Office Action 1: Non-Final Rejection (2024-10-17)
Rejection types: Double Patenting

CLAIMS:
CLAIMS We claim:

1\. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and a humanized monoclonal anti-human-PD-1 antibody or an antigen-binding fragment thereof comprising:
(i) a heavy chain variable domain comprising HCDR1 comprising SEQ ID NO: 1, HCDR2 comprising SEQ ID NO: 2 and HCDR3 comprising SEQ ID NO: 9, and
(ii) a light chain variable domain comprising LCDR1 comprising SEQ ID NO: 12, LCDR2 comprising SEQ ID NO: 13 and LCDR3 comprising SEQ ID NO: 14.

2\. The pharmaceutical composition of claim 1, wherein the humanized monoclonal anti-human-PD-1 antibody or an antigen-binding fragment thereof comprises:
(i) a heavy chain variable domain comprising HCDR1 consisting of SEQ ID NO: 1, HCDR2 consisting of SEQ ID NO: 2 and HCDR3 consisting of SEQ ID NO: 9, and
(ii) a light chain variable domain comprising LCDR1 consisting of SEQ ID NO: 12, LCDR2 consisting of SEQ ID NO: 13 and LCDR3 consisting of SEQ ID NO: 14.

3\. The pharmaceutical composition of claim 1, wherein the antibody or antigen- binding fragment thereof comprises a light chain constant domain derived from a human kappa light chain constant domain and a heavy chain constant domain derived from a human IgG1, IgG2, IgG3 or IgG4 heavy chain constant domain.

4\. The pharmaceutical composition of claim 1, wherein the antibody or antigen- binding fragment thereof comprises a light chain constant domain derived from a human kappa light chain constant domain and a heavy chain constant domain derived from a human IgG1 heavy chain constant domain, optionally with a substitution or a combination of substitutions selected from the group consisting of T250Q/M428L; M252Y/S254T/T256E + H433K/N434F;
E233P/L234V/L235A/G236A + A327G/A330S/P331 S; E333A; S239D/A330L/1332E;
P2571/Q311; K326W/E333S; S239D/1332E/G236A; N297A; L234A/L235A; N297A + M252Y/S254T/T256E; K322A and K444A, preferably selected from the group consisting of N297A optionally in combination with M252Y/S254T/T256E, and L234A/L235A.
5\. The pharmaceutical composition of claim 1, wherein the antibody or antigen- binding fragment thereof comprises a light chain constant domain derived from a human kappa light chain constant domain and a heavy chain constant domain derived from a human IgG4 heavy chain constant domain, optionally with a substitution or a combination of substitutions selected from the group consisting of S228P; L234A/L235A, S228P + M252Y/S254T/T256E and K444A.

6\. A method of treating cancer in a subject comprising administering a composition according to claim 1 to a subject having cancer, said cancer being selected from the group consisting of malignant mesothelioma, colorectal cancer and hepatocellular carcinoma.

7\. The method of claim 6, wherein the cancer has tumor cells which are PD-L1 negative.

8\. The method of claim 6, wherein said composition is administered in combination with radiotherapy or an additional therapeutic agent.

9\. The method of claim 6, wherein the subject to be treated is immunosuppressed, immunocompromised or immunodepressed.

OFFICE ACTION:
DETAILED ACTION
Notice of Pre-AIA  or AIA  Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Status of the claims
Claims 1-9 are pending and are examined.
Specification
35 U.S.C. 112(a) or pre-AIA  35 U.S.C.  112, requires the specification to be written in “full, clear, concise, and exact terms.” The specification is replete with terms which are not clear, concise and exact. The specification should be revised carefully in order to comply with 35 U.S.C. 112(a) or pre-AIA  35 U.S.C.  112. Examples of some unclear, inexact or verbose terms used in the specification are presented in the Certificate of correction accompanying U.S. Pat. No. 11,352,430.
