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 .
Effective Filing Date
The present application was filed on August 12, 2022 and does not claim the benefit of, or priority to, any previously filed applications.
Claim Status and Action summary
Claims 1-5, 7-11, 14-18, 21-24, and 26 are pending in the present application. Claims 6, 12-13, 19-20, and 25 were canceled by applicant. Claims 21-23 were newly added in the amendment filed on August 26, 2025. Claims 24 (now canceled) and 25 were newly added in the amendment filed on February 2, 2026. Claim 26 was newly added in the amendment filed on June 26, 2026.
Claims 1-5, 7-11, 14-18, 21-24, and 26 are under examination.
This action is in response to the papers filed June 24, 2026.
Any objections and rejections not reiterated below are hereby withdrawn.
Applicant is reminded that no new matter may be added.
Claim Rejections - 35 USC § 112(a) – Written Description
The following is a quotation of the first paragraph of 35 U.S.C. 112(a):
(a) IN GENERAL.—The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor or joint inventor of carrying out the invention.
Claims 1, 17-18, and 26 are rejected under 35 U.S.C. 112(a) as failing to comply with the written description requirement. The claims contain subject matter which was not described in the specification in such a way as to reasonably convey to one skilled in the relevant art that the inventor or a joint inventor, or for applications subject to pre-AIA 35 U.S.C. 112, the inventor(s), at the time the application was filed, had possession of the claimed invention.
This is a new matter rejection.
This is a new grounds of rejection necessitated by the amendments to the claims.
Regarding claim 1, the claim as amended includes the new limitation “detecting by a length-based method a sample containing Y chromosome DNA when presence of only one amplicon from SRY, DAZ2 or TSPY1 is detected”. The specification teaches “methods for detecting the single copy SRY gene are not sensitive enough to accurately determine gender.” (Specification, page 3). Therefore, the specification as originally filed does not provide support for an embodiment wherein the presence of Y chromosome DNA is detected in a sample when only one amplicon is detected.
Regarding claim 1, the claim as amended includes the new limitation “wherein the lengths of said first, second, third, fourth, fifth, sixth, seventh, and eighth nucleotide primers range consist of 15 to 30 nucleotides”. The disclosed oligonucleotide sequences of SEQ ID NO: 1, 2, 7, and 8 consist of 34, 35, 39, and 36 nucleotides, respectively. The limitation that the sequences “consist of” a range of nucleotide lengths from 15 to 30 nucleotides is therefore not supported by the disclosure as originally filed.
Regarding claims 17, 18, and 26, the claims as amended now recite a step of “treating the subject from whom the sample was obtained for hemophilia”. However, the specification as originally filed only discloses a genus of potential “prenatal regimen or treatment for a pregnant woman or fetus based on gender”. There is no disclosure of treating “a subject from whom the sample was obtained” encompassing post-natal treatment for hemophilia or Duchenne muscular dystrophy. Furthermore, the specification teaches no method steps of “treating… for Duchenne muscular dystrophy”.
Claim Rejections - 35 USC § 112(a) –Scope of Enablement
The following is a quotation of the first paragraph of 35 U.S.C. 112(a):
(a) IN GENERAL.—The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor or joint inventor of carrying out the invention.
Claim 26 is rejected under 35 U.S.C. 112(a) because the specification, while being enabling for symptomatic treatment of Duchenne muscular dystrophy with glucocorticoids in post-natal children, does not reasonably provide enablement for administering any treatment (e.g. encompassing gene-therapy) to any subject (e.g. fetuses, neonates, children) so long as the fetal sample was obtained from the subject. The specification does not enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the invention commensurate in scope with these claims.
This is a new grounds of rejection necessitated by the addition of new claim 26.
Factors to be considered in determining whether a disclosure meets the enablement requirements of 35 USC 112, first paragraph, have been described by the court in In re Wands, 8 USPQ2d 1400 (CA FC 1988). Wands states at page 1404, “Factors to be considered in determining whether a disclosure would require undue experimentation have been summarized by the board in Ex parte Forman. They include (1) the quantity of experimentation necessary, (2) the amount of direction or guidance presented, (3) the presence or absence of working examples, (4) the nature of the invention, (5) the state of the prior art, (6) the relative skill of those in the art, (7) the predictability or unpredictability of the art, and (8) the breadth of the claims.”
The nature of the invention and the breadth of the claims:
Claim 26 is broadly drawn to a method for simultaneous detection of Y chromosome-specific target DNA of the SRY, DAZ2, and TSPY1 genes and control DNA of the ACTB gene comprising steps of simultaneously amplifying the recited genes by PCR, detecting the presence or absence of corresponding PCR amplicons, selecting a sample based on the presence or absence of the PCR amplicons, and “treating the subject from which the sample was obtained for Duchenne Muscular Dystrophy” (DMD). As is evident from the claim language (and the remarks dated June 26, 2026), the scope of the claimed invention “does not exclude treatment of a fetal subject after birth (e.g., a neonate or older child)” (response, June 24, 2026, page 10).
As such, the scope of claim 26 “treating the subject from which the sample was obtained” encompasses administering any treatment “for DMD” to any of: a fetus, a neonate, a child that is older than a neonate, or an adult for DMD, so long as the sample was obtained from said subject prior to the birth of said subject.
The state of the art:
The invention is in a class of invention which the CAFC has characterized as “the unpredictable arts such as chemistry and biology.” Mycogen Plant Sci., v. Monsanto Co., 243 F.3d 1316, 1330 (Fed. Cir. 2001).
The post-filing date art teaches that “muscular dystrophy” is a very large genus of genetic disorders comprising diverse phenotypic presentations involving at least 50 genes (Bozzi et al., “Diagnosis, Pathogenesis and Treatment of Muscular Dystrophy” Biomedicines 2025, 13, 1820). The art further teaches that at least 110 distinct “muscular dystrophies” are recognized, not all of which have single genes associated with the phenotype within the chromosome band segregating with the disease phenotype (Benarroch et al., “The 2025 version of the gene table of neuromuscular disorders (nuclear genome)” Neuromuscular Disorders 46 (2025), 105261). Furthermore, Benarroch et al. teach that of the 110 distinct “muscular dystrophies”, only 3 are known to be X-linked (“Duchenne muscular dystrophy; Becker muscular dystrophy”, “Emery-Dreifuss muscular dystrophy, X-linked, type 1”, and “Emery-Dreifuss muscular dystrophy 6, X-linked”), while the other members of the genus exhibit autosomal patterns of inheritance and are attributed to genes/loci on autosomes (Benarroch et al., Gene table of monogenic neuromuscular disorders (nuclear genome only), see Group 1 and 2 in the table).
The post-filing date art (Bozzi (2025)) teaches that there is no definitive cure for any of the known muscular dystrophies. Bozzi teaches that there are experimental/supportive therapies (Glucocorticoid therapy, and several drugs in clinical trials). However, Bozzi teaches glucocorticoid therapy, while helpful for delaying disease progression, has significant side effects including adrenal suppression, growth delay, weakened bones, and metabolic syndrome. Bozzi additionally teaches that the experimental drugs in clinical trials demonstrated broad safety profiles, but with limited demonstrated effectiveness (Bozzi, page 1, paragraphs 3-4). Furthermore, none of the experimental treatments (or glucocorticoid treatment) reviewed by Bozzi appear to have been investigated for safety or effectiveness for fetal subjects.
