DETAILED ACTION
Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Election/Restrictions
It is acknowledged that the Applicant elected claims 1, 5-8, 10-12, 14-16, 18-27 and 72 without traverse in the reply filed June 29, 2026.
Claims 1, 5-8, 10-12, 14-16, 18-28, 49, and 72 are currently pending.
Claims 28 and 49 are withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to nonelected subject matter, there being no allowable generic or linking claim.
Claims 1, 5-8, 10-12, 14-16, 18-27 and 72 are examined herein.
Priority
It is acknowledged that the instant application in a continuation of International PCT Application No. PCT/EP2022/079299, filed October 20, 2022, and claims benefit of provisional 63/257,954, filed October 20, 2021. The effective filing date is considered to be October 20, 2021.
Nucleotide and/or Amino Acid Sequence Disclosures
REQUIREMENTS FOR PATENT APPLICATIONS CONTAINING NUCLEOTIDE AND/OR AMINO ACID SEQUENCE DISCLOSURES
Items 1) and 2) provide general guidance related to requirements for sequence disclosures.
37 CFR 1.821(c) requires that patent applications which contain disclosures of nucleotide and/or amino acid sequences that fall within the definitions of 37 CFR 1.821(a) must contain a "Sequence Listing," as a separate part of the disclosure, which presents the nucleotide and/or amino acid sequences and associated information using the symbols and format in accordance with the requirements of 37 CFR 1.821 - 1.825. This "Sequence Listing" part of the disclosure may be submitted:
In accordance with 37 CFR 1.821(c)(1) via the USPTO patent electronic filing system (see Section I.1 of the Legal Framework for Patent Electronic System (https://www.uspto.gov/PatentLegalFramework), hereinafter "Legal Framework") as an ASCII text file, together with an incorporation-by-reference of the material in the ASCII text file in a separate paragraph of the specification as required by 37 CFR 1.823(b)(1) identifying:
the name of the ASCII text file;
ii) the date of creation; and
iii) the size of the ASCII text file in bytes;
In accordance with 37 CFR 1.821(c)(1) on read-only optical disc(s) as permitted by 37 CFR 1.52(e)(1)(ii), labeled according to 37 CFR 1.52(e)(5), with an incorporation-by-reference of the material in the ASCII text file according to 37 CFR 1.52(e)(8) and 37 CFR 1.823(b)(1) in a separate paragraph of the specification identifying:
the name of the ASCII text file;
the date of creation; and
the size of the ASCII text file in bytes;
In accordance with 37 CFR 1.821(c)(2) via the USPTO patent electronic filing system as a PDF file (not recommended); or
In accordance with 37 CFR 1.821(c)(3) on physical sheets of paper (not recommended).
When a “Sequence Listing” has been submitted as a PDF file as in 1(c) above (37 CFR 1.821(c)(2)) or on physical sheets of paper as in 1(d) above (37 CFR 1.821(c)(3)), 37 CFR 1.821(e)(1) requires a computer readable form (CRF) of the “Sequence Listing” in accordance with the requirements of 37 CFR 1.824.
If the "Sequence Listing" required by 37 CFR 1.821(c) is filed via the USPTO patent electronic filing system as a PDF, then 37 CFR 1.821(e)(1)(ii) or 1.821(e)(2)(ii) requires submission of a statement that the "Sequence Listing" content of the PDF copy and the CRF copy (the ASCII text file copy) are identical.
If the "Sequence Listing" required by 37 CFR 1.821(c) is filed on paper or read-only optical disc, then 37 CFR 1.821(e)(1)(ii) or 1.821(e)(2)(ii) requires submission of a statement that the "Sequence Listing" content of the paper or read-only optical disc copy and the CRF are identical.
Specific deficiencies and the required response to this Office Action are as follows:
Specific deficiency – Nucleotide and/or amino acid sequences appearing in the specification are not identified by sequence identifiers in accordance with 37 CFR 1.821(d).
Required response – Applicant must provide:
A substitute specification in compliance with 37 CFR 1.52, 1.121(b)(3) and 1.125 inserting the required sequence identifiers, consisting of:
A copy of the previously-submitted specification, with deletions shown with strikethrough or brackets and insertions shown with underlining (marked-up version);
A copy of the amended specification without markings (clean version); and
A statement that the substitute specification contains no new matter.
Specific deficiency – Nucleotide and/or amino acid sequences appearing in the drawings are not identified by sequence identifiers in accordance with 37 CFR 1.821(d). Sequence identifiers for nucleotide and/or amino acid sequences must appear either in the drawings or in the Brief Description of the Drawings.