Double Patenting
The nonstatutory double patenting rejection is based on a judicially created doctrine grounded in public policy (a policy reflected in the statute) so as to prevent the unjustified or improper timewise extension of the “right to exclude” granted by a patent and to prevent possible harassment by multiple assignees. A nonstatutory double patenting rejection is appropriate where the conflicting claims are not identical, but at least one examined application claim is not patentably distinct from the reference claim(s) because the examined application claim is either anticipated by, or would have been obvious over, the reference claim(s). See, e.g., In re Berg, 140 F.3d 1428, 46 USPQ2d 1226 (Fed. Cir. 1998); In re Goodman, 11 F.3d 1046, 29 USPQ2d 2010 (Fed. Cir. 1993); In re Longi, 759 F.2d 887, 225 USPQ 645 (Fed. Cir. 1985); In re Van Ornum, 686 F.2d 937, 214 USPQ 761 (CCPA 1982); In re Vogel, 422 F.2d 438, 164 USPQ 619 (CCPA 1970); In re Thorington, 418 F.2d 528, 163 USPQ 644 (CCPA 1969).
A timely filed terminal disclaimer in compliance with 37 CFR 1.321(c) or 1.321(d) may be used to overcome an actual or provisional rejection based on nonstatutory double patenting provided the reference application or patent either is shown to be commonly owned with the examined application, or claims an invention made as a result of activities undertaken within the scope of a joint research agreement. See MPEP § 717.02 for applications subject to examination under the first inventor to file provisions of the AIA  as explained in MPEP § 2159. See MPEP § 2146 et seq. for applications not subject to examination under the first inventor to file provisions of the AIA . A terminal disclaimer must be signed in compliance with 37 CFR 1.321(b).
The filing of a terminal disclaimer by itself is not a complete reply to a nonstatutory double patenting (NSDP) rejection. A complete reply requires that the terminal disclaimer be accompanied by a reply requesting reconsideration of the prior Office action. Even where the NSDP rejection is provisional the reply must be complete. See MPEP § 804, subsection I.B.1. For a reply to a non-final Office action, see 37 CFR 1.111(a). For a reply to final Office action, see 37 CFR 1.113(c). A request for reconsideration while not provided for in 37 CFR 1.113(c) may be filed after final for consideration. See MPEP §§ 706.07(e) and 714.13.
The USPTO Internet website contains terminal disclaimer forms which may be used. Please visit www.uspto.gov/patent/patents-forms. The actual filing date of the application in which the form is filed determines what form (e.g., PTO/SB/25, PTO/SB/26, PTO/AIA /25, or PTO/AIA /26) should be used. A web-based eTerminal Disclaimer may be filled out completely online using web-screens. An eTerminal Disclaimer that meets all requirements is auto-processed and approved immediately upon submission. For more information about eTerminal Disclaimers, refer to www.uspto.gov/patents/apply/applying-online/eterminal-disclaimer.
Claims 1-9 are rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1-6 and 11-15 of U.S. Patent No. 11,352,430. Although the claims at issue are not identical, they are not patentably distinct from each other because the U.S Patent is claiming the same antibody that is comprised in the instant pharmaceutical composition and for the same treatments. it would have been obvious for a person of ordinary skill in the art at the time that the invention was filed to have used the antibody of the Patent to formulate a pharmaceutical composition since the use of the antibody is as a therapeutical and the art was highly developed in formulation of pharmaceutical compositions for antibodies to be used in treatment.
Conclusion
No claims are allowed.

APPLICANT REBUTTAL: Remarks
Claims 1-9 are pending in the subject application. By this Amendment, Applicant has amended the application. It is respectfully submitted that no new matter is introduced by this amendment. Entry and consideration of the amendments presented herein is respectfully requested. Accordingly, claims 1-9 are currently before the Examiner. Favorable consideration of the pending claims is respectfully requested.