Similarly, the pre-filing date art, Zhang et al., “Recent advances of glucocorticoids in the treatment of Duchenne muscular dystrophy (Review)”. Experimental and Therapeutic Medicine 21.5 (March 1, 2021): 447 teaches “although glucocorticoid-dependent medication is the mainstay treatment for DMD, a standard treatment regimen has yet to be determined” (Zhang et al., Abstract), “glucocorticoids… prolong autonomous walking, reduce scoliosis, and improve cardiopulmonary function, quality of life, and the survival rate of patients” (Zhang et al., page 1, column 2, paragraph 1)
Other treatment approaches in the post-filing date art such as in utero gene editing using lipid nanoparticles (LNPs) as delivery vehicles for Cas9 mRNAs and sgRNAs have been attempted in mouse models (Gao et al., “In utero delivery of mRNA to the heart, diaphragm, and muscle with lipid nanoparticles”, Bioactive Materials (2023) 25, pp. 387-398) and (Palanki et al., “In utero delivery of targeted ionizable lipid nanoparticles facilitates in vivo gene editing of hematopoietic stem cells” PNAS (2024) 121 (32) e2400783121). However, these studies caution that further study is needed to “achieve clinically meaningful mRNA delivery rates” (Gao et al., page 388, column 2, paragraph 2), and “further work is necessary to fully characterize… safety of… LNP[s]” (Palanki et al., page 9, column 1, paragraph 2).
Furthermore, several examples in the art teach examples of Muscular Dystrophies for which there are no effective clinical treatments, much less treatments suitable for administration to a fetus.
Zhong et al., 2021 teaches Duchenne Muscular Dystrophy (DMD) is a fatal X-linked muscular disease for which there is no effective clinical treatment (Zhong, background). Furthermore, Kihara et al., “In utero transplantation of myoblasts and adipose-derived mesenchymal stem cells to murine models of Duchenne muscular dystrophy does not lead to engraftment and frequently results in fetal death” Regenerative Therapy (2022) Oct20;21:486-493 teach experimental in utero cell therapies in mouse models comprising fetal intraperitoneal transplantation and transplacental transplantation with allogenic myoblasts or adipose-derived stem cells (ASCs). Kihara et al. found that in-utero transplanted cells were not viable, and that the interventions showed high fetal mortality rates (Kihara et al., table 1 and page 490-491 bridging paragraph).
More recently, Farmer et al., “Feasibility and safety of cellular therapy for in-utero repair of myelomeningocele (CuRe Trial): a first-in-human, phase 1, single-arm study” The Lancet (2026) 407, pp.867-875 demonstrated improved immediate safety findings of administration of allogenic placenta-derived stem cells (PMSCs) onto exposed fetal spinal cords during experimental fetal surgical repair of myelomeningocele (spina bifida). However, it is not clear whether transplanted PMSCs will remain safe in longer term follow up studies of the very small trial cohort (6 patients), as Farmer et al. only assessed birth outcomes and acknowledged the possibility of malignant or proliferative transformation of the transplanted PMSCs (Farmer et al., page 873, column 1-2 bridging paragraph). Furthermore, Farmer et al. teach that other genetic disorders requiring long-term engraftment of therapeutic stem cells following fetal administration have not been successful and continue (as of 2026) to be optimized (Farmer et al., page 873, column 2, paragraph 2).
Most recently, Travis “Second gene-editing death in China disclosed after long delay” Science Briefs. https://www.science.org/content/article/second-gene-editing-death-china-disclosed-after-long-delay; 6 August 2026, reported a study evaluating an experimental gene-editing therapy for DMD was halted after a delayed report of a death of a child resulting from the failed treatment attempt.
Guidance in the specification and Working Examples
The specification broadly teaches that prenatal treatments may comprise: maternal dietary supplementation with amino acids or sugars, fetal blood transfusion, administration of thyroxin or other enzymes or hormones, or in utero stem cell transplantation (Specification, page 38, line 10-16).
The specification does not teach any working examples of treating a fetus, a neonate, or a subject for any condition, much less DMD.
Quantity of Experimentation
Claim 26 is broadly drawn to a method comprising treating “the subject from whom the sample was obtained (i.e. a fetus, a neonate, a child who is older than a neonate, or an adult) for Duchenne muscular dystrophy”. The specification teaches “Over a hundred X-linked inherited diseases have been discovered in humans” (specification, page 1, line 19-20). However, the specification does not teach any exemplary treatments that may be administered to a fetus. The prior art teaches that “muscular dystrophy” is a very large genus of known heritable phenotypes comprising at least 110 distinct “muscular dystrophies”, not all of which have single genes associated with the phenotype within the chromosome band segregating with the disease phenotype (Benarroch et al.). The prior art further recognizes that there are no effective cures or effective treatments for any of the known muscular dystrophies (Bozzi, page 1, paragraphs 3-4) at any developmental stage, much less during fetal development. Furthermore, even in early-stage mouse models, an attempted prenatal treatment for Duchenne muscular dystrophy failed to correct the disease, as donor myoblast or adipose-derived stem cells failed to engraft into fetal muscle tissue and high fetal mortality was observed for the attempted surgical intervention. Muscular dystrophies are not treated with any routine therapies. Therefore, it is unpredictable whether particular effective treatments for the broadly claimed “treating… for Duchenne muscular dystrophy” can be administered to a fetus.
The art also teaches several studies where the skilled artisan was unable to treat Duchenne Muscular Dystrophy (see above, Bozzi et al., Kihara et al., and Travis). The specification teaches that prenatal therapies encompass maternal dietary supplementation with amino acids or sugars, fetal blood transfusion, administration of thyroxin or other enzymes or hormones, or in utero stem cell transplantation (Specification, page 38, line 10-16). The art further suggests that gene therapy or gene editing based therapies may eventually be able to treat Duchenne Muscular Dystrophy (Zhong, 2021 page 8, column 1, paragraph 5). Furthermore, the post-filing date art (Bozzi et al., Gao et al., Palanki et al., and Travis) demonstrate that while in utero delivery of lipid nanoparticles comprising CRISPR-Cas9 components for activating reporter gene expression is possible in mice (Gao et al., 2023), this line of research remains active, there is no demonstration of therapeutic editing during fetal development for any muscular dystrophy, and the therapies have not been shown to be safe or effective in human fetuses. Even more, the post-filing date comprises negative examples of failed trials for DMD treatments that resulted in fetal death and the death of at least one child (Kihara et al., and Travis).