Required response – Applicant must provide:
Replacement and annotated drawings in accordance with 37 CFR 1.121(d) inserting the required sequence identifiers;
AND/OR
A substitute specification in compliance with 37 CFR 1.52, 1.121(b)(3) and 1.125 inserting the required sequence identifiers into the Brief Description of the Drawings, consisting of:
A copy of the previously-submitted specification, with deletions shown with strikethrough or brackets and insertions shown with underlining (marked-up version);
A copy of the amended specification without markings (clean version); and
A statement that the substitute specification contains no new matter.
Claim Rejections - 35 USC § 112
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, 5-8, 10-12, 14-16, 18-27 and 72 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, 5-8, 10-12, 14-16, 18-27 and 72 are rejected for the recitation of “hemiprobe sequence” in claims 1, 6, 7, 15, 16, and 72, as indefinite. A limiting definition of ‘hemiprobe sequence’ is not given in either the claims or the specification and there is no clear definition of the term in the art. It is not clear whether the term as used in the claims is intended to limit the stem-loop primer (e.g. by requiring specific structural features) or if it is simply the term used by the Applicant to describe the 5’ and 3’ sequences of the stem-loop primer, with all structural requirements explicitly set forth in the claims. As a result, one of skill in the art would not be able to determine the metes and bounds of the claimed subject matter.
Claims 1, 5-8, 10-12, 14-16, 18-27 and 72 are rejected because the requirements for the potential variant DNA sequence are not clear. Claim 1 recites a generic ‘DNA sequence,’ but requires that the reaction mixture contain ‘a reverse primer configured to hybridize to a genomic region’ within the DNA sequence. Claim 14 limits the DNA sequence to one comprising genomic DNA. Is ‘genomic region’ in claim 1 intended to limit the potential variant DNA sequence to one comprised of genomic DNA? If so, it is not clear how claim 14 provides further limitations. If the DNA of claim 1 is not limited to genomic DNA, it is unclear what is meant by ‘genomic region’ and how the reverse primer is intended to be configured. As a result, one of skill in the art would not be able to determine the metes and bounds of the claimed subject matter.
Claims 1, 5-8, 10-12, 14-16, 18-27 and 72 are rejected for the recitation of “genomic” in claims 1 and 14 as indefinite. The claims and specification do not provide a limiting definition of ‘genomic DNA’ or ‘genomic region’ and it is unclear what is intended to be excluded by the term. The specification allows that genomic DNA may be an example of double-stranded DNA (par. 33), and the plain meaning of genomic DNA encompasses at least any DNA derived from an organism’s genome. It is not clear if the limitations are intended to exclude mitochondrial DNA, cell-free DNA, single-stranded DNA, plasmid DNA, amplified product, or some other combination of DNA. As a result, one of skill in the art would not be able to determine the metes and bounds of the claimed subject matter. For the purposes of compact prosecution, ‘genomic’ is interpreted to mean DNA derived from an organism’s genome.
Claims 1, 5-8, 10-12, 14-16, 18-27 and 72 are rejected for the recitation of “a reverse primer configured to hybridize to a genomic region 3’ from said variation of said at least one nucleotide” as indefinite. The variant is expected to exist in the products of amplification as well as the input DNA sequence itself, and the variation is expected to occur as corresponding sets of nucleotides on either strand. Therefore, it is unclear if the limitation is intended to restrict the hybridization target of the reverse primer to a particular template (either the input DNA sequence or amplified product) or to a particular strand of a particular template (either the plus or minus strand). As a result, one of skill in the art would not be able to determine the metes and bounds of the claimed subject matter. Clarification is requested.
Claim 19 is rejected because it is unclear which elements are meant to have a Tm of 55-75 ºC. Tm is a characteristic of binding between at least partially complementary nucleic acid molecules. In this case, for example, it is unclear which of the following are intended to have a Tm of 55-75ºC:
The stem-loop sequence and its complement in the extension product
Self-complementary internal sequences of the stem-loop sequence which form the stem
If what is intended is that some self-complementary portion of the stem-loop sequence of the primer has a Tm of 55-75ºC, the claim must be amended to reflect that meaning.
