DETAILED ACTION
Notice of Pre-AIA or AIA Status
The present application is being examined under the pre-AIA first to invent provisions.
Status of the Claims
The claim set received 24 March 2026 has been entered into the application.
Claims 3, 7, 12, 16, 19, 22-24, and 28 are amended.
Claims 31-34 are new.
Claims 1-2, 4-6, 9-11, 14-15, 17-18, 20, and 27 previously are canceled.
Claim(s) 3, 7-8, 12-13, 16, 19, 21-26, and 28-34 are pending.
Election/Restrictions
The Applicant has elected Group II, claims 3-19 drawn to a system determining the state of tissue of interest in a subject based on principle component analysis. Election was made without traverse in the reply filed on 09 July 2024.
Priority
Acknowledgment is made of applicant’s claim for priority to U.S Provisional Application filed 27 January 2012. Application 17/187,298 is a continuation of abandoned application 16/373,996 filed 03 April 2019 which is a continuation of 15/377,894 (U.S Patent 10,287,632) filed 13 December 2019 which is a continuation of 14/861,650 (U.S Patent 10,240,200) filed 26 March 2019 which is a divisional of 13/752,131 (Abandoned) filed 28 January 20213 which claims priority benefit to U.S Provisional Application 61/591,642 filed 27 January 2012.
Information Disclosure Statement
The information disclosure statement (IDS) submitted on 24 March 2026 is in compliance with the provisions of 37 CFR 1.97. Accordingly, the information disclosure statement is being considered by the examiner.
Claim Rejections - 35 USC § 101
The instant rejection is maintained for reason for record in the Office Action mailed 05 November 2025 and modified in view of the amendments filed 24 March 2026. It is noted the amendments received 24 March 2026 necessitated new ground(s) of rejection.
35 U.S.C. 101 reads as follows:
Whoever invents or discovers any new and useful process, machine, manufacture, or composition of matter, or any new and useful improvement thereof, may obtain a patent therefor, subject to the conditions and requirements of this title.
Claims 3, 7-8, 12-13, 16, 19, 21-26, and 28-34 are rejected under 35 U.S.C. 101 because the claimed invention is directed to non-statutory subject matter.
Claims Analysis
Under broadest reasonable interpretation, the claims are direct towards a system for analyzing and evaluating nucleic acid data, more specifically, cell-free messenger ribonucleic acids (cf-mRNAs) for deconvolving tissue types.
Following the flowchart of the MPEP 2106
Step I - Process, Machine, Manufacture or Composition
Claims 3, 7-8, 12-13, 16, 19, 21-26, and 28-34 are drawn to a method, so a process.
2A Prong I - Identification of an Abstract Idea
Claim 3 recites
(h) computer processing the collection of sequencing data wherein the computer processing comprises deconvoluting the test fractional contributions of tissues types from plasma derived from the test blood sample of the test subject against a reference cell-free transcriptome in a reference blood sample of a reference subject.
This step can be performed in the human mind by following instructions to deconvolute sequencing data into test fractional contributions of tissue types against a reference cell free transcriptome and is therefore an abstract idea. This step encompasses performing mathematical/statistical computations to deconvolute test fraction contribution of tissue types against a reference cell free transcriptome and is therefore an abstract idea. The step encompasses organizing and manipulating information (i.e., sequencing data) through mathematical correlations (i.e., deconvolution and/or test fractional contributions) and is therefore an abstract idea. Here, the claims take existing data (i.e., sequencing data) and manipulate the data using mathematical correlations by deconvolution to produce test fractional contributions of cells and/or tissue types. See MPEP 2106.04(a)(2)(I)(A)(iv). The term “test fractional contributions” is interpreted as proportion of cells of a specific tissue type(s).
wherein deconvolving the test fractional contributions of tissue types further comprises: (1) identifying a panel of tissue-associated transcripts; (2) determining total ribonucleic acid (RNA) in plasma derived from the test blood sample of the test subject; and (3) assessing the total RNA of (2) against the panel of tissue-associated transcripts of (1), wherein the total RNA is considered a summation of the tissue-associated transcripts.
