 1, 3, 6-9, 11-15, 19, 21, 24-27 and 29-31 are indefinite over the recitation of “the reference polynucleotide” at (ii) of claims 1 and 19 because this phrase lacks proper antecedent basis. Claims 1 and 19 should be amended at (ii) to recite “a reference polynucleotide.”
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 and because 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. The claims are also vague and indefinite over the recitation that the primers don’t interfere with the hybridization of the blocking oligonucleotide per se. Claims 1 and 19 should be amended at (iv) to recite “a pair of primers that flank a genetic alteration in the target polynucleotide and do not interfere with the hybridization of the blocking oligonucleotide to the reference polynucleotide, wherein the sample comprises the double-stranded target polynucleotide and a double-stranded reference polynucleotide, wherein the target polynucleotide has the genetic alteration relative to the reference polynucleotide.
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) because 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. Rather, the claims recite a final step of enriching “the target polynucleotide.” It is unclear as to whether the enriched target polynucleotide is the same as or distinct from the amplification products containing a target polynucleotide.
Double Patenting
4. The nonstatutory double patenting rejection is based on a judicially created doctrine grounded in public policy (a policy reflected in the statute) so as to prevent the unjustified or improper timewise extension of the “right to exclude” granted by a patent and to prevent possible harassment by multiple assignees. A nonstatutory double patenting rejection is appropriate where the conflicting claims are not identical, but at least one examined application claim is not patentably distinct from the reference claim(s) because the examined application claim is either anticipated by, or would have been obvious over, the reference claim(s). See, e.g., In re Berg, 140 F.3d 1428, 46 USPQ2d 1226 (Fed. Cir. 1998); In re Goodman, 11 F.3d 1046, 29 USPQ2d 2010 (Fed. Cir. 1993); In re Longi, 759 F.2d 887, 225 USPQ 645 (Fed. Cir. 1985); In re Van Ornum, 686 F.2d 937, 214 USPQ 761 (CCPA 1982); In re Vogel, 422 F.2d 438, 164 USPQ 619 (CCPA 1970); In re Thorington, 418 F.2d 528, 163 USPQ 644 (CCPA 1969).
A timely filed terminal disclaimer in compliance with 37 CFR 1.321(c) or 1.321(d) may be used to overcome an actual or provisional rejection based on nonstatutory double patenting provided the reference application or patent either is shown to be commonly owned with the examined application, or claims an invention made as a result of activities undertaken within the scope of a joint research agreement. See MPEP § 717.02 for applications subject to examination under the first inventor to file provisions of the AIA  as explained in MPEP § 2159. See MPEP § 2146 et seq. for applications not subject to examination under the first inventor to file provisions of the AIA . A terminal disclaimer must be signed in compliance with 37 CFR 1.321(b).
The filing of a terminal disclaimer by itself is not a complete reply to a nonstatutory double patenting (NSDP) rejection. A complete reply requires that the terminal disclaimer be accompanied by a reply requesting reconsideration of the prior Office action. Even where the NSDP rejection is provisional the reply must be complete. See MPEP § 804, subsection I.B.1. For a reply to a non-final Office action, see 37 CFR 1.111(a). For a reply to final Office action, see 37 CFR 1.113(c). A request for reconsideration while not provided for in 37 CFR 1.113(c) may be filed after final for consideration. See MPEP §§ 706.07(e) and 714.13.
The USPTO Internet website contains terminal disclaimer forms which may be used. Please visit www.uspto.gov/patent/patents-forms. The actual filing date of the application in which the form is filed determines what form (e.g., PTO/SB/25, PTO/SB/26, PTO/AIA /25, or PTO/AIA /26) should be used. A web-based eTerminal Disclaimer may be filled out completely online using web-screens. An eTerminal Disclaimer that meets all requirements is auto-processed and approved immediately upon submission. For more information about eTerminal Disclaimers, refer to www.uspto.gov/patents/apply/applying-online/eterminal-disclaimer.