Claim 72 is rejected for the recitation of “for processing a DNA sequence having or suspected of having a methylated cytosine at a particular residue” and “wherein said DNA sequence comprises a uridine residue” as indefinite. It is unclear whether the residues recited in the two lines are intended to be at the same position (i.e. intended to be the same residue). If the DNA sequence is only suspected of having a methylated cytosine at a particular residue, it is apparent that the residue may be instead an unmethylated cytosine (or any other type of residue). In the case that there is an unmethylated cytosine, a process such as bisulfite sequencing (par. 97) may result in an altered DNA sequence possessing a uridine residue at the same position. The claim requires that the DNA sequences are the same. Therefore, it is unclear how the DNA sequence may have simultaneously an unmethylated cytosine and a uridine residue at the same position. In the case that the DNA sequence has a methylated cytosine at a particular residue, bisulfite treatment would not alter the identity of the residue. Therefore, the recitation that the DNA sequence has a methylated cytosine is also contradictory to the recitation that the DNA sequence comprises a uridine residue. If the claim is intended to recite a step of bisulfite treatment, it must be amended to include said step. If the claim is intended to recite ‘a method for processing a DNA sequence to determine methylation status at a particular position’, it must be amended to reflect that meaning.
Claim Rejections - 35 USC § 102
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 the appropriate paragraphs of pre-AIA 35 U.S.C. 102 that form the basis for the rejections under this section made in this Office action:
A person shall be entitled to a patent unless –
(a) the invention was known or used by others in this country, or patented or described in a printed publication in this or a foreign country, before the invention thereof by the applicant for a patent.
(b) the invention was patented or described in a printed publication in this or a foreign country or in public use or on sale in this country, more than one year prior to the date of application for patent in the United States.
Claims 1, 5-7, 10, 14-16, 21-23, and 27 are rejected as anticipated by Marras et al. (published Aug. 14, 2014; International Publication No. WO 2014/124290).
Regarding claim 1, Marras recites a method for processing a DNA sequence having or suspected of having sequence variation relative to a wild-type sequence (pg. 5, 1st par.). Marras recites that the method comprises combining reagents in a reaction mixture suitable for processing said DNA sequence (pg. 12, last par.). Marras recites that the reaction mixture comprises:
said DNA sequence, wherein said DNA sequence comprises a variation of at least one nucleotide relative to said wild-type sequence (pg. 5, 1st par.);
a stem-loop primer (for example, Fig. 4: last three panels)
a reverse primer configured to hybridize to a genomic region 3' from said variation of said at least one nucleotide relative to said wild-type sequence (pg. 18, last par.; Fig. 2, element ‘203’);
The variation of at least one nucleotide is represented by the ‘x’ in Figure 1, relative to the ‘y’ of a wildtype sequence (Fig. 1; pg. 7, ln. 5-10). Here, Marras is directed to the use of genomic DNA (pg. 25, 2nd par.). For clarity, Marras’s Figure 2 (see below) shows the DNA sequence as element 101, the stem-loop primer as element 103, and the reverse primer as element 203. In Marras, the stem-loop primer is called a ‘multi-part primer.’
Figure 2:
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Marras states: “In the 5’-to-3’ direction, the primer includes anchor sequence 104, bridge sequence 105, and foot sequence 106” (pg. 7, ln. 11-12). Consistent with this orientation, Fig. 2 shows the extension product ‘202’ (extended from the stem-loop primer), which hybridizes to the reverse primer ‘203’ at a region which is 3’ relative to the variant. This satisfies the limitation that the reverse primer is configured to hybridize to a region 3’ from a variant sequence (and is consistent with the instant specification – see instant Fig. 1).
Marras recites that the stem-loop primer comprises (Fig 1):
a 5' hemiprobe sequence configured to hybridize to a complementary first end region of said DNA sequence;
a stem-loop sequence (Fig. 4; pg. 20, ln. 5-27); and
a 3' hemiprobe sequence configured to hybridize to a second end region of said DNA sequence,
wherein a 3' portion of said 3' hemiprobe sequence comprises a nucleotide sequence complementary to said variation of said at least one nucleotide relative to said wild-type sequence but not complementary to said wild-type sequence;
For clarity, in Marras’s Figure 1 (see below; pg. 7), the 5’ hemiprobe sequence may be element 104 (‘the anchor sequence’) and the 3’ hemiprobe sequence may be element 106 (‘the foot sequence’). The variation of at least one nucleotide is represented by the ‘x’ in Figure 1, relative to the ‘y’ of a wildtype sequence (Fig. 1; pg. 7, ln. 21-22).
Figure 1:
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The stem-loop sequence could be the hairpins variants 404 and 409 comprised in the primer, as shown in the last three diagrams of Fig. 4 (see below; pg. 20, ln. 5-27).