Therefore, the skilled artisan would be required to perform additional undue and unpredictable experimentation to determine an effective prenatal treatment for the broad genus of “treating the subject from whom the sample was obtained for Duchenne muscular dystrophy”. The skilled artisan would have to perform additional unpredictable and undue experimentation for each known candidate treatment (e.g. various gene-editing treatments, stem cell transplantation, etc.) for each class of “subject” encompassed by the present claim (i.e. fetus, neonate, a child who is older than a neonate, or an adult from whom the fetal sample was obtained) and further resolve unpredictable factors such as dosing, effective route of administration, safety, and long-term efficacy, with no expectation of success at the clinical stage, as demonstrated by the failed in utero cell therapy for Duchenne muscular dystrophy in mouse models taught by Kihara et al. and the fatal, failed gene-editing human experiments for DMD reported by Travis. Furthermore, the art teaches Duchenne Muscular Dystrophy (DMD) (i.e. “muscular dystrophy” is a fatal X-linked muscular disease for which there is no effective clinical treatment (Zhong, background). Even more, despite the improved safety demonstrated by the 2026 spina bifida trial (Farmer et al.), the in-utero LNP gene editing platform taught by Gao et al., 2023, does not appear to have been followed up on by the study’s authors in the intervening 2+ years between the publication of Gao et al. and the writing of this office action (March 2026), much less as a demonstrated therapeutic for in-utero treatment of any muscular dystrophy or hemophilia. In fact, Farmer et al. caution that, regarding other recent studies, “in-utero intravenous transplantation of maternal bone marrow-derived hematopoietic stem cells for thalassaemia and of allogeneic fetal liver-derived stem cell for osteogenesis imperfecta appeared safe, but long-term engraftment for genetic conditions is challenging and continues to be optimised. In contrast, long-term engraftment is not warranted for myelomeningocele, which is a structural birth defect with a defined anatomical area requiring transient regeneration and repair.” (Farmer et al., page 873, column 2, paragraph 2).
The quantity of experimentation in this area is very large because there are many unknown, and unpredictable variables for each of the possible “treatments” encompassed by the claim as presently written. Each candidate treatment experimentation to discover, test, and administer candidate therapies without an expectation of success at each intervening step. Furthermore, there is no guidance in the specification or art that supports the claim scope: “treating the subject from whom the sample was obtained” as broadly claimed.
Level of Skill in the art
The level of skill in the art is deemed to be high.
Conclusion
In the instant case, given the breadth of the claim to treating any subject (i.e. a fetus, a neonate, a child, or an adult) for DMD with any treatment (e.g., glucocorticoids, gene-therapy, stem-cell transplantation), the lack of guidance provided in the specification and prior art as to how the skilled artisan might treat a subject with many of the treatments within the claimed genus, the very large quantity of experimentation in the art, lack of demonstrated working examples, the presence of negative teachings in the art within the broadly claimed genus, and the unpredictability of the art, balanced only against the high level of skill in the art, it is the position of the examiner that it would require undue experimentation for one of skill in the art to perform the method of claim 26 as broadly written.
Response to arguments
The response traverses the scope of enablement rejection under 112(a) on the grounds that “the rejection is moot for claims 17 and 18 which have been directed to hemophilia for which there are known treatments. It would not apply to new claim 26 which is directed to Duchenne muscular dystrophy for which there are treatments.”
The new grounds of rejection above addresses newly added claim 26, which now encompasses a step of “treating the subject from whom the sample was obtained for Duchenne muscular dystrophy”. As is discussed in detail above, the claim encompasses methods of administering any treatment to any subject so long as the fetal sample was obtained from the subject. As is discussed in detail above, the disclosure and the art demonstrate only symptomatic treatment of Duchenne muscular dystrophy with glucocorticoids in post-natal children, and that the full scope of “treatment” (e.g. encompassing gene-therapy) administered to the full scope of “subjects” (e.g. fetuses, neonates, children) is not enabled.
Claim Rejections - 35 USC § 112(b) – Indefiniteness
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
Claims 1, 17-18 and 26 are rejected under 35 U.S.C. 112(b) as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor regards as the invention.
This is a new grounds of rejection necessitated by the amendments to the claims.
Where applicant acts as his or her own lexicographer to specifically define a term of a claim contrary to its ordinary meaning, the written description must clearly redefine the claim term and set forth the uncommon definition so as to put one reasonably skilled in the art on notice that the applicant intended to so redefine that claim term. Process Control Corp. v. HydReclaim Corp., 190 F.3d 1350, 1357, 52 USPQ2d 1029, 1033 (Fed. Cir. 1999). The term “nucleotide primers” in claim 1 is used by the claim to mean “polynucleotide primers” or “oligonucleotide primers” while the accepted meaning of “nucleotide” is “A nucleic acid monomer consisting of a ribose or deoxyribose sugar, a nitrogenous base linked to the 1’ carbon of the ribose or deoxyribose sugar by an N-glycosidic linkage, and a triphosphate group linked to the 5’ carbon of the ribose or deoxyribose sugar.” The term is indefinite because the specification does not clearly redefine the term “nucleotide” to encompass polymers comprising multiple nucleotide monomers.
Claim 1 recites: “detecting… a sample containing Y chromosome DNA when presence of only one amplicon from SRY, DAZ2, or TSPY1 is detected”. However, the instant specification teaches “methods for detecting the single copy SRY gene are not sensitive enough to accurately determine gender.” (Specification, page 3). It is unclear whether the claim is intended to require something more that circumvents said lack of sensitivity, and it appears that the plain meaning of the claim language is contradicted by the present specification.
Claim 1 requires a set of primers “comprising” specific SEQ ID NOs (i.e. the claimed primers encompass polynucleotides having the recited sequence and may comprise additional polynucleotide sequence). Claim 1 further recites the limitation, “wherein the lengths of said [set of primers] range consist of 15 to 30 nucleotides.”
It is noted that, among the recited set of 8 polynucleotide primers, SEQ ID NO 1 is a polynucleotide sequence of length 34, SEQ ID NO 2 is a polynucleotide sequence of length 35, SEQ ID NO 3 is a polynucleotide sequence of length 25, SEQ ID NO 4 is a polynucleotide sequence of length 24, SEQ ID NO 5 is a polynucleotide sequence of length 21, SEQ ID NO 6 is a polynucleotide sequence of length 24, SEQ ID NO 7 is a polynucleotide sequence of length 39, and SEQ ID NO 8 is a polynucleotide sequence of length 36. Because the claimed primers comprise polynucleotide sequences ranging in length from 21 to 29 nucleotides, It is unclear how the primers “comprising” (i.e. having at least the polynucleotide sequence of) the recited sequences are intended to have lengths consisting of 15 to 30 nucleotides. There are no recited sequences having lengths consisting of 15 to 20 nucleotides and the recited polynucleotides SEQ ID NOs: 1, 2, 7 and 8 each comprise (i.e. have at least) 34, 35, 39, and 36 nucleotides, respectively.
Claims 17-18 and 26 each depend from claim 1 and each recite the limitation "the subject from whom the sample was obtained". There is insufficient antecedent basis for this limitation in the claim, as claim 1 does not recite “a subject” nor a step of “obtaining a sample from a subject”, but merely “a sample containing DNA”.
Claims 17-18 and 26 each depend from claim 1 and recite the limitation: “wherein the sample is a… fetal sample, and wherein said method further comprises treating the subject from whom the sample was obtained…”.
It is noted that the scope of “a subject” encompasses any (human) subject from which a sample containing DNA can be obtained (e.g. a fetus, an adult, a neonate, a pregnant woman, etc.). Furthermore, the scope of “a fetal sample” encompasses fetal cells or DNA that are obtained from the blood of a pregnant woman (i.e. the fetal sample is obtained from a pregnant woman). It is therefore unclear whether the claim as presently written is intended to further require: a) treating the fetus, b) treating a subject who is not a fetus (e.g. a neonate, etc.), whose Y-chromosome specific target DNA was detected in a fetal sample by the method of claim 1 (note: claim 1 does not recite a “subject”, “individual”, or “obtaining a sample”), c) treating a pregnant woman (i.e. the subject from whom a sample of maternal blood containing fetal DNA was obtained), or something else entirely.