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). Although the claims at issue are not identical, they are not patentably distinct from each other because the present claims and the claims of ‘325 are both inclusive of methods for detecting a target polynucleotide comprising a genetic alteration relative to a reference polynucleotide lacking the genetic alteration in a sample comprising a mixture of the target polynucleotide and the reference polynucleotide, the methods comprising:  performing a polymerase chain reaction (PCR) amplification to obtain an amplification product in a reaction mixture comprising: (i) the sample; (ii) a DNA polymerase that lacks 5'->3' nuclease activity and comprises 3'->5' nuclease activity; (iii) a pair of primers that flank the genetic alteration; and (iv) a blocking oligonucleotide that hybridizes selectively to the reference polynucleotide lacking the genetic alteration to form a blocking duplex; and performing multiple cycles of PCR amplification (e.g., claim 4 of ‘325); and sequencing the amplification product and detecting the genetic alteration in the amplification product, thereby detecting the target polynucleotide comprising the genetic alteration in the sample.
The claims of ‘325 do not recite that the pair of primers that flank the genetic alteration do not interfere with hybridization of the blocking oligonucleotide. However, since the method of ‘325 requires primers that flank the genetic alteration and a blocking oligonucleotide that hybridizes to a region of the reference nucleic acid that lacks the genetic alteration to form a duplex, such primers would include primers that have the property that they do not interfere with the hybridization of the blocking oligonucleotide to the reference nucleic acid so that the duplex could be formed, as required by the claims of ‘325. Regarding the recitations in (b), (c) and (d) of the present claims, it is a property of PCR that it includes a step in which the temperature is increased to denature double stranded DNA (and thereby above a melting temperature of double-stranded target nucleic acid comprising the genetic alteration and double-stranded reference nucleic acid); a step of reducing the temperature so that the primer pair can hybridize to the target nucleic acid comprising the genetic alteration; and a step of extending the primers annealed to the target polynucleotide. Further, when read in light of the specification of ‘325, it is clear that the method claimed in ’325 in which PCR is performed to produce an amplification product encompasses the heating / denaturing, primer annealing and primer extension steps of the present claims.
Regarding present claims 3 and 21, claim 5 of ‘325 encompasses methods wherein the DNA polymerase has an error rate of less than 10-5.
The present claims (6-8 and 24-26) and the claims of ‘325 also include methods wherein the blocking oligonucleotide comprises one or more locked nucleic acids (LNAs) comprising a sugar moiety having a 2'-O conjugated to 4'-C via a methylene group (e.g., claims 12 and 13 of ‘325); methods wherein the blocking oligonucleotides are no more than 20 nucleotides (claim 14 of ‘325), which meets the limitation of the present claims of a blocking oligonucleotide of 20 nucleotides; methods wherein the biological sample is a blood sample (claim 21 of ‘325), and thereby a liquid biopsy; and methods wherein the genetic alteration is a substitution, deletion or insertion (e.g., claims 15-17 of ‘325). Further, regarding present claims 8 and 26, when read in light of the specification of ‘325, it is clear that the blocking oligonucleotides comprising LNAs include blocking oligonucleotides in which all of the nucleotides are LNAs. Regarding present claims 30 and 31, claim 1 of ‘325 recites sequencing the amplification product.
This is a provisional nonstatutory double patenting rejection because the patentably indistinct claims have not in fact been patented.
5. 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).
The claims of ‘325 are discussed above.
Regarding claims 27 and 29, Makrigiorgos teaches methods for enriching a sample for a double-stranded target nucleic acid that comprises a genetic alteration wherein the method comprises performing a PCR assay in the presence of a blocking oligonucleotide that binds to a reference / wildtype nucleic acid and not to the target nucleic acid (e.g., claim 1; Figure 2 and para [0012-0015] and [0061-0064]).
Makrigiorgos teaches that the amplification products may be detected using SYBR Green dye or LC-dye (i.e., a DNA binding fluorophore) or a fluorescently labeled probe (e.g., para [0046] and [0060] and claims 21 and 23). In particular, Makrigiorgos (para [0060]) states:
In this embodiment, the reaction mixture may include a nucleic acid detection agent (e.g., nucleic acid detection dye such as SYBR Green dye or LC-Green dye or a probe operatively coupled to a fluorescent dye) for quantifying and/or monitoring the amplification products of the reaction. Once the enrichment of the target sequence is complete the sample may be further processed, e.g., subjected to a sequencing reaction. The enriched alleles may be further processed by a variety of procedures including: MALDI-TOF, HR-Melting, Di-deoxy-sequencing, Single-molecule sequencing, second generation high throughput sequencing, pyrosequencing, RFLP, digital PCR and quantitative-PCR (See FIG. 1B).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method claimed in ‘325 so as to have included in the reaction mixture a DNA binding fluorophore, such as SYBR Green dye or LC-dye, or a fluorescently labeled probe, in order to have achieved the advantage set forth by Makrigiorgos of facilitating quantifying and/or monitoring the amplification products.