Figure 4:
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Marras recites incubating said reaction mixture under conditions suitable to extend a product (pg. 17, last par.) from the 3’ hemiprobe sequence. In Marras, this extension product would be element 201, or the ‘extension product 201’ (pg. 18, last par; Fig. 2).
Regarding claim 5, Marras recites incubating the reaction mixture under conditions suitable to produce extension products from the reverse primer (pg. 18, last par.; Fig. 2). In Marras, the extension product from the reverse primer is ‘extension product 204.’
Regarding claims 6 and 10, Marras recites combining in said reaction mixture or a second reaction mixture said extension products produced from said reverse primer and a forward primer configured to hybridize to a region of said extension products complementary to: (i) a portion of said 5' hemiprobe sequence; and (ii) a portion of a stem of said stem-loop sequence (pg. 45, ln. 13-24; Fig. 2 and 18). Here, the ‘forward primer’ is the same as the stem-loop primer, and the primer binds to the extension product of the reverse primer, including the full lengths of the 5’ hemiprobe sequence and the stem-loop sequence). Marras recites incubating the reaction under conditions suitable to produce extension products from the forward primer (pg. 45, ln. 13-24).
Regarding claim 7, Marras teaches that the primer (here, the forward and stem-loop primer are the same) comprises at least 15-40 nucleotides (pg. 9, 1st full par.). As discussed above, the primer binds to the full length of the 5’ hemiprobe sequence as well as other elements of the stem-loop primer, and so must be at least the length of the 5’ hemiprobe sequence (or ‘anchor sequence’ - 15-40 nucleotides). This overlaps the claimed ranges (i.e. “comprises at least 12 nucleotides, and comprises at least 7 nucleotides complementary to a complement of said portion of said 5' hemiprobe sequence”) with sufficient specificity that the limitation is considered to have been met.
Regarding claim 14, Marras recites that the DNA sequence may comprise genomic DNA (pg. 25, ln. 5-14).
Regarding claim 15, Marras recites that the 5' hemiprobe sequence (or ‘anchor sequence’) is 15-40 nucleotides in length (pg. 9, 1st full par.). This overlaps with the claimed range of ‘at least 7 nucleotides in length’ with sufficient specificity that the limitation is considered to have been met.
Regarding claim 16, Marras recites that the 3' hemiprobe sequence (or ‘foot sequence’) is 5-8 nucleotides in length (pg. 10, 1st full par.). This overlaps with the claimed range of ‘at least 3 nucleotides in length’ with sufficient specificity that the limitation is considered to have been met.
Regarding claim 21, Marras teaches that a loop of a stem-loop sequence comprises a unique structure which is distinct in sequence, including the sequence of the loop (pg. 44, ln. 29-32; Fig. 18, elements 405A/405B). This unique sequence/structure is considered to be fall within the plain meaning of the term ‘barcode,’ and is consistent with the lack of limiting definition in the instant specification. Therefore, the limitation is considered to have been met
Regarding claim 22 and 23, Marras teaches that said reaction mixture or said second reaction mixture further comprises an oligonucleotide probe comprising a detectable moiety, wherein said oligonucleotide probe is configured to hybridize to a complement of at least part of a stem-loop primer (Fig. 4; pg. 20, ln. 22-27). Here, the detectable oligonucleotide probe is a component of the primer which binds to the complement of itself at the part of the stem-loop primer which comprises the stem of the stem-loop (see Fig. 4, last panel).
Regarding claim 27, Marras teaches performing a PCR reaction or a dPCR reaction (pg. 16, last par. – pg. 17, 1st par.).
Claim Rejections - 35 USC § 103
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 pre-AIA 35 U.S.C. 103(a) which forms the basis for all obviousness rejections set forth in this Office action:
(a) A patent may not be obtained though the invention is not identically disclosed or described as set forth in section 102, if the differences between the subject matter sought to be patented and the prior art are such that the subject matter as a whole would have been obvious at the time the invention was made to a person having ordinary skill in the art to which said subject matter pertains. Patentability shall not be negated by the manner in which the invention was made.
This application currently names joint inventors. In considering patentability of the claims under pre-AIA 35 U.S.C. 103(a), the examiner presumes that the subject matter of the various claims was commonly owned at the time any inventions covered therein were made absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and invention dates of each claim that was not commonly owned at the time a later invention was made in order for the examiner to consider the applicability of pre-AIA 35 U.S.C. 103(c) and potential pre-AIA 35 U.S.C. 102(e), (f) or (g) prior art under pre-AIA 35 U.S.C. 103(a).