The application was filed with Figures 1-14. The Office Action Summary page did not indicate that the drawings were accepted or objected to by the Examiner. Applicant respectfully requests that the Examiner consider the figures and indicate their status in the next communication.
The Examiner indicates that 35 U.S.C. § 1.12(a) requires the specification to be written in "full, clear, concise, and exact terms". The Office Action points to examples of some unclear, inexact or verbose terms used in the specification that were presented in the Certificate of Correction filed in the parent patent. Applicant notes that the Certificate of Correction that was filed (and subsequently issued) in U.S. Patent No. 11,352,430 identified errors that were introduced by the Patent Office when printing the patent. Regardless, Applicant has made amendments to the specification to correct typographical issues and grammatical issues. Entry of the amendments is respectfully requested.
Claims 1-9 are rejected under the judicially created doctrine of "obviousness-type" double patenting over claims 1-6 and 11-15 of U.S. Patent No. 11,352,430. Applicant respectfully asserts that the claims are not obvious over the cited patent. However, in order to expedite prosecution of the subject application, Applicant has submitted a Terminal Disclaimer with this Amendment which obviates this rejection. Accordingly, reconsideration and withdrawal of the rejection is respectfully requested.
It should be understood that the amendments presented herein have been made
solely
to expedite prosecution of the subject application to completion and should not be construed as an indication of Applicant's agreement with or acquiescence to the Examiner's position. Applicant expressly reserves the right to pursue the invention(s) disclosed in the subject application, including any subject matter canceled or not pursued during prosecution of the subject application, in a related application.
In view of the foregoing remarks, Applicant believes that the currently pending claims are in condition for allowance, and such action is respectfully requested.
The Commissioner is hereby authorized to charge any fees under 37 C.F.R. § § 1.16 or 1.17 as required by this paper to Deposit Account No. 19-0065.
Applicant invites the Examiner to call the undersigned if clarification is needed on any of this response, or if the Examiner believes a telephonic interview would expedite the prosecution of the subject application to completion.
The undersigned hereby certifies that this correspondence is being transmitted by EFS-Web to the United States Patent and Trademark Office on January 17, 2025.
Respectfully submitted, /FRANK C. EISENSCHENK/Frank C. Eisenschenk, Ph.D.Patent Attorney

NEXT OFFICE ACTION: EXAMINER'S AMENDMENT
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 .
An examiner’s amendment to the record appears below. Should the changes and/or additions be unacceptable to applicant, an amendment may be filed as provided by 37 CFR 1.312. To ensure consideration of such an amendment, it MUST be submitted no later than the payment of the issue fee.
The application has been amended as follows:
In the title, after: “antibody”, please add:
-- and methods of using them for cancer treatment --.
In the Remarks submitted on 01/172025, Applicant requested that the status of the drawings submitted on 06/02/2022 should be indicated. Examiner positively affirm that the drawings were considered and accepted by the Examiner.
The Terminal Disclaimer over the U.S. Patent 11,352,430 submitted on 01/17/2025 was considered and approved by the Office.
The allowed claims are: 1-9.
Reasons for Allowance
The following is an examiner’s statement of reasons for allowance: Applicant submitted an amended Specification and a terminal disclaimed thus obviating the objections and rejections in the previous Office action.