This is a provisional nonstatutory double patenting rejection because the patentably indistinct claims have not in fact been patented.
Claim Rejections - 35 USC § 103
6.  In the event the determination of the status of the application as subject to AIA  35 U.S.C. 102 and 103 (or as subject to pre-AIA  35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA  to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
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.
Claim(s) 1, 3, 6, 7, 9, 11-15, 19, 21, 24, 25, 27 and 29-31  is/are rejected under 35 U.S.C. 103 as being unpatentable over Makrigiorgos, G. (U.S. 20110217714) in view of Huang et al (Molecular and Cellular Probes. 2010. 24: 376-380), as evidenced by Cline et al (Nucleic Acids Research. 1996. 24(18): 3546-3551).
Makrigiorgos teaches a method comprising the steps of: (a) preparing a reaction mixture comprising: (i) a sample comprising a double-stranded reference polynucleotide (e.g., a wildtype sequence) and a double-stranded target polynucleotide that comprises a genetic alteration relative to the reference polynucleotide; (ii) a DNA polymerase; (iii) a blocking oligonucleotide that hybridizes selectively to the reference polynucleotide and not to the target polynucleotide; and (iv) a pair of primers that flank a genetic alteration present in the target polynucleotide and do not interfere with the hybridization of the blocking oligonucleotide to the reference polynucleotide; (b) increasing the temperature of the reaction mixture above the melting temperatures of the double-stranded reference polynucleotide and the double-stranded target polynucleotide; (c) reducing the temperature of the reaction mixture to a temperature that is below the melting temperature of the blocking oligonucleotide to the reference polynucleotide but is above the melting temperature of the primer oligonucleotide, wherein the blocking oligonucleotide and the reference polynucleotide form a blocking duplex; (d) reducing the temperature of the reaction mixture, wherein the primer pair hybridizes to the target polynucleotide; (e) extending the primers hybridized to the target polynucleotide; and (f) repeating steps (b) through (e) for two or more cycles so as to enrich the target polynucleotide (e.g., claim 1; Figure 2 and para [0012-0015] and [0061-0064]).
Note that the present claims recite the open claim language of “comprising” and thereby may include additional steps, including additional steps of raising or reducing the temperature of the reaction mixture.
Makrigiorgos does not exemplify as a single embodiment the above method wherein the DNA polymerase used to extend the primers hybridized to the target polynucleotide is a DNA polymerase that lacks 5’-3’ exonuclease activity and comprises 3’-5’ exonuclease activity.
However, Makrigiorgos does teach that the DNA polymerase used in the PCR / amplification step may be Pyrococcus furiosus (Pfu) DNA polymerase (para [0045]). It is a property of Pfu DNA polymerase that it lacks 5’-3’ exonuclease activity and comprises 3’-5’ exonuclease activity.
Further, Huang teaches methods of performing PCR using LNA oligonucleotide blockers (“WTB”) to suppress amplification of wild-type nucleic acids (e.g., abstract, Figure 1 and p. 377). It is stated that “In the present WTB-PCR system, a wild-type blocker (WTB) which was the chimeras of locked nucleic acid (LNA) and DNA oligonucleotides was used to specifically inhibit the amplification of wild-type KRAS gene. The results showed that the present WTB-PCR was a reliable and simple method to detect rare mutated KRAS in clinical FFPE sections with high sensitivity (0.01%) and a single copy level” (p. 377, col. 1).
Huang teaches that PCR is performed using Pfu DNA polymerase (p. 377 section “2.3”). Huang states “to avoid hydrolysis of WTB by 5’-3’ exonuclease activities of traditional DNA polymerase (e.g., Taq DNA polymerase), a Pfu DNA polymerase, which is defect of such activities as the introductions of manufacturer (Promega), was used” (p. 377, col. 2).