Claims 8 and 24-26 are rejected as unpatentable over Marras et al. (published Aug. 14, 2014; International Publication No. WO 2014/124290), as applied to claims 1, 6, 7, and 10 above, and further in view of Whitman et al. (published Dec. 30, 2010; Patent Application Publication No. US 20100330574).
Marras teaches the limitations of claims 1, 6, 7, and 10, as discussed above in the rejections under 35 U.S.C. 102(a)(1).
Regarding claim 8, Marras teaches that the forward primer comprises nucleotides complementary to a complement of the full length of the stem-loop sequence. As discussed in the rejections of claims 6, 7, and 10 above: in Marras, the forward primer is the same as the stem-loop primer, and the primer binds to the extension product of the reverse primer, including the full length of the stem-loop sequence. Marras meets the limitation that the forward primer be comprised of at least 9 nucleotides, because the primer is comprised of at least 15 nucleotides (the length of just one of the primer’s elements, the 5’ hemiprobe or ‘anchor’ sequence; pg. 9, 1st full par.).
Regarding claim 24, Marras teaches that said reaction mixture or said second reaction mixture further comprises an oligonucleotide probe comprising a detectable moiety (Fig. 19). Marras teaches that the probe can hybridize to a loop sequence for detection (pg. 43, 2nd par).
Regarding claims 25 and 26, Marras teaches detectable moieties comprising a fluorophore and a quencher (for example, pg. 20, ln. 5-10; Fig. 18). Marras teaches that they may be separate from the primer molecule (Fig. 19, elements 408A/408B), as well as integrated (Fig. 4, last panel: element 416; Fig. 18, elements 1910A/B). Consistent with the orientation of the primer molecule, as discussed for claim 1 (see page 10-11 of this office action), the fluorophore may be 5’ (see Fig. 4, last panel: element 416; Fig. 18, element 408A/B).
Regarding claim 8, Marras does not explicitly teach that the forward primer comprises at least 9 nucleotides complementary to a complement of a portion of the stem of the stem-loop sequence. That is, Marras does not explicitly teach a length of the stem of the stem-loop sequence of at least 9 nucleotides, as required implicitly by claim 8.
Whitman teaches stem-loop sequences wherein the stem comprises 11, 14, or 16 nucleotides (par. 143-144) as a matter of optimization.
It would have been obvious to a person with ordinary skill in the art before the effective filing date of the instant invention to optimize the length of the stem-loop’s stem to be greater than 9 nucleotides because Whitman demonstrates that the length of the stem is a results-effective variable. One would have had reasonable expectation of success because Whitman demonstrates that primers with hairpin stems of such lengths successfully amplify nucleic acids (Fig. 8). Because Marras teaches a forward primer which is fully complementary to a complement of a stem-loop sequence, it flows naturally from the combination of Whitman and Marras that that the forward primer comprises at least 9 nucleotides complementary to a complement of a portion of the stem of the stem-loop sequence.
Regarding claim 24, Marras does not explicitly teach that the reaction mixture comprises an oligonucleotide probe comprising a detectable moiety which is configured to hybridize to a complement of the loop sequence within the stem-loop sequence.
Whitman teaches the inclusion of stem-loop sequences in primers which are formed through the hybridization of regions termed ‘tag’ and ‘anti-tag’ regions, thereby increasing assay sensitivity (par. 62-63). Whitman teaches oligonucleotide probes comprising a detectable moiety which are configured to hybridize to a complement of these stem-loop sequences (par. 29, 65).
It would have been obvious to a person with ordinary skill in the art before the effective filing date of the instant invention to include a stem-loop having the hybridization sequence for a detectable probe within the bridge element of Marras, in order to reduce competition for probe binding (Whitman: par. 63-64; Marras: pg. 44, 1st par.). In this case, sequestering the oligonucleotide probe hybridization sequence in the loop of a hairpin would allow for sensitive detection of amplified product. It would have been obvious to include the hybridization sequence specifically in the loop of the stem-loop because Marras explains that a loop has fewer design constraints, allowing for relatively long unique sequences for multiplexed detection (pg. 43, par. 2). One would have had reasonable expectation of success because Whitman provides guidance for primer hairpin design (par. 14) and shows that these type of hairpin primers may outperform standard primers (par. 132; Fig. 8), and Marras sets forth principles relevant for the design of loops having optional detectable elements (pg. 47-48).