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 US20220205050: Claims:
A method for identifying, in a sample, one or more parent nucleic acid molecules containing a target nucleotide sequence differing from nucleotide sequences of other parent nucleic acid molecules in the sample by one or more nucleotides, one or more copy numbers, one or more transcript sequences, and/or one or more methylated or hydroxymethylated residues, said method comprising: providing a sample containing one or more parent nucleic acid molecules potentially containing the target nucleotide sequence differing from the nucleotide sequences of other parent nucleic acid molecules by one or more nucleotides, one or more copy numbers, one or more transcript sequences, and/or one or more methylated or hydroxymethylated residues; providing one or more enzymes capable of digesting deoxyuracil (dU)-containing nucleic acid molecules; providing one or more primary oligonucleotide primer sets, each primary oligonucleotide primer set comprising (a) a first primary oligonucleotide primer that comprises a nucleotide sequence that is complementary to a sequence in the parent nucleic acid molecule adjacent to the target nucleotide sequence and (b) a second primary oligonucleotide primer that comprises a nucleotide sequence that is complementary to a portion of an extension product formed from the first primary oligonucleotide primer, wherein the first or second primary oligonucleotide primer further comprises of a 5â² primer-specific portion; blending the sample, the one or more first primary oligonucleotide primers of the primer sets, the one or more enzymes capable of digesting deoxyuracil (dU)-containing nucleic acid molecules, a deoxynucleotide mix, and a DNA polymerase to form one or more polymerase extension reaction mixtures; subjecting the one or more polymerase extension reaction mixtures to conditions suitable for digesting deoxyuracil (dU)-containing nucleic acid molecules present in the polymerase extension reaction mixtures and for carrying out one or more polymerase extension reaction cycles comprising a denaturation treatment, a hybridization treatment, and an extension treatment, thereby forming primary extension products comprising nucleotide sequences complementary to the target nucleotide sequence; blending the one or more polymerase extension reaction mixtures comprising the primary extension products, the one or more second primary oligonucleotide primers of the primary oligonucleotide primer sets, the one or more enzymes capable of digesting deoxyuracil (dU)-containing nucleic acid molecules in the reaction mixtures, a deoxynucleotide mix including dUTP, and a DNA polymerase to form one or more first polymerase chain reaction mixtures; subjecting the one or more first polymerase chain reaction mixtures to conditions suitable for digesting deoxyuracil (dU)-containing nucleic acid molecules present in the polymerase chain reaction mixtures and for carrying out one or more first polymerase chain reaction cycles comprising a denaturation treatment, a hybridization treatment, and an extension treatment, thereby forming first polymerase chain reaction products comprising the target nucleotide sequence or a complement thereof; providing one or more oligonucleotide probe sets, each probe set comprising (a) a first oligonucleotide probe having a 5â² primer-specific portion and a 3â² target sequence-specific portion, and (b) a second oligonucleotide probe having a 5â² target sequence-specific portion and a 3â² primer-specific portion, wherein the first and second oligonucleotide probes of a probe set are configured to hybridize, in a base specific manner, on a complementary target nucleotide sequence of a first polymerase chain reaction product; blending the first polymerase chain reaction products with a ligase, and the one or more oligonucleotide probe sets to form one or more ligation reaction mixtures; subjecting the one or more ligation reaction mixtures to one or more ligation reaction cycles whereby the first and second oligonucleotide probes of the one or more oligonucleotide probe sets are ligated together, when hybridized to their complementary sequence, to form ligated product sequences in the ligation reaction mixtures wherein each ligated product sequence comprises the 5â² primer-specific portion, the target-specific portions, and the 3â² primer-specific portion; providing one or more secondary oligonucleotide primer sets, each secondary oligonucleotide primer set comprising (a) a first secondary oligonucleotide primer comprising the same nucleotide sequence as the 5â² primer-specific portion of the ligated product sequence and (b) a second secondary oligonucleotide primer comprising a nucleotide sequence that is complementary to the 3â² primer-specific portion of the ligated product sequence; blending the ligated product sequences, the one or more secondary oligonucleotide primer sets, the one or more enzymes capable of digesting deoxyuracil (dU)-containing nucleic acid molecules, a deoxynucleotide mix including dUTP, and a DNA polymerase to form one or more second polymerase chain reaction mixtures; subjecting the one or more second polymerase chain reaction mixtures to conditions suitable for digesting deoxyuracil (dU)-containing nucleic acid molecules present in the second polymerase chain reaction mixtures and for carrying out one or more polymerase chain reaction cycles comprising a denaturation treatment, a hybridization treatment, and an extension treatment thereby forming second polymerase chain reaction products; and detecting and distinguishing the second polymerase chain reaction products in the one or more second polymerase chain reaction mixtures to identify the presence of one or more parent nucleic acid molecules containing target nucleotide sequences differing from nucleotide sequences in other parent nucleic acid molecules in the sample by one or more nucleotides, one or more copy numbers, one or more transcript sequences, and/or one or more methylated or hydroxymethylated residues.