In view of the teachings of Huang, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have specifically performed the method of Makrigiorgos using Pfu DNA polymerase as the DNA polymerase in the amplification step in order to have accomplished the objective set forth by Huang of avoiding hydrolysis of the blocking oligonucleotide which can occur when using alternative DNA polymerases which have 5’-3’ exonuclease activity.
Regarding claims 3 and 21, it is also a property of Pfu DNA polymerase that it has an error rate of less than 10-5. This fact is evidenced by the teachings of Cline which discloses that Pfu DNA polymerase has an error rate of 1.3 x 10-6 (see abstract and p. 3550, col. 1). Note that Cline is cited only to establish what is inherent to Pfu DNA polymerase and the method of Makrigiorgos.
Regarding claims 6 and 24, Makrigiorgos teaches that the blocking oligonucleotides may include locked nucleic acids (LNA; e.g., para [0018]).
Regarding claims 7 and 25, Makrigiorgos teaches that the blocking oligonucleotides incorporate LNAs to enhance the enhance the selective binding of the blocking oligonucleotide to the reference nucleic acid. Makrigiorgos does not teach that the LNAs comprise a sugar moiety having a 2’-O conjugated to a 4’-C via a methylene group.
However, Huang teaches:
“LNA is a nucleic acid analog with a 2’-O, 4’-C methylene bridge.
This bicyclic structure locks the ribose moiety into a C3’-endo conformation, which significantly increases binding affinity (i.e., stabilizes the duplex in A-form conformation) [20]. LNA can bind to natural DNA according to standard base-pairing rules, but
with much greater affinity than standard oligonucleotides [8,20]. Only one base mismatch between an LNA oligonucleotide and its complementary strand can significantly lower the melting temperatures, thereby allowing an LNA oligonucleotide to
discriminate a single base mismatch” (p. 379, col. 2).
In view of the teachings of Huang, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have used LNAs having a 2’-O, 4’-C methylene bridge in the method of Makrigiorgos since Huang teaches that these are the types of LNAs that can be incorporated into wildtype / reference blocking oligonucleotides to increase the affinity of the binding of the blocking oligonucleotide to the wildtype / reference nucleic acid, thereby enhancing the suppression / blocking of amplification of wildtype / reference nucleic acid.
Regarding claim 9, Makrigiorgos exemplifies blocking oligonucleotides that are 19 or 20 nucleotides in length (see e.g., Table 1).
Regarding claims 11-13, Makrigiorgos teaches that the mutation (i.e., genetic alteration) in the target nucleic acid may be a substitution, deletion or insertion (e.g., para [0034 and [0055]).
Regarding claims 14 and 15, Makrigiorgos teaches methods wherein the sample is a blood sample (e.g., para [0047-0048] and [0051]), which is considered to be a liquid biopsy biological fluid.
Regarding claims 27, and 29-31, Makrigiorgos (para [0060]) states:
In this embodiment, the reaction mixture may include a nucleic acid detection agent (e.g., nucleic acid detection dye such as SYBR Green dye or LC-Green dye or a probe operatively coupled to a fluorescent dye) for quantifying and/or monitoring the amplification products of the reaction. Once the enrichment of the target sequence is complete the sample may be further processed, e.g., subjected to a sequencing reaction. The enriched alleles may be further processed by a variety of procedures including: MALDI-TOF, HR-Melting, Di-deoxy-sequencing, Single-molecule sequencing, second generation high throughput sequencing, pyrosequencing, RFLP, digital PCR and quantitative-PCR (See FIG. 1B).
Accordingly, Makrigiorgos teaches that the amplification products may be detected using SYBR Green dye or LC-dye (i.e., a DNA binding fluorophore) or a fluorescently labeled probe, or by sequencing the amplification products (see also para [0046] and claims 19, 21 and 23).7. Claim(s) 8 and 26 is/are rejected under 35 U.S.C. 103 as being unpatentable over Makrigiorgos, G. (U.S. 20110217714) in view of Huang et al (Molecular and Cellular Probes. 2010. 24: 376-380), as evidenced by Cline et al (Nucleic Acids Research. 1996. 24(18): 3546-3551), and further in view of Albitar et al (J visualized Experiments. 2017. 121: e55130, 8 pages).
The teachings of Makrigiorgos and Huang are presented above.