Claim 26 recites “treating… for Duchenne muscular dystrophy”, however the specification does not define any particular treatment for DMD. It is unclear whether the claim is intended to be limited to “what is well-known [that] is best omitted”, or is somehow intended to require an unspecified particular treatment for DMD.
Claim Rejections - 35 USC § 103
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
Claims 1-4, 9, 11, 14, 16, 21, and 24 are/remain rejected under 35 U.S.C. 103 as being unpatentable over Kolialexi et al., “Early non-invasive detection of fetal Y chromosome sequences in maternal plasma using multiplex PCR” European Journal of Obstetrics and Gynecology and Reproductive Biology. 161 34-37, 2012 in view of Fernandez-Martinez et al., “Noninvasive fetal sex determination in maternal plasma: a prospective feasibility study” Genetics in Medicine. 14, 1, 101-106 (2012), Xie et al., “Designing highly multiplex PCR primer sets with Simulated Annealing Design using Dimer Likelihood Estimation (SADDLE)” Nature Communications, published April 11, 2022, Rychlik (Nucleic Acids Research, Vol. 17, No. 21, Pg 8543-8551, 1989), Buck (Biotechniques, Vol. 27, Pg. 528-536, 1999), and Genbank. Bethesda (MD): National Library of Medicine (US), National Center for Biotechnology Information; 2004 – [cited March 18, 2025].
This rejection has been updated as necessitated by the amendments to the claims.
Regarding claims 1 and 2, Kolialexi teaches multiplex PCR detection of SRY, DYS14 (i.e. TSPY1), and ACTB (Kolialexi, page 35, column 2, paragraph 3) for early non-invasive detection of fetal Y chromosome DNA in maternal plasma (i.e. detecting the presence of amplicons of different lengths (Kolialexi, Table 1), and selecting a sample containing Y chromosome DNA or a sample not containing Y chromosome DNA (Kolialexi, Fig 1).
Kolialexi does not teach amplifying and detecting DAZ2 with the multiplexed markers of SRY, TSPY1, and ACTB.
However, Fernandez-Martinez teaches amplifying DAZ in addition to the two Y-specific markers of Kolialexi. Fernandez-Martinez teaches: “we chose to use a combination of 3 Y chromosome sequences to maximize the accuracy of the test… [and] demonstrate that the detection of 3 markers is highly accurate for clinical use” (Fernandez-Martinez, page 105, column 2, paragraph 1).
Therefore, it would have been prima facie obvious prior to the effective filing date of the claimed invention for one of ordinary skill in the art to modify the multiplex PCR sex-determination method taught by Kolialexi by amplifying and detecting DAZ2 in addition to SRY and TSPY1, as taught by Fernandez-Martinez. The ordinary artisan would have been motivated to modify the method of Kolialexi with additional primers to detect DAZ2 in addition to the other Y specific markers SRY and TSPY1 and the control sequence ACTB because Fernandez-Martinez teaches that detection of only two Y chromosome markers (SRY and TSPY1) is prone to error (Fernandez-Martinez, page 105, column 1, paragraph 2) and that inclusion of a third marker (DAZ2) improves the accuracy of sex determination (Fernandez-Martinez, page 105, column 2, paragraph 1). The ordinary artisan would have been reasonably confident that addition of an additional set of primers specific to DAZ2, as taught by Fernandez-Martinez, to the multiplex sex-determination method taught by Kolialexi would have improved the accuracy of sex determination.
Kolialexi does not teach primers comprising or consisting of the nucleotide sequences recited by claim 1 as amended. However, Genbank teaches the complete nucleotide sequences of the human genes SRY (GenBank: L10102.1, published 1993), ACTB (GenBank: M10277.1, published 1985), TSPY1 (GenBank: X74029.1, published 1993), and DAZ2 (AC000021.1, published 1996). Rychlik teaches it is routine and predictable to make primers for DNA amplification wherein primers are designed to a known oligonucleotide sequence. Rychlik teaches criteria to design/choose primers for DNA amplification (see whole document and Abstract). Buck provides evidence of the equivalence of primers. Specifically, Buck invited primer submissions from a number of labs (Pg. 532, column 3), with 69 different primers being submitted (Pg 530, column 1). Buck also tested 95 primers spaced at 3 nucleotide intervals along the entire sequence at issue, thereby testing more than 1/3 of all possible 18-mer primers on the 300 base pair sequence (Pg 530, column 1). When Buck tested each of the primers selected by the methods of the different labs, Buck found that every single primer worked (Pg. 533, column 1). Further, every single control primer functioned as well (Pg. 533, column 1). Buck expressly states, “The results of the empirical sequencing analysis were surprising in that nearly all of the primers yielded data of extremely high quality” (Pg. 535, column 2). Therefore, Buck provides direct evidence that all primers would be expected to function, and in particular, all primers selected according to the ordinary criteria. This clearly shows that every primer would have a reasonable expectation of success. Finally, Xie teaches a publicly available computer program for selecting highly multiplexed combinations of primer pairs for qPCR assays. (Xie, page 2, column 2) Xie demonstrates a successful example of a set of 60 primers designed by their program to detect 56 gene fusions that frequently occur in non-small cell lung cancer. (Xie, page 6, column 2) Xie teaches that highly multiplex qPCR assays, designed by the program disclosed by their paper, are useful for highly multiplex molecular diagnostics where the cost of next generation sequencing assays cannot be economically justified (Xie, page 8, column 2).
Therefore, it would have been prima facie obvious prior to the effective filing date of the claimed invention for one of ordinary skill in the art to design primers capable of amplifying segments of the known sequences of the Y specific DNA target genes, taught by Kolialexi in view of Fernandez-Martinez by the ordinary methods for multiplex primer design taught by Rychlik, Buck, and Xie. The ordinary artisan would have been motivated to design multiplex compatible primers by the methods of Rychlik, Buck, and Xie because of the teaching of Xie that that highly multiplex qPCR assays, are useful for molecular diagnostics where the cost of next generation sequencing assays cannot be economically justified (Xie, page 8, column 2). The ordinary artisan would have been reasonably confident that a multiplex primer set, designed by the method of Xie, for the four target genes taught by Kolialexi in view of Fernandez-Martinez would have successfully detected the presence or absence of the target genes because Xie teaches a successful 60 primer multiplex qPCR panel targeting 56 gene fusions.
Regarding claim 3, Kolialexi teaches DNA is stored at -80°C (i.e. is frozen) (Kolialexi, page 35, column 2, paragraph 2).
Regarding claim 4, Kolialexi teaches the sample comprises fetal DNA (Kolialexi, abstract, paragraph 1) or maternal cell-free plasma (Kolialexi, page 35, column 2, paragraph 2).
Regarding claim 9, Kolialexi teaches isolating target DNA from other components (Kolialexi, page 35, column 2, paragraph 2).
Regarding claim 11, Fernandez-Martinez teaches contacting a sample with TaqMan minor-groove binding probes (i.e. sequence-specific DNA probes to SRY, DAZ2, and TSPY1) (Fernandez-Martinez, page 104, column 2, paragraph 1-2).
Regarding claim 14, Kolialexi teaches the fetal DNA is extracted form maternal blood where one or both parents have or are carriers of an X-linked genetic disease, namely hemophilia, Duchenne muscular dystrophy, or chronic granulomatous disease (Kolialexi, page 35, column 1, paragraph 4).