providing a sample containing one or more parent nucleic acid molecules potentially containing the target nucleotide sequence differing from the nucleotide sequences of other parent nucleic acid molecules by one or more nucleotides, one or more copy numbers, one or more transcript sequences, and/or one or more methylated or hydroxymethylated residues;
providing one or more enzymes capable of digesting deoxyuracil (dU)-containing nucleic acid molecules;
providing one or more primary oligonucleotide primer sets, each primary oligonucleotide primer set comprising (a) a first primary oligonucleotide primer that comprises a nucleotide sequence that is complementary to a sequence in the parent nucleic acid molecule adjacent to the target nucleotide sequence and (b) a second primary oligonucleotide primer that comprises a nucleotide sequence that is complementary to a portion of an extension product formed from the first primary oligonucleotide primer, wherein the first or second primary oligonucleotide primer further comprises of a 5â² primer-specific portion;
blending the sample, the one or more first primary oligonucleotide primers of the primer sets, the one or more enzymes capable of digesting deoxyuracil (dU)-containing nucleic acid molecules, a deoxynucleotide mix, and a DNA polymerase to form one or more polymerase extension reaction mixtures;
subjecting the one or more polymerase extension reaction mixtures to conditions suitable for digesting deoxyuracil (dU)-containing nucleic acid molecules present in the polymerase extension reaction mixtures and for carrying out one or more polymerase extension reaction cycles comprising a denaturation treatment, a hybridization treatment, and an extension treatment, thereby forming primary extension products comprising nucleotide sequences complementary to the target nucleotide sequence;
blending the one or more polymerase extension reaction mixtures comprising the primary extension products, the one or more second primary oligonucleotide primers of the primary oligonucleotide primer sets, the one or more enzymes capable of digesting deoxyuracil (dU)-containing nucleic acid molecules in the reaction mixtures, a deoxynucleotide mix including dUTP, and a DNA polymerase to form one or more first polymerase chain reaction mixtures;
subjecting the one or more first polymerase chain reaction mixtures to conditions suitable for digesting deoxyuracil (dU)-containing nucleic acid molecules present in the polymerase chain reaction mixtures and for carrying out one or more first polymerase chain reaction cycles comprising a denaturation treatment, a hybridization treatment, and an extension treatment, thereby forming first polymerase chain reaction products comprising the target nucleotide sequence or a complement thereof;
providing one or more oligonucleotide probe sets, each probe set comprising (a) a first oligonucleotide probe having a 5â² primer-specific portion and a 3â² target sequence-specific portion, and (b) a second oligonucleotide probe having a 5â² target sequence-specific portion and a 3â² primer-specific portion, wherein the first and second oligonucleotide probes of a probe set are configured to hybridize, in a base specific manner, on a complementary target nucleotide sequence of a first polymerase chain reaction product;
blending the first polymerase chain reaction products with a ligase, and the one or more oligonucleotide probe sets to form one or more ligation reaction mixtures;
subjecting the one or more ligation reaction mixtures to one or more ligation reaction cycles whereby the first and second oligonucleotide probes of the one or more oligonucleotide probe sets are ligated together, when hybridized to their complementary sequence, to form ligated product sequences in the ligation reaction mixtures wherein each ligated product sequence comprises the 5â² primer-specific portion, the target-specific portions, and the 3â² primer-specific portion;
providing one or more secondary oligonucleotide primer sets, each secondary oligonucleotide primer set comprising (a) a first secondary oligonucleotide primer comprising the same nucleotide sequence as the 5â² primer-specific portion of the ligated product sequence and (b) a second secondary oligonucleotide primer comprising a nucleotide sequence that is complementary to the 3â² primer-specific portion of the ligated product sequence;
blending the ligated product sequences, the one or more secondary oligonucleotide primer sets, the one or more enzymes capable of digesting deoxyuracil (dU)-containing nucleic acid molecules, a deoxynucleotide mix including dUTP, and a DNA polymerase to form one or more second polymerase chain reaction mixtures;
subjecting the one or more second polymerase chain reaction mixtures to conditions suitable for digesting deoxyuracil (dU)-containing nucleic acid molecules present in the second polymerase chain reaction mixtures and for carrying out one or more polymerase chain reaction cycles comprising a denaturation treatment, a hybridization treatment, and an extension treatment thereby forming second polymerase chain reaction products; and
detecting and distinguishing the second polymerase chain reaction products in the one or more second polymerase chain reaction mixtures to identify the presence of one or more parent nucleic acid molecules containing target nucleotide sequences differing from nucleotide sequences in other parent nucleic acid molecules in the sample by one or more nucleotides, one or more copy numbers, one or more transcript sequences, and/or one or more methylated or hydroxymethylated residues.