Regarding claims 8 and 26, Makrigiorgos does not teach that all of the nucleotides in the blocking oligonucleotides are LNAs.
However, Albitar teaches methods of performing wild-type blocking PCR (WTB-PCR) using blocking oligonucleotides that comprise LNAs (p. 2). Albitar provides guidance as to how to design the most effective blocking oligonucleotides. For example, Albitar (p. 2) teaches:
PNG
media_image1.png
246
868
media_image1.png
Greyscale
In view of the teachings of Albitar, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have optimized the blocking oligonucleotides used in the method of Makrigiorgos so as to have selected the appropriate number of LNA nucleotides to be included in the blocking oligonucleotides, including using a LNA at each position in the blocking oligonucleotide, so as to ensure the highest level of affinity of the blocking oligonucleotide to the reference / wildtype nucleic acid and distinction between the melting temperature of the blocking oligonucleotide bound to reference / wildtype nucleic acid as compared to target / mutant nucleic acid. Albitar teaches the parameters of the LNA blocking oligonucleotide which affect its ability to suppress amplification of reference / wildtype nucleic acids, including length of the blocking oligonucleotide, sequence of the blocking oligonucleotide and concentration of the blocking oligonucleotide (e.g., p. 2). One would have been motivated to have incorporated an optimum number of LNAs in the blocking oligonucleotide, including using a LNA for each of the nucleotides in the blocking oligonucleotide, in order to have accomplished the objective set forth by Albitar of optimizing the blocking of amplification of the reference / wildtype nucleic acid to ensure the sensitive detection of small quantities of mutated nucleic acids in a sample.

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

NEXT 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.”

CITED REFERENCE SUMMARIES: Ref US20110217714A1: Claims:
A method for enriching a target nucleic acid sequence in an amplification reaction mixture, said method comprising: a. preparing an amplification reaction mixture including at least the following constituents: a nucleic acid sample having a reference sequence and also suspected of having one or more target sequences that are at least 50% homologous to said reference sequence and are also amplifiable by the same primer pair as said reference sequence, and an excess amount of reference blocking nucleic acid sequence which is fully complementary with at least a portion of the sequence of one of the strands of the reference sequence between its primer sites; b. subjecting the reaction mixture suspected of having said target sequence to a first denaturing temperature that is above the melting temperature (T m ) of the reference sequence so as to form denatured reference strands and denatured target strands; c. reducing the temperature of the reaction mixture so as to permit formation of heteroduplexes of the reference blocking sequence and the complementary reference strand and heteroduplexes of the reference blocking sequence and target strands; d. subjecting said reaction mixture to a critical temperature (T c ) 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 reference strand; e. 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; and f. extending said primer pair so as to enrich said target sequence relative to said reference sequence.
a. preparing an amplification reaction mixture including at least the following constituents: a nucleic acid sample having a reference sequence and also suspected of having one or more target sequences that are at least 50% homologous to said reference sequence and are also amplifiable by the same primer pair as said reference sequence, and an excess amount of reference blocking nucleic acid sequence which is fully complementary with at least a portion of the sequence of one of the strands of the reference sequence between its primer sites;
a nucleic acid sample having a reference sequence and also suspected of having one or more target sequences that are at least 50% homologous to said reference sequence and are also amplifiable by the same primer pair as said reference sequence, and
an excess amount of reference blocking nucleic acid sequence which is fully complementary with at least a portion of the sequence of one of the strands of the reference sequence between its primer sites;
b. subjecting the reaction mixture suspected of having said target sequence to a first denaturing temperature that is above the melting temperature (T m ) of the reference sequence so as to form denatured reference strands and denatured target strands;
c. reducing the temperature of the reaction mixture so as to permit formation of heteroduplexes of the reference blocking sequence and the complementary reference strand and heteroduplexes of the reference blocking sequence and target strands;
d. subjecting said reaction mixture to a critical temperature (T c ) 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 reference strand;
e. 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; and
f. extending said primer pair so as to enrich said target sequence relative to said reference sequence.
The method of claim 1 wherein a 3′-end on the reference blocking sequence is blocked to inhibit extension.
The method of claim 1 wherein a 5′-end on the reference blocking sequence strands comprises a nucleotide that prevents 5′ to 3′ exonucleolysis by Taq DNA polymerases.