Regarding claim 16, Kolialexi teaches further testing the fetus for a causative mutation in male fetuses at risk for hemophilia (Kolialexi, page 37, column 1, paragraph 3)
Regarding claim 21, Kolialexi teaches sequencing the amplicons (Kolialexi, page 35, column 2, paragraph 3).
Regarding claim 24, Fernandez-Martinez teaches that detection of only two Y chromosome markers (SRY and TSPY1) is prone to error (Fernandez-Martinez, page 105, column 1, paragraph 2) and that inclusion of a third marker (DAZ2) improves the accuracy of sex determination (Fernandez-Martinez, page 105, column 2, paragraph 1) (i.e. male gender is determined when amplicons for SRY, TSPY1, and DAZ2 are detected).
Claims 5, 7, 10, and 15 are/remain rejected under 35 U.S.C. 103 as being unpatentable over Kolialexi et al., in view of Fernandez-Martinez et al., “Noninvasive fetal sex determination in maternal plasma: a prospective feasibility study” Genetics in Medicine. 14, 1, 101-106 (2012), Xie et al., “Designing highly multiplex PCR primer sets with Simulated Annealing Design using Dimer Likelihood Estimation (SADDLE)” Nature Communications, published April 11, 2022, Rychlik (Nucleic Acids Research, Vol. 17, No. 21, Pg 8543-8551, 1989), Buck (Biotechniques, Vol. 27, Pg. 528-536, 1999), and Genbank. Bethesda (MD): National Library of Medicine (US), National Center for Biotechnology Information; 2004 – [cited March 18, 2025] as applied to claims 1-4, 9, 11, 14, 16, 21, and 24 above, and further in view of Zhang et al., “Frequency-enhanced transferrin receptor antibody-labelled microfluidic chip (FETAL-Chip) enables efficient enrichment of circulating nucleated red blood cells for non-invasive prenatal diagnosis” Lab Chip, 2018, 18, 2749-2756 (2018).
Regarding claim 5, 7, and 10, the method of non-invasive prenatal sex determination by multiplex PCR of Y chromosome-specific genes from fetal DNA isolated from peripheral maternal blood, taught by Kolialexi in view of Fernandez-Martinez, Xie et al., Rychlik, Buck, and Genbank does not teach additional steps of isolating fetal cells expressing CD71 from maternal blood. However, Zhang teaches a method for enriching fetal nucleated red blood cells from maternal circulation for non-invasive prenatal genetic testing, including prenatal sex determination by qPCR of the SRY gene (Zhang, page 2750, column 1, paragraph 3-column 2, paragraph 1 and figure 7) (i.e. Zhang teaches the isolating fetal cells expressing CD71 from peripheral maternal blood). Zhang further teaches that “highly fragmented cffDNA and a huge background of maternal DNA affect the accuracy of [non-invasive prenatal testing] and limit the application in other genetic disorders such as microdeletions and duplications” (Zhang, page 2749, column 2, paragraph 1) and circulating mononuclear fetal red blood cells hold complete genetic information as well as proteins which can be used for fetal analysis (Zhang, page 2750, column 1, paragraph 3).
Therefore, it would have been prima facie obvious prior to the effective filing date of the claimed invention for one of ordinary skill in the art to combine the method of multiplex PCR detection of SRY, DAZ2, TSPY1, and ACTB in peripheral maternal blood for non-invasive prenatal sex determination, taught by Kolialexi in view of Fernandez-Martinez, Xie et al., Rychlik, Buck, and Genbank, with the method of isolating CD71 positive fetal cells from peripheral maternal blood for non-invasive prenatal diagnosis, comprising an SRY qPCR assay for sex determination, taught by Zhang. The ordinary artisan would have been motivated to combine the method of Kolialexi in view of Fernandez-Martinez, Xie et al., Rychlik, Buck, and Genbank with the method of Zhang because of the teaching of Zhang that assays based on cell-free fetal DNA detection are limited in their application to detecting genetic disorders because of the presence of maternal DNA and the highly fragmented nature of cffDNA (Zhang, page 2749, column 2, paragraph 1). The ordinary artisan would have been reasonably confident that purifying CD71-positive fetal cells from peripheral maternal blood by the method of Zhang would have improved the assay taught Kolialexi in view of Fernandez-Martinez, Xie et al., Rychlik, Buck, and Genbank by decreasing background due to maternal DNA.
Regarding claim 15, Zhang teaches imaging DAPI-stained captured fetal cells (Zhang, fig 5) (i.e. determining the karyotype of the fetus).
Claim 8 is rejected under 35 U.S.C. 103 as being unpatentable over Kolialexi et al., in view of Fernandez-Martinez et al., “Noninvasive fetal sex determination in maternal plasma: a prospective feasibility study” Genetics in Medicine. 14, 1, 101-106 (2012), Xie et al., “Designing highly multiplex PCR primer sets with Simulated Annealing Design using Dimer Likelihood Estimation (SADDLE)” Nature Communications, published April 11, 2022, Rychlik (Nucleic Acids Research, Vol. 17, No. 21, Pg 8543-8551, 1989), Buck (Biotechniques, Vol. 27, Pg. 528-536, 1999), and Genbank. Bethesda (MD): National Library of Medicine (US), National Center for Biotechnology Information; 2004 – [cited March 18, 2025] as applied to claims 1-4, 9, 11, 14, 16, 21, and 24 above, and further in view of McMichael et al., “DNA from Buccal Swabs Suitable for High-Throughput SNP Multiplex Analysis” Journal of Biomolecular Techniques 20:232-235 (2009).
Regarding claim 8, Kolialexi in view of Fernandez-Martinez, Xie et al., Rychlik, Buck, and Genbank do not teach isolating maternal and paternal DNA from buccal cells.
However, McMichael et al. teach buccal swabs (i.e. collecting buccal cells) are a “convenient and reliable method for collection of genetic material that is inexpensive and noninvasive and suitable for self-collection” providing “DNA of sufficient quantity and quality for high-throughput SNP multiplex analysis” (McMichael et al., Abstract). McMichael et al. demonstrate successful multiplex amplification and analysis using three different multiplex PCR assays comprising 25, 11, or 3 SNPs (McMichael et al., page 233, column 2, paragraph 2 and page 235, column 1, paragraph 4).
Therefore, it would have been prima facie obvious prior to the effective filing date of the claimed invention for one of ordinary skill in the art to modify the method of multiplex PCR detection of SRY, DAZ2, TSPY1, and ACTB in peripheral maternal blood for non-invasive prenatal sex determination, taught by Kolialexi in view of Fernandez-Martinez, Xie et al., Rychlik, Buck, and Genbank, with the method of multiplex amplification, detection, and analysis of SNPs comprising cheap, convenient, and rapid collection of high-quality DNA from buccal samples of adults, taught by McMichael et al. The ordinary artisan would have been motivated to modify the method taught by Kolialexi in view of Fernandez-Martinez, Xie et al., Rychlik, Buck, and Genbank to detect the claimed Y chromosome-specific markers in maternal and paternal buccal cell samples, rather than in peripheral maternal blood, because of the relative ease with which buccal cell samples may be collected compared to peripheral blood. The ordinary artisan would have been relatively confident that parental buccal swab samples comprising buccal cells would have been a suitable sample for multiplex PCR detection of Y chromosome specific markers because of the teaching of McMichael et al that multiplex detection of 3, 11, or 25 SNPs was readily attainable from buccal samples.