Claim 11 is rejected as unpatentable over Marras et al. (published Aug. 14, 2014; International Publication No. WO 2014/124290), as applied to claims 1, 5, and 6, and further in view of Ongagna-Yhombi et al. (published Feb. 22, 2013; Ongagna-Yhombi et al. Malar J. 2013 Feb 22;12:74).
Marras recites the limitations of claims 1, 5, and 6, as discussed in the rejection under 35 U.S.C. 102(a)(1) above.
Regarding claim 11, Marras does not recite that the concentrations of reverse and forward primer are in excess of the concentration of stem-loop primer. Marras does not recite a forward primer which is distinct from the stem-loop primer, as required implicitly by the limitation.
Ongagna-Yhombi teaches semi-nested asymmetric PCR, wherein three primers U1, U2, and U4 are utilized in a single tube (figure 1B; pg. 6, col. 2, 1st par.). U1 would be the equivalent of the reverse primer, U2 would be the equivalent of the stem-loop primer, and U4 would be the equivalent of the separate forward primer. In a first phase of the assay, the amplification product of U1 and U2 is favored. In a second phase, this amplification product serves as template for U1 and U4 to generate a second amplicon. Ongagna-Yhombi teaches that the concentration of U1 and U4 in the reaction mixture are in excess of the concentration of U2.
It would have been obvious to a person with ordinary skill in the art before the effective filing date of the instant invention to combine the teachings of Marras with the teachings of Ongagna-Yhombi. One would have been motivated to do so in order to reduce assay time without compromising sensitivity and specificity (Abstract). One would have had reasonable expectation of success because Ongagna-Yhombi demonstrates proof-of-principle (Figure 3; pg. 6, col. 1, 1st par.).
Claim 12 is rejected as unpatentable over Marras et al. (published Aug. 14, 2014; International Publication No. WO 2014/124290), as applied to claims 1 and 6, and further in view of Lorenz (published May 22, 2012; Lorenz TC. J Vis Exp. (63):e3998).
Regarding claim 12, Marras teaches that the concentration of the stem-loop primer is ‘very high.’ The concentration of the stem-loop primer is intended to be sufficient to incorporate with the exponentially abundant amplicons generated in reactions involving the initial DNA template (pg. 32, ln. 14-19), and in PCR, the amount of generated amplicons would be greater than the initial concentration of DNA.
Regarding claim 12, Marras does not explicitly teach that the concentration of stem-loop primer in a reaction mixture is in excess of the concentration of the DNA sequence.
Lorenz teaches primer concentrations in excess of the concentration of a DNA sequence (pg. 4, part 7, point 2).
It would have been obvious to a person with ordinary skill in the art, with reasonable expectation of success, to optimize the concentration of the stem-loop primer to be in excess of the concentration of DNA sequence in a reaction mixture during the course of routine experimentation because Lorenz demonstrates that the ratio of template to primer is a results-effective variable in amplification reactions.
Claims 18-20 are rejected as unpatentable over Marras et al. (published Aug. 14, 2014; International Publication No. WO 2014/124290), as applied to claim 1 above, and further in view of Jonstrup et al (published May 10, 2013; Jonstrup et al. Sensors (Basel). 2013 May 10;13(5):5937-44).
Marras recites the limitations of claim 1, as discussed in the rejections under 35 U.S.C. 102(a)(1) above.
Regarding claims 18-20, Marras does not explicitly teach that the stem-loop sequence comprises 15 nucleotides (i.e. is at least 15 nucleotides in length), a stem-loop sequence which is configured to have a specific Tm, or that a loop of said stem-loop is 1 nucleotide to 20 nucleotides in length.
Regarding claims 18-20, Jonstrup explicitly teaches stem-loop sequences comprising 15-25 nucleotides, stem-loop sequences configured to have a Tm ranging from about 30ºC to about 70ºC, and loops ranging between 10-20 nucleotides in length (Figure 3).
It would have been obvious to a person with ordinary skill in the art before the effective filing date of the instant invention to optimize the stem-loop sequences of Marras to have the claimed lengths and/or melting temperatures because Jonstrup establishes that overall length, loop length, and melting temperature are results-effective variables in the design of hairpin sequences (pg. 5939, 2nd full par.). One would have had reasonable expectation of success because Jonstrup demonstrates the effects of such variables on fluorescence intensity (for example, Figure 3) in relevant contexts such as PCR systems (pg. 5938: Fluorescent measurements; pg. 5939, last full par.).