The method of claim 1 wherein reference blocking sequence provided in step a) is a single-stranded nucleic acid reference blocking sequence.
The method of claim 1 wherein reference blocking sequence is provided in step a) as double-stranded nucleic acid reference blocking sequence which denatures to form single strand reference blocking sequences in step b) when the reaction mixture is subject to the first denaturing temperature.
The method of claim 1 wherein the reference blocking sequence is one of single stranded DNA, RNA, peptide nucleic acid or locked nucleic acid.
The method of claim 1 wherein the reference blocking sequence is a chimera between single stranded DNA, RNA, peptide nucleic acid or locked nucleic acid or another modified nucleotide.
The method of claim 7 wherein the position of the peptide nucleic acid or locked nucleic acid on the chimera sequence are selected to match positions where mutations are suspected to be present, thereby maximizing the difference between the temperature needed to denature heteroduplexes of the reference blocking sequence and target strands and the temperature needed to denature heteroduplexes of the reference blocking sequence and the complementary reference strand.
The method of claim 1 wherein the reference blocking sequence is fully complementary with one of the strands of the reference sequence between its primer binding sites, or overlapping at either end the primer binding sites.
The method of claim 1 wherein the reference blocking sequence is equal to or shorter than the reference sequence.
The method of claim 1 wherein the cooling step c) is less than one minute.
The method of claim 1 wherein the reference blocking sequence is present in the reaction mixture in an amount substantially equal to 25 nM.
The method of claim 1 wherein the melting temperature of the double-stranded target sequence is greater than or equal to the melting temperature of the double-stranded reference sequence.
The method of claim 1 wherein said reference and target sequences are first amplified by subjecting the reaction mixture to PCR and then subjecting at least a portion of the reaction mixture to the enrichment method of claim 1 .
The method of claim 1 wherein said target sequence comprises a homozygous mutation.
The method of claim 1 wherein said target sequence is differentially methylated from the reference sequence, and prior to implementing the method of claim 1 on the reaction mixture, the reaction mixture is treated with sodium bisulfite.
The method of claim 1 wherein said reference and target sequences comprise at least 25 base pairs.
The method of claim 1 wherein said method is repeated for two or more cycles.
The method of claim 1 further comprising the step of analyzing said reaction mixture with enriched target sequence using one or more of the methods selected from the group consisting of: MALDI-TOF, HR-Melting, Di-deoxy-sequencing, Single-molecule sequencing, pyrosequencing, Second generation high-throughput sequencing, SSCP, RFLP, dHPLC, CCM, digital PCR and quantitative-PCR.
The method of claim 1 wherein said T c is applied for 1 second-60 seconds.
The method of claim 1 wherein said reaction mixture contains a nucleic acid detection dye.
The method of claim 14 wherein said method is performed in a real-time PCR device.
The method of claim 1 performed under real-time reaction conditions utilizing a labeled probe.
A computer readable medium comprising program instructions for performing the method of claim 1 .
Ref US20110217714: Claims:
1. A method for enriching a target nucleic acid sequence in an amplification reaction mixture, said method comprising:
a. preparing an amplification reaction mixture including at least the following constituents:
a nucleic acid sample having a reference sequence and also suspected of having one or more target sequences that are at least 50% homologous to said reference sequence and are also amplifiable by the same primer pair as said reference sequence, and
an excess amount of reference blocking nucleic acid sequence which is fully complementary with at least a portion of the sequence of one of the strands of the reference sequence between its primer sites;
 b. 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; c. reducing the temperature of the reaction mixture so as to permit formation of heteroduplexes of the reference blocking sequence and the complementary reference strand and heteroduplexes of the reference blocking sequence and target strands; d. 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 reference strand; e. 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; and f. extending said primer pair so as to enrich said target sequence relative to said reference sequence.
2. The method of claim 1 wherein a 3′-end on the reference blocking sequence is blocked to inhibit extension.
3. The method of claim 1 wherein a 5′-end on the reference blocking sequence strands comprises a nucleotide that prevents 5′ to 3′ exonucleolysis by Taq DNA polymerases.
4. The method of claim 1 wherein reference blocking sequence provided in step a) is a single-stranded nucleic acid reference blocking sequence.