A method for identifying, in a sample, one or more parent nucleic acid molecules containing a target nucleotide sequence differing from nucleotide sequences of other parent nucleic acid molecules in the sample by one or more nucleotides, one or more copy numbers, one or more transcript sequences, and/or one or more methylated or hydroxymethylated residues, said method comprising: providing a sample containing one or more parent nucleic acid molecules potentially containing the target nucleotide sequence differing from the nucleotide sequences of other parent nucleic acid molecules by one or more nucleotides, one or more copy numbers, one or more transcript sequences, and/or one or more methylated or hydroxymethylated residues; providing one or more enzymes capable of digesting deoxyuracil (dU)-containing nucleic acid molecules; providing one or more nucleases capable of digesting nucleic acid molecules present not comprising polymerase-incorporated modified nucleotides; providing one or more primary oligonucleotide primer sets, each primary oligonucleotide primer set comprising (a) a first primary oligonucleotide primer that comprises a nucleotide sequence that is complementary to a sequence in the parent nucleic acid molecule adjacent to the target nucleotide sequence and (b) a second primary oligonucleotide primer that comprises a nucleotide sequence that is complementary to a portion of an extension product formed from the first primary oligonucleotide primer, wherein the first or second primary oligonucleotide primer further comprises a 5â² primer-specific portion; blending the sample, the one or more first primary oligonucleotide primers, the one or more enzymes capable of digesting deoxyuracil (dU)-containing nucleic acid molecules, a deoxynucleotide mix that comprises one or more modified nucleotides that protect extension products but not other DNA from nuclease digestion, and a DNA polymerase to form one or more polymerase extension reaction mixtures; subjecting the one or more polymerase extension reaction mixtures to conditions suitable for digesting deoxyuracil (dU)-containing nucleic acid molecules present in the polymerase extension reaction mixtures and for carrying out one or more polymerase extension reaction cycles comprising a denaturation treatment, a hybridization treatment, and an extension treatment, thereby forming primary extension products comprising modified nucleotide bases and comprising the complement of the target nucleotide sequence; blending the one or more polymerase extension reaction mixtures comprising the primary extension products with the one or more nucleases; subjecting the one or more polymerase extension reaction mixtures comprising the primary extension products with the one or more nucleases to conditions suitable for digesting nucleic acid molecules but not primary extension products comprising modified nucleotides to produce one or more partially digested polymerase extension reaction mixtures; blending the one or more partially digested polymerase extension reaction mixtures, the one or more secondary primary oligonucleotide primers of the primer sets, a deoxynucleotide mix, and a DNA polymerase to form one or more first polymerase chain reaction mixtures; subjecting the one or more first polymerase chain reaction mixtures to conditions suitable for carrying out two or more first polymerase chain reaction cycles comprising a denaturation treatment, a hybridization treatment, and an extension treatment, thereby forming first polymerase chain reaction products comprising the target nucleotide sequence or a complement thereof; providing one or more secondary oligonucleotide primer sets, each secondary oligonucleotide primer set comprising (a) a first secondary oligonucleotide primer having a 3â² portion that is complementary to a portion of an extension product formed from the first primary oligonucleotide primer and (b) a second secondary oligonucleotide primer having a 3â² portion that comprises a nucleotide sequence that is complementary to a portion of an extension product formed from the first secondary oligonucleotide primer; blending the