Claim 72 is rejected as unpatentable over Marras et al. (published Aug. 14, 2014; International Publication No. WO 2014/124290) and in view of Herman et al. (published Sep. 3, 1996; Herman et al. Proc Natl Acad Sci U S A. 1996 Sep 3;93(18):9821-6).
Regarding claim 72, Marras recites a method for processing a DNA sequence having or suspected of having sequence variation relative to a wild-type sequence (pg. 5, 1st par.). Marras recites that the method comprises combining reagents in a reaction mixture suitable for processing said DNA sequence (pg. 12, last par.). Marras recites that the reaction mixture comprises:
said DNA sequence, wherein said DNA sequence comprises a variation of at least one nucleotide relative a wild-type sequence (pg. 5, 1st par.);
a stem-loop primer (for example, Fig. 4: last three panels)
For clarity, Marras’s Figure 2 (see below) shows the DNA sequence as element 101 and the stem-loop primer as element 103. In Marras, the stem-loop primer is called a ‘multi-part primer’ or a forward primer.
Figure 2:
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Marras recites that the stem-loop primer comprises (Fig 1):
a 5' hemiprobe sequence configured to hybridize to a complementary first end region of said DNA sequence;
a stem-loop sequence (Fig. 4; pg. 20, ln. 5-27); and
a 3' hemiprobe sequence configured to hybridize to a second end region of said DNA sequence,
wherein a 3' portion of said 3' hemiprobe sequence comprises a nucleotide sequence complementary to a variant sequence but not complementary to said wild-type sequence;
In Marras, the 5’ hemiprobe sequence may be element 104 (‘the anchor sequence’) and the 3’ hemiprobe sequence may be element 106 (‘the foot sequence’), and they are oriented in the 5’-to-3’ direction (pg. 7, ln. 11-12; Fig. 1). The variation is represented by the ‘x’ relative to the ‘y’ of a wildtype sequence, and is located 3’ within the 3’ hemiprobe sequence (Fig. 1; pg. 7, ln. 21-22).
Figure 1:
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The stem-loop sequence could be the hairpins variants 404 and 409 comprised in the primer, as shown in the last three diagrams of Fig. 4 (see below; pg. 20, ln. 5-27).
Figure 4:
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Regarding claim 72, Marras does not teach that the DNA sequence has or is suspected of having a methylated cytosine at a particular residue, nor that the DNA sequence comprises a uridine residue, nor that the 3’ hemiprobe sequence comprises a nucleotide complementary to the uridine residue but not complementary to a cytidine residue.
Herman teaches DNA sequences having or suspected of having a methylated cytosine at a particular residue, wherein the DNA sequences may comprise a uridine residue after bisulfite treatment (Abstract). Herman teaches a primer binding sequence comprising a nucleotide complementary to a uridine residue but not complementary to a cytidine residue (pg. 9821, col. 2, last full par.; pg. 9823, col. 1)
It would have been obvious to a person with ordinary skill in the art before the effective filing date of the instant invention to combine the teachings of Marras and Herman. One would have been motivated to so do in order to rapidly assess methylation status in order to understand biological processes such as tumor suppression in cancer (Abstract). One would have had reasonable expectation of success because Herman successfully demonstrates primer sequence-based discrimination between methylated and unmethylated sequences (Fig. 2).
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, 5-8, 10-12, 14-16, 18-27 and 72 are rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1-21 of Kubista et al. (U.S. Patent No. 11,421,268) in view of Marras et al. (published Aug. 14, 2014; International Publication No. WO 2014/124290).
Although the claims at issue are not identical, they are not patentably distinct from one another. Both sets of claims are drawn to methods of processing nucleic acids. Both sets of claims require:
combining nucleic acid in a reaction mixture suitable for processing nucleic acid and incubating under conditions to product extension products from a stem-loop primer and a reverse primer (claim 1, 2)
a stem-loop primer comprising a 5’ hemiprobe sequence configured to hybridize to a complementary first end of a nucleic acid, a stem-loop sequence, and a 3’ hemiprobe sequence (claim 1)
a reverse primer configured to hybridize to a genomic region distinct from a target region (claim 2)
The reference does not explicitly teach DNA, nor that the DNA have or be suspected of having a sequence variation relative to wild-type sequences. The reference does not explicitly teach that a 3’ portion of the 3’ hemiprobe sequence comprises a nucleotide sequence complementary to the variant and not to wild-type, nor that the reverse primer hybridizes to a sequence 3’ to a variant in the target.