5. The method of claim 1 wherein reference blocking sequence is provided in step a) as double-stranded nucleic acid reference blocking sequence which denatures to form single strand reference blocking sequences in step b) when the reaction mixture is subject to the first denaturing temperature.
6. The method of claim 1 wherein the reference blocking sequence is one of single stranded DNA, RNA, peptide nucleic acid or locked nucleic acid.
7. The method of claim 1 wherein the reference blocking sequence is a chimera between single stranded DNA, RNA, peptide nucleic acid or locked nucleic acid or another modified nucleotide.
8. The method of claim 7 wherein the position of the peptide nucleic acid or locked nucleic acid on the chimera sequence are selected to match positions where mutations are suspected to be present, thereby maximizing the difference between the temperature needed to denature heteroduplexes of the reference blocking sequence and target strands and the temperature needed to denature heteroduplexes of the reference blocking sequence and the complementary reference strand.
9. The method of claim 1 wherein the reference blocking sequence is fully complementary with one of the strands of the reference sequence between its primer binding sites, or overlapping at either end the primer binding sites.
10. The method of claim 1 wherein the reference blocking sequence is equal to or shorter than the reference sequence.
11. The method of claim 1 wherein the cooling step c) is less than one minute.
12. The method of claim 1 wherein the reference blocking sequence is present in the reaction mixture in an amount substantially equal to 25 nM.
13. The method of claim 1 wherein the melting temperature of the double-stranded target sequence is greater than or equal to the melting temperature of the double-stranded reference sequence.
14. The method of claim 1 wherein said reference and target sequences are first amplified by subjecting the reaction mixture to PCR and then subjecting at least a portion of the reaction mixture to the enrichment method of claim 1.
15. The method of claim 1 wherein said target sequence comprises a homozygous mutation.
16. The method of claim 1 wherein said target sequence is differentially methylated from the reference sequence, and prior to implementing the method of claim 1 on the reaction mixture, the reaction mixture is treated with sodium bisulfite.
17. The method of claim 1 wherein said reference and target sequences comprise at least 25 base pairs.
18. The method of claim 1 wherein said method is repeated for two or more cycles.
19. The method of claim 1 further comprising the step of analyzing said reaction mixture with enriched target sequence using one or more of the methods selected from the group consisting of: MALDI-TOF, HR-Melting, Di-deoxy-sequencing, Single-molecule sequencing, pyrosequencing, Second generation high-throughput sequencing, SSCP, RFLP, dHPLC, CCM, digital PCR and quantitative-PCR.
20. The method of claim 1 wherein said Tc is applied for 1 second-60 seconds.
21. The method of claim 1 wherein said reaction mixture contains a nucleic acid detection dye.
22. The method of claim 14 wherein said method is performed in a real-time PCR device.
23. The method of claim 1 performed under real-time reaction conditions utilizing a labeled probe.
24. A computer readable medium comprising program instructions for performing the method of claim 1.
Ref APP-18_691325: Claims:
A method for detecting a target polynucleotide comprising a genetic alteration relative to a reference polynucleotide lacking the genetic alteration in a sample comprising a mixture of the target polynucleotide and the reference polynucleotide, the method comprising: (a) performing a polymerase chain reaction (PCR) amplification to obtain an amplification product in a reaction mixture comprising: (i) the sample; (ii) a high fidelity DNA polymerase that lacks 5′→3′ nuclease activity and comprises 3′→5′ nuclease activity; (iii) a pair of primers that flank the genetic alteration; and (iv) a blocking oligonucleotide that hybridizes selectively to the reference polynucleotide lacking the genetic alteration to form a blocking duplex; and (b) sequencing the amplification product and detecting the genetic alteration in the amplification product, thereby detecting the target polynucleotide comprising the genetic alteration in the sample.
(a) performing a polymerase chain reaction (PCR) amplification to obtain an amplification product in a reaction mixture comprising: (i) the sample; (ii) a high fidelity DNA polymerase that lacks 5′→3′ nuclease activity and comprises 3′→5′ nuclease activity; (iii) a pair of primers that flank the genetic alteration; and (iv) a blocking oligonucleotide that hybridizes selectively to the reference polynucleotide lacking the genetic alteration to form a blocking duplex; and
(ii) a high fidelity DNA polymerase that lacks 5′→3′ nuclease activity and comprises 3′→5′ nuclease activity;
(iii) a pair of primers that flank the genetic alteration; and
(iv) a blocking oligonucleotide that hybridizes selectively to the reference polynucleotide lacking the genetic alteration to form a blocking duplex; and
(b) sequencing the amplification product and detecting the genetic alteration in the amplification product, thereby detecting the target polynucleotide comprising the genetic alteration in the sample.