first polymerase chain reaction products, the one or more secondary oligonucleotide primer sets, the one or more enzymes capable of digesting deoxyuracil (dU) containing nucleic acid molecules, a deoxynucleotide mix including dUTP, and a DNA polymerase to form one or more second polymerase chain reaction mixtures; subjecting the one or more second polymerase chain reaction mixtures to conditions suitable for digesting deoxyuracil (dU)-containing nucleic acid molecules present in the second polymerase chain reaction mixtures and for carrying out two or more second polymerase chain reaction cycles comprising a denaturation treatment, a hybridization treatment, and an extension treatment thereby forming second polymerase chain reaction products; and detecting and distinguishing the second polymerase chain reaction products in the one or more second polymerase chain reaction mixtures to identify the presence of one or more parent nucleic acid molecules containing target nucleotide sequences differing from nucleotide sequences in other parent nucleic acid molecules in the sample by one or more nucleotides, one or more copy numbers, one or more transcript sequences, and/or one or more methylated or hydroxymethylated residues.
providing one or more nucleases capable of digesting nucleic acid molecules present not comprising polymerase-incorporated modified nucleotides;
providing one or more primary oligonucleotide primer sets, each primary oligonucleotide primer set comprising (a) a first primary oligonucleotide primer that comprises a nucleotide sequence that is complementary to a sequence in the parent nucleic acid molecule adjacent to the target nucleotide sequence and (b) a second primary oligonucleotide primer that comprises a nucleotide sequence that is complementary to a portion of an extension product formed from the first primary oligonucleotide primer, wherein the first or second primary oligonucleotide primer further comprises a 5â² primer-specific portion;
blending the sample, the one or more first primary oligonucleotide primers, the one or more enzymes capable of digesting deoxyuracil (dU)-containing nucleic acid molecules, a deoxynucleotide mix that comprises one or more modified nucleotides that protect extension products but not other DNA from nuclease digestion, and a DNA polymerase to form one or more polymerase extension reaction mixtures;
subjecting the one or more polymerase extension reaction mixtures to conditions suitable for digesting deoxyuracil (dU)-containing nucleic acid molecules present in the polymerase extension reaction mixtures and for carrying out one or more polymerase extension reaction cycles comprising a denaturation treatment, a hybridization treatment, and an extension treatment, thereby forming primary extension products comprising modified nucleotide bases and comprising the complement of the target nucleotide sequence;
blending the one or more polymerase extension reaction mixtures comprising the primary extension products with the one or more nucleases;
subjecting the one or more polymerase extension reaction mixtures comprising the primary extension products with the one or more nucleases to conditions suitable for digesting nucleic acid molecules but not primary extension products comprising modified nucleotides to produce one or more partially digested polymerase extension reaction mixtures;
blending the one or more partially digested polymerase extension reaction mixtures, the one or more secondary primary oligonucleotide primers of the primer sets, a deoxynucleotide mix, and a DNA polymerase to form one or more first polymerase chain reaction mixtures;
subjecting the one or more first polymerase chain reaction mixtures to conditions suitable for carrying out two or more first polymerase chain reaction cycles comprising a denaturation treatment, a hybridization treatment, and an extension treatment, thereby forming first polymerase chain reaction products comprising the target nucleotide sequence or a complement thereof;
providing one or more secondary oligonucleotide primer sets, each secondary 
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