Claims 1 and 17-18 are rejected under 35 U.S.C. 103 as being unpatentable over Kolialexi et al., in view of Fernandez-Martinez et al., “Noninvasive fetal sex determination in maternal plasma: a prospective feasibility study” Genetics in Medicine. 14, 1, 101-106 (2012), Xie et al., “Designing highly multiplex PCR primer sets with Simulated Annealing Design using Dimer Likelihood Estimation (SADDLE)” Nature Communications, published April 11, 2022, Rychlik (Nucleic Acids Research, Vol. 17, No. 21, Pg 8543-8551, 1989), Buck (Biotechniques, Vol. 27, Pg. 528-536, 1999), and Genbank. Bethesda (MD): National Library of Medicine (US), National Center for Biotechnology Information; 2004 – [cited March 18, 2025] as applied to claims 1-4, 9, 11, 14, 16, 21, and 24 above, and further in view of Iizuka et al., “Neonatal Gene Therapy for Hemophilia B by a Novel Adenovirus Vector Showing Reduced Leaky Expression of Viral Genes” (Molecular Therapy-Methods Clinical Development. 2017 Jul 8; 6:183-193).
This obviousness rejection is over the scope of claims 17 and 18 identified as enabled in the 112(a) rejection above.
Regarding claim 1, as described above, Kolialexi et al. in view of Fernandez-Martinez et al., Xie et al., Rychlik, Buck, and Genbank teach methods comprising multiplex detection of the recited Y-chromosome specific and control DNA markers for detecting the presence of Y-chromosome DNA in a (male or female) fetal sample.
Regarding claims 17-18, Kolialexi et al. in view of Fernandez-Martinez et al., Xie et al., Rychlik, Buck, and Genbank do not teach further treating a fetus for hemophilia.
However, Iizuka et al. teach approaches for treating hemophilia in neonatal animal models comprising gene transfer for hemophilia B (Iizuka et al., abstract).
Therefore, it would have been prima facie obvious prior to the effective filing date of the claimed invention for one of ordinary skill in the art to have combined the methods taught by Kolialexi et al. in view of Fernandez-Martinez et al., Xie et al., Rychlik, Buck, and Genbank comprising detecting Y-chromosome specific DNA sequences in fetal samples with the methods taught by Iizuka et al. comprising treating neonatal animal models for hemophilia. The ordinary artisan would have been motivated to treat a fetus diagnosed with hemophilia and determined as having, or lacking, Y-chromosome DNA by the teachings of Kolialexi et al. that fetal sex and hemophilia status can be determined from cell free fetal DNA in the first trimester of pregnancy and by the teachings of Iizuka et al. that a single administration of a gene therapy vector for Hemophilia B rescued the bleeding phenotypes of neonatal hemophilia B mice for more than 100 days.
Claim 22 is rejected under 35 U.S.C. 103 as being unpatentable over Kolialexi et al., in view of Fernandez-Martinez et al., “Noninvasive fetal sex determination in maternal plasma: a prospective feasibility study” Genetics in Medicine. 14, 1, 101-106 (2012), Xie et al., “Designing highly multiplex PCR primer sets with Simulated Annealing Design using Dimer Likelihood Estimation (SADDLE)” Nature Communications, published April 11, 2022, Rychlik (Nucleic Acids Research, Vol. 17, No. 21, Pg 8543-8551, 1989), Buck (Biotechniques, Vol. 27, Pg. 528-536, 1999), and Genbank. Bethesda (MD): National Library of Medicine (US), National Center for Biotechnology Information; 2004 – [cited March 18, 2025] as applied to claims 1-4, 9, 11, 14, 16, 21, and 24 above, and further in view of EP 1201768 A2 (Laird, published 2002).
Kolialexi in view of Fernandez-Martinez, Xie et al., Rychlik, Buck, and Genbank do not teach that primers for multiplex PCR comprise one or more modified nucleotides.
However, Laird teaches PCR amplification using primers modified with 2’-O-methyl-, 2’-fluoro-, 2’-amino- or arabinose nucleotides (Laird, paragraph 0032-0033) reduce the formation of non-specific amplification, such as primer dimers (Laird, paragraph 0037).
Xie teaches that one of the major factors in designing multiplex compatible primer sets is reducing the frequency of primer dimer formation (Xie, Abstract).
Therefore, it would have been prima facie obvious prior to the effective filing date of the claimed invention for one of ordinary skill in the art to modify the method of multiplex PCR amplification of SRY, DAZ2, TSPY1, and ACTB, taught by Kolialexi in view of Fernandez-Martinez, Xie, Rychlik, Buck, and Genbank by including 2’-O-methyl-, 2’-fluoro-, 2’-amino- or arabinose nucleotides in the primers, as taught by Laird. The ordinary artisan would have been motivated to include modified nucleotides in the primers as taught by Laird by the suggestion of Xie that reduction of primer dimer formation is a major factor in designing multiplex-optimized primer sets (Xie, Abstract), and by the teaching of Laird that inclusion of modified nucleotides reduces the formation of primer dimers and off-target primer binding (Laird, paragraph 0037). The ordinary artisan would have been reasonably confident that inclusion of 2’-O-methyl-, 2’-fluoro-, 2’-amino- or arabinose nucleotides in the multiplex optimized primers designed by the method of Xie would have successfully further reduced the formation of primer dimers and off target amplification, thus reducing background signal in the multiplex Y chromosome detection method taught by Kolialexi in view of Fernandez-Martinez, Xie, Rychlik, Buck, and Genbank.
Claim 23 is rejected under 35 U.S.C. 103 as being unpatentable over Kolialexi et al., in view of Fernandez-Martinez et al., “Noninvasive fetal sex determination in maternal plasma: a prospective feasibility study” Genetics in Medicine. 14, 1, 101-106 (2012), Xie et al., “Designing highly multiplex PCR primer sets with Simulated Annealing Design using Dimer Likelihood Estimation (SADDLE)” Nature Communications, published April 11, 2022, Rychlik (Nucleic Acids Research, Vol. 17, No. 21, Pg 8543-8551, 1989), Buck (Biotechniques, Vol. 27, Pg. 528-536, 1999), and Genbank. Bethesda (MD): National Library of Medicine (US), National Center for Biotechnology Information; 2004 – [cited March 18, 2025] as applied to claims 1-4, 9, 11, 14, 16, 21, and 24 above, and further in view of Tounta et al., “A Multiplex PCR for Non-invasive Fetal RHD Genotyping Using Cell-free Fetal DNA” In vivo May 2011, 25(3) 411-417 and Piovesan et al., “On the length, weight, and GC content of the human genome” BMC Research Notes 2019 Feb 27; 12:106.
Kolialexi in view of Fernandez-Martinez, Xie et al., Rychlik, Buck, and Genbank do not teach the multiplex PCR method detects 0.2 ng of Y chromosome.
However, Tounta et al. teach multiplex PCR of 5 loci in circulating cell free fetal DNA comprising SRY and ACTB (Tounta et al., abstract). Tounta et al. further teach that multiplex amplification of the target loci were observed from single cells isolated from blood (Tounta et al., page 414, column 1, paragraph 1) after optimization of parameters comprising annealing temperatures and primer concentrations (Tounta et al., page 412, column 2, paragraph 2). Piovesan et al. teach the (human) male nuclear diploid genome weighs approximately 6.41 pg (Piovesan et al., abstract) and the Y chromosome weighs approximately 0.06 pg (Piovesan et al., table 2). Therefore, the teaching of Tounta et al. of optimized multiplex PCR detection of multiple targets comprising Y chromosome specific SRY sequences from a single circulating cell teaches multiplex PCR detection of 0.06 pg of Y chromosome DNA (i.e. far less than the recited 0.2 ng of Y chromosome DNA).