The method of claim 1, wherein the target polynucleotide represents no more than about 3% of the mixture.
The method of claim 1 or 2, wherein the target polynucleotide is present in the sample at a copy number less than about 30.
The method of claim 1, wherein the PCR amplification is performed for no more than 60 cycles.
The method of claim 1, wherein the DNA polymerase has an error rate of less than 10−5.
The method of claim 1, the amplification product is sequenced to obtain multiple reads, and the genetic alteration is present in at least about 90% of the multiple reads.
The method of claim 1, wherein the genetic alteration is present in at least about 90% of the amplification product.
The method of claim 1, the method comprising removing the blocking oligonucleotide from the amplification product prior to sequencing the amplification product.
The method of claim 1, further comprising determining the minimum number of PCR cycles necessary for detecting a target polynucleotide that is present in the mixture at a percentage of no more than 0.1%, 0.05%, 0.02%, or 0.01%.
The method of claim 1, wherein the blocking oligonucleotide comprises one or more modified nucleotides that enhance the stability of the duplex formed by the blocking oligonucleotide and the reference polynucleotide.
The method of claim 1, 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.
The method of claim 1, wherein the blocking oligonucleotide comprises one or more locked nucleic acids (LNAs) comprising a sugar moiety having a 2′-O conjugated to 4′-C via a methylene group.
The method of claim 12, wherein all of the nucleotides of the blocking oligonucleotide are LNAs.
The method of claim 1, wherein the blocking oligonucleotide has a length that is no more than about 20 nucleotides.
The method of claim 1, wherein the genetic alteration comprises a substitution.
The method of claim 1, wherein the genetic alteration comprises a deletion.
The method of claim 1, wherein the genetic alteration comprises an insertion.
The method of claim 1, wherein the genetic alteration is present in a genetic loci selected from the group consisting of PIK3CA, KRAS, APC, FAT4, KMT2D, KMT2C, and BRAF.
The method of claim 1, wherein the sample is a biological sample from a subject.
The method of claim 1, wherein the sample is a blood sample from a subject.
The method of claim 21, wherein the blood sample is a cell-free blood sample and/or the blood sample is treated to remove cells prior to performing PCR amplification.
The method of claim 1, wherein prior to the method being performed the sample was treated with a reagent that selectively modifies non-methylated cytosine residues to produce detectable modified residues but which does not modify methylated cytosine residues.
The method of claim 1, wherein the genetic alteration is associated with cancer in a subject.
The method of claim 24, wherein prior to performing step (a), the method comprises sequencing a cancer sample from the subject and detecting the genetic alteration in the cancer sample.
The method of claim 1, wherein the reference polynucleotide comprises a wild-type sequence with respect to the genetic alteration and the genetic alteration is a genetic mutation present in the target polynucleotide.
The method of claim 1, wherein the sample is obtained from a subject who has cancer or is at risk for developing cancer.
The method of claim 1, wherein the subject has been diagnosed with cancer and the subject currently is in remission.
A kit for performing a method for detecting a target polynucleotide comprising a genetic alteration relative to a reference polynucleotide lacking the genetic alteration in a sample comprising a mixture of the target polynucleotide and the reference polynucleotide, wherein the kit comprises: (i) a DNA polymerase that lacks 5′→3′ nuclease activity and comprises 3′→5′ nuclease activity; (ii) a pair of primers that flank the genetic alteration; and (iii) a blocking oligonucleotide that hybridizes selectively to the reference polynucleotide lacking the genetic alteration to form a blocking duplex and obtaining an amplified fragment sample.
The kit of claim 30, wherein the genetic alteration is in a genetic loci selected from PIK3CA, KRAS, APC, FAT4, KMT2D, KMT2C, and BRAF.
The kit of claim 30, wherein the blocking oligonucleotide consists of LNAs and the blocking oligonucleotide is no longer than 20 nucleotides.