Marras teaches that DNA as a polynucleotide of interest (pg. 5, par. 1). Marras teaches that the DNA have or be suspected of having a sequence variation relative to wild-type sequences (Fig. 1, ‘x’ vs. ‘y’). Marras teaches a reverse primer configured to hybridize to a genomic region 3' from a sequence variant relative to wild-type (pg. 18, ln. 16-31; Fig. 2, element ‘203’). Marras recites a 3’ portion of the 3’ hemiprobe sequence comprises a nucleotide sequence complementary to the variant and not to wild-type (Fig. 1).
It would have been obvious to combine the teachings of Marras and the reference patent in order to distinguish and quantitate different rare target sequences (Marras: pg. 44). One would have had reasonable expectation of success because the reference and Marras are in the same field of endeavor and Marras demonstrates successful use of equivalent stem-loop primer systems for variant detection (Fig. 7).
Claims 1, 5-8, 10-12, 14-16, 18-27 and 72 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1, 2, 4-8, 10-16 of co-pending Application No. 17/813,273 in view of Marras et al. (published Aug. 14, 2014; International Publication No. WO 2014/124290).
Although the claims at issue are not identical, they are not patentably distinct from one another. Both sets of claims are drawn to methods. Both sets of claims require:
nucleic acid having or be suspected of having a sequence variation relative to wild-type sequences (claim 1, 13)
combining nucleic acid in a reaction mixture suitable for processing nucleic acid and incubating under conditions to product extension products from a stem-loop primer and a reverse primer (claim 1, 2, 13, 15)
a stem-loop primer comprising a 5’ hemiprobe sequence configured to hybridize to a complementary first end of a nucleic acid, a stem-loop sequence, and a 3’ hemiprobe sequence (claim 1, 13, 14)
the 3’ hemiprobe sequence comprises a nucleotide sequence complementary to the variant and not to wild-type (claim 2)
Although the reference does not explicitly require that the nucleotide complementary to the variant is located 3’ within the 3’ hemiprobe sequence, such a configuration would be an obvious design choice (see Marras: Fig. 1; pg. 7, ln. 21-22). Although the reference patent does not explicitly require a reverse primer configured to hybridize to a genomic region 3’ to a primer binding region in the target, it would be obvious to use such a reverse primer in order to amplify the stem-loop primer extension product (see Marras: pg. 18, ln. 16-31). This is a provisional nonstatutory double patenting rejection because the patentably indistinct claims have not in fact been patented.
Claims 1, 5-8, 10-12, 14-16, 18-27 and 72 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 58-76 of co-pending Application No. 18/556,183 in view of Marras et al. (published Aug. 14, 2014; International Publication No. WO 2014/124290).
Although the claims at issue are not identical, they are not patentably distinct from one another. Both sets of claims are drawn to methods (claim 67). Both sets of claims require:
a DNA sequence (claim 72)
combining nucleic acid in a reaction mixture suitable for processing nucleic acid and incubating under conditions to product extension products from a stem-loop primer (claim 67, 73)
a stem-loop primer comprising a 5’ hemiprobe sequence configured to hybridize to a complementary first end of a nucleic acid, a stem-loop sequence, and a 3’ hemiprobe sequence (claim 58, 67)
The reference does not explicitly require that the DNA have or be suspected of having a sequence variation relative to wild-type sequences, nor that 3’ portion of the 3’ hemiprobe sequence comprises a nucleotide sequence complementary to the variant and not to wild-type, nor a reverse primer which hybridizes to a sequence 3’ to a primer binding region in the target.
Marras teaches that the DNA have or be suspected of having a sequence variation relative to wild-type sequences (Fig. 1, ‘x’ vs. ‘y’). Marras teaches a reverse primer configured to hybridize to a genomic region 3' from said variation of said at least one nucleotide relative to said wild-type sequence (pg. 18, ln. 16-31; Fig. 2, element ‘203’). Marras recites a 3’ portion of the 3’ hemiprobe sequence comprises a nucleotide sequence complementary to the variant and not to wild-type (Fig. 1).
It would have been obvious to combine the teachings of Marras and the reference patent in order to distinguish and quantitate different rare target sequences (pg. 44). One would have had reasonable expectation of success because the reference and Marras are in the same field of endeavor and Marras demonstrates successful use of equivalent stem-loop primer systems for variant detection (Fig. 7). This is a provisional nonstatutory double patenting rejection because the patentably indistinct claims have not in fact been patented
Conclusion
No claims are allowed.
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/C.M.J./Examiner, Art Unit 1682
/WU CHENG W SHEN/Supervisory Patent Examiner, Art Unit 1682
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