Therefore, it would have been prima facie obvious prior to the effective filing date of the claimed invention for one of ordinary skill in the art to have optimized the method comprising multiplex detection of the Y chromosome specific- and internal control- markers taught by Kolialexi in view of Fernandez-Martinez, Xie et al., Rychlik, Buck, and Genbank using the teachings of Tounta et al. and Piovesan et al. that multiplex PCR for circulating fetal cell free DNA for detection of target loci comprising Y-chromosome specific markers can be optimized to detect an amount of DNA equivalent to as little as a single diploid genome equivalent. The ordinary artisan would have been motivated to have optimized the multiplex PCR assay taught by Kolialexi in view of Fernandez-Martinez, Xie et al., Rychlik, Buck, and Genbank using the optimizable parameters taught by Tounta et al. to minimize the amount of genetic material required as input to perform the assay.
Claim 26 is rejected under 35 U.S.C. 103 as being unpatentable over Kolialexi et al., in view of Fernandez-Martinez et al., “Noninvasive fetal sex determination in maternal plasma: a prospective feasibility study” Genetics in Medicine. 14, 1, 101-106 (2012), Xie et al., “Designing highly multiplex PCR primer sets with Simulated Annealing Design using Dimer Likelihood Estimation (SADDLE)” Nature Communications, published April 11, 2022, Rychlik (Nucleic Acids Research, Vol. 17, No. 21, Pg 8543-8551, 1989), Buck (Biotechniques, Vol. 27, Pg. 528-536, 1999), and Genbank. Bethesda (MD): National Library of Medicine (US), National Center for Biotechnology Information; 2004 – [cited March 18, 2025] as applied to claims 1-4, 9, 11, 14, 16, 21, and 24 above, and further in view of Zhang et al., “Recent advances of glucocorticoids in the treatment of Duchenne muscular dystrophy (Review)”. Experimental and Therapeutic Medicine 21.5 (March 1, 2021): 447.
This is a new grounds of rejection necessitated by the addition of claim 26.
This rejection is to the scope of claim 26 identified as enabled in the 112(a) scope of enablement rejection above, wherein a non-fetal subject is given symptomatic treatment for DMD with glucocorticoids.
Regarding claim 26, Kolialexi et al. and Fernandez-Martinez et al. teach sex determination of fetuses by the methods described in the rejections above from maternal blood samples obtained from pregnant women at high risk of carrying a fetus affected with… Duchenne muscular dystrophy (Kolialexi et al., page 35, column 1, paragraph 4) (Fernandez-Martinez et al., page 104, column 1).
Kolialexi in view of Fernandez-Martinez, Xie et al., Rychlik, Buck, and Genbank do not teach treating a subject for Duchenne muscular dystrophy.
However, Zhang et al., teach “although glucocorticoid-dependent medication is the mainstay treatment for DMD, a standard treatment regimen has yet to be determined” (Zhang et al., Abstract), “glucocorticoids… prolong autonomous walking, reduce scoliosis, and improve cardiopulmonary function, quality of life, and the survival rate of patients” (Zhang et al., page 1, column 2, paragraph 1).
Therefore, it would have been prima facie obvious prior to the effective filing date of the claimed invention for one of ordinary skill in the art to have combined the methods of non-invasive prenatal diagnosis for sex determination and Duchenne muscular dystrophy, taught by Kolialexi in view of Fernandez-Martinez, Xie et al., Rychlik, Buck, and Genbank and described in detail in the rejections above, with the methods of symptomatic treatment for DMD comprising administration of glucocorticoids to affected children. The ordinary artisan would have been motivated to combine the methods of sex determination and non-invasive prenatal diagnosis taught by Kolialexi in view of Fernandez-Martinez, Xie et al., Rychlik, Buck, and Genbank with the methods of treatment taught by Zhang et al. by the teaching of Zhang et al. that “glucocorticoids… prolong autonomous walking, reduce scoliosis, and improve cardiopulmonary function, quality of life, and the survival rate of patients” (Zhang et al., page 1, column 2, paragraph 1).
Response to arguments
The response asserts that the specific sequences of the primers recited by claim 1 are distinct from the longer sequences of the genes from which they are derived, which are known in the art on the grounds that the ordinary artisan would not have been motivated to select the specific primers claimed and that the particular primers claimed provide a specific, non-obvious solution for amplifying portions of the recited genes and asserts that the primers claimed result with superior detection properties compared to the cited prior art. The response further asserts that: a) consumption of polymerase and nucleotides, b) the possibility of introducing primer-primer interactions, and c) the repetitive nature of the Y chromosome constitutes unpredictable factors. These arguments have been reviewed but are not persuasive.
As described above, Buck, and Rychlik teach first principles and demonstrate interchangeability of primers for a given known target sequence. Xie et al. teach publicly available software for searching for sets of compatible multiplex primers and demonstrate examples wherein as many as 60 primer pairs are successfully used in multiplex PCR. Furthermore, the claimed primer sequences are simply fragments of the extremely well-known Y-chromosome specific sequences taught by the prior art (SRY, DAZ2, TSPY1) and the well-known and widely used internal control sequence ACTB, the complete sequences of which are taught by Genbank. Therefore, to summarize the detailed rejections above, the prior art, taken together, renders obvious the claimed invention as follows. It would have been obvious to the ordinary artisan to make a simple combination of markers used by Kolialexi et al. and Fernandez-Martinez et al. in multiplex PCR assays for prenatal diagnosis for which a number of suitable primers could have been selected using the known sequences in Genbank using the known methods taught by Xie, Buck, and Rychlik. Absent some unexpected result present from the specific claimed primers, it is the position of the examiner that selection of any combination of primers specific to the well-known sequences of the genetic markers widely used for fetal sex determination would have been obvious to the ordinary artisan. Even in the instance that some “manual” screening or “adjustment” (i.e. BLAST search to confirm no significant homology to off-target sequences or inputting specific desired melting temperatures into the program taught by Xie et al.) was performed to optimize the specific primers, optimization of these parameters are well known and routine to those of ordinary skill in the art.
To further demonstrate that the claimed primers are merely obvious variants of those known in the art, the examiner has prepared comparative alignments of each of the amplicons that may reasonably be expected to be produced by the claimed primers and those expected to be produced by the primers taught in the art (see below).
Comparing the ACTB amplicons expected from the claimed primers to those from the primers taught by Kolialexi: it is clear that the naturally occurring sequences of claimed primers are simply an alternative to those known in the art readily selected by the ordinary artisan by the methods discussed above, absent evidence of unexpected results.
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Comparing the SRY amplicons expected from the claimed primers to those from the primers taught by Kolialexi: it is clear that the naturally occurring sequences of claimed primers are simply an alternative to those known in the art readily selected by the ordinary artisan by the methods discussed above, absent evidence of unexpected results.
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Comparing the DAZ amplicons (repeats amplified in DAZ 1-4 by both primer sets) expected from the claimed primers to those from the primers taught by Fernandez-Martinez et al., it is clear that the naturally occurring sequences of claimed primers are simply an alternative to those known in the art readily selected by the ordinary artisan by the methods discussed above, absent evidence of unexpected results.
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Comparing the TSPY amplicons (repeats amplified in TSPY1-4, 6P, 7P, 8-10, and 11P by both primer sets) expected from the claimed primers to those from the primers taught by Kolialexi et al., it is clear that the naturally occurring sequences of claimed primers are simply an alternative to those known in the art readily selected by the ordinary artisan by the methods discussed above, absent evidence of unexpected results.
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The response dated June 24, 2026 asserts that the claimed method differs from mere selection of primers (that is well known and routine in the art) because “in multiplex PCR, primers must be selected to avoid hairpins, self-dimers, and cross-dimers” and “the examiner has not met their burden…[to demonstrate] that prior art methods would select the specific primer pairs… and explained bow the prior art methods provided a reasonable expectation of successfully avoiding known problems and obtaining the specific multiplex primers required by the claims” and “further rebuts… arguments… that any and all complementary fragments of a target gene are functional equivalents, “obvious variants” or “alternatives” for use in multiplex PCR…”
The response further argues that Rychlik or Buck cannot be relied upon for teaching the equivalence of the claimed primers and prior art primers because Buck does not use the claimed Y chromosome markers, “cherry picks” primer sequences selected by other labs, and evaluate avoidance of hairpins and primer dimers.
These assertions and arguments have been thoroughly reviewed and are not persuasive.
First, as described above, Buck does not merely cherry-pick primers, but rather invited multiple labs to independently design primers by their own methods, which were known in the art at the time. Furthermore, Buck tested 95 primers spaced at 3 nucleotide intervals along the entire template sequence, testing more than 1/3 of all possible 18-mer primers along the template sequence without regard to pre-selection or design criteria. When Buck tested each of the primers selected by the methods of the different labs, Buck found that every single primer worked (Pg. 533, column 1). Further, every single control primer (i.e. the 95 primers spaced at 3 nucleotide intervals) functioned as well (Pg. 533, column 1). Buck expressly states, “The results of the empirical sequencing analysis were surprising in that nearly all of the primers yielded data of extremely high quality” (Pg. 535, column 2). Therefore, Buck provides direct evidence that all primers would be expected to function, and in particular, all primers selected according to the ordinary criteria. This clearly shows that every primer would have a reasonable expectation of success. Additionally, as described above, Rychlik teaches ordinary, routine, and predictable criteria for selection of primer and probe sequences from known sequences including checking nucleic acid sequences for off-target priming, formation of secondary structures, and primer dimerization.
The response asserts that selection of the claimed set of primers differs from the combination of prior art primers because “in multiplex PCR, primers must be selected to avoid hairpins, self-dimers, and cross-dimers” and “the examiner has not met their burden…[to demonstrate] that prior art methods would select the specific primer pairs… and explained bow the prior art methods provided a reasonable expectation of successfully avoiding known problems and obtaining the specific multiplex primers required by the claims” and “further rebuts… arguments… that any and all complementary fragments of a target gene are functional equivalents, “obvious variants” or “alternatives” for use in multiplex PCR…” (i.e. the claimed primers are asserted not to be functional equivalents, obvious variants… of the cited combination of prior art primers because the prior art primers would not have been reasonably expected to function in the claimed multiplex PCR method).
In order to evaluate these arguments and assertions, the examiner utilized publicly available web-tools for evaluating candidate primer sets for self- and cross- primer hybridization (ThermoFisherScientific Multiple Primer Analyzer, https://www.thermofisher.com/us/en/home/brands/thermo-scientific/molecular-biology/molecular-biology-learning-center/molecular-biology-resource-library/thermo-scientific-web-tools/multiple-primer-analyzer.html, accessed August 13, 2026) to compare expected self- and cross- primer hybridization products between the claimed combination of primers and the combination of cited prior art primers.
From the results which follow, it appears that the claimed primers are predicted to form a greater number of said self- and cross- primer hybridization products which are asserted to be undesirable for multiplex PCR relative to the combination of cited prior art primers.
For the claimed primer sequences SEQ ID NO: 1-8:
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Seq ID Nos 1, 4, 7, and 8 are predicted to form self-hybridization dimers, and the following four cross primer dimers are predicted: 1 with 3, 1 with 4, 3 with 4, and 3 with 8.
For the combination of cited prior art primers:
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No self-hybridization primer dimers are predicted for the prior art primers. Two cross primer dimers are predicted for the prior art primers (Kolialexi_ACTB_forward with Kolialexi_SRY_reverse) and (Kolialexi_ACTB_forward with Fernandez-Martinez_DAZ_forward).
Absent unexpected results, and contrary to the arguments in the response, it appears that the claimed primers and the combination of prior art primers would both have been expected to function in multiplex PCR. In fact, based upon the number of predicted self- and cross- primer dimers, it appears that the combination of prior art primers would have had greater support for “expected success” than the claimed combination of primers absent experimental data comparing the two combinations of primers under the same conditions.
Quoting MPEP 716.01(c):
“
Objective evidence which must be factually supported by an appropriate affidavit or declaration to be of probative value includes evidence of unexpected results, commercial success, solution of a long-felt need, inoperability of the prior art, invention before the date of the reference, and allegations that the author(s) of the prior art derived the disclosed subject matter from the inventor or at least one joint inventor. See, for example, In re De Blauwe, 736 F.2d 699, 705, 222 USPQ 191, 196 (Fed. Cir. 1984) ("It is well settled that unexpected results must be established by factual evidence." "[A]ppellants have not presented any experimental data showing that prior heat-shrinkable articles split. Due to the absence of tests comparing appellant’s heat shrinkable articles with those of the closest prior art, we conclude that appellant’s assertions of unexpected results constitute mere argument."). See also In re Lindner, 457 F.2d 506, 508, 173 USPQ 356, 358 (CCPA 1972); Ex parte George, 21 USPQ2d 1058 (Bd. Pat. App. & Inter. 1991).
Arguments presented by the applicant cannot take the place of evidence in the record. In re Schulze, 346 F.2d 600, 602, 145 USPQ 716, 718 (CCPA 1965) and In re De Blauwe, 736 F.2d 699, 705, 222 USPQ 191, 196 (Fed. Cir. 1984). Examples of statements which are not evidence and which must be supported by an appropriate affidavit or declaration include statements regarding unexpected results, commercial success, solution of a long-felt need, inoperability of the prior art, invention before the date of the reference, and allegations that the author(s) of the prior art derived the disclosed subject matter from the inventor or at least one joint inventor.
”
Therefore, for the reasons detailed in the rejections and summarized here, the claims are/remain rejected under U.S.C. 103 over the cited combinations of prior art references.
Conclusion
No claim is allowed.
Applicant's amendment necessitated the new ground(s) of rejection presented in this Office action. Accordingly, THIS ACTION IS MADE FINAL. See MPEP § 706.07(a). Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any nonprovisional extension fee (37 CFR 1.17(a)) pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to ZACHARY MARK TURPIN whose telephone number is (703)756-5917. The examiner can normally be reached Monday-Friday 8:00 am - 5:00 pm.
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/Z.M.T./Examiner, Art Unit 1682
/WU CHENG W SHEN/Supervisory Patent Examiner, Art Unit 1682
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