This step can be performed in the human mind by following instruction to (1) identify a panel of tissue-associated transcripts and (2) determine the total RNA of step (2) against the panel of tissue-associated transcripts of step (1) and observing, comparing, assessing, and evaluating the identified panel of tissue-associated transcripts and determined (i.e., summed) total RNA to deconvolve test fractional of tissues types and is therefore an abstract idea.
wherein the total RNA is considered a summation of the tissue-associated transcripts.
This step describes the total RNA as a summation of the tissue-associated transcription which encompasses mathematical concepts of summation with respect to summing the tissue-associated transcription to yield a total RNA which reads on abstract ideas/mathematical concepts.
Claims 3, 7-8, 12-13, 16, 19, 21-26, 28, and 33-34 are further drawn to limitations that describe the abstract ideas of claim 3 and are therefore also abstract ideas.
2A Prong II - Consideration of Practical Application
Claim 3 does not recite any additional element which integrates the recited judicial exception into a practical application. Here, in the instant case, the claims merely set forth a method of data analysis for analyzing sequence data to produce test fraction contributions of cells and/or tissue types for deconvoluting cell into tissue types. Such a result only produces information (i.e., deconvolved test fraction contribution from sequencing data) and does not provide for a practical application in the real-world realm of physical things and acts, i.e., the claims do not utilize the data generated by the judicial exception to affect any type of change. See MPEP 2106.04(a)(2)(A)(iv). Therefore, the claims do not utilize the isolated cfRNA, obtained cDNA, amplified cDNA, sequenced data, and deconvoluted test fractional contributions and the abstract ideas to "transform the nature of the claim” in order to construct a practical application such as treating a subject, transformation of matter, or improving upon an existing technology.
This judicial exception is not integrated into a practical application because the claims do not meet any of the following criteria:
an additional element reflects an improvement in the functioning of a computer, or an improvement to other technology or technical field;
an additional element that applies or uses a judicial exception to effect a particular treatment or prophylaxis for a disease or medical condition;
an additional element implements a judicial exception with, or uses a judicial exception in conjunction with, a particular machine or manufacture that is integral to the claim;
an additional element effects a transformation or reduction of a particular article to a different state or thing; and
an additional element applies or uses the judicial exception in some other meaningful way beyond generally linking the use of the judicial exception to a particular technological environment, such that the claim as a whole is more than a drafting effort designed to monopolize the exception.
Step 2B: Consideration of Additional Elements and Significantly More
The claimed method also recites "additional elements" that are not limitations drawn to an abstract idea.
The recited additional element of centrifugation of claims 3 step (a) does not add significantly more than the recited judicial because centrifuging samples to separate blood elements (i.e., cfRNA, cfDNA, plasma, proteins) is well-known and conventional. See MPEP 2106.05(d)(II)(i). To provide evidence of conventionality of utilizing centrifuges of claim 3 step (a), Bischoff et al. (Bischoff) discloses centrifuging blood samples using an initial 800g for 10 and 16,000g for 10 to remove contaminating cellular particles [Bischoff, page 1 right col para 0008] (US Patent Pub No.: US 20110183338, Patent Pub Date: 28 Jul 2011).
The recited additional element of isolating cfRNA by adding cell-membrane stabilizing agent of claim 3 step (b) does not add significantly more to the recited judicial exception because adding membrane stabilizers for further collecting nucleic acid data that is subsequently analyzed by the abstract ideas is well-known and conventional. See MPEP 2106.05(d)(II)(i, iii, v, vii). Adding cell-membrane stabilizers to blood samples to prevent cell degradation and preserve the integrity of cells and their components for accurate testing and analysis is well known. To provide evidence of conventionality, Dhallan et al. (Dhallan) discloses collecting free fetal DNA (cfDNa) and that agents were added to samples to inhibit cell lysis such as membrane stabilizers, a cross-linker, or a cell lysis inhibitor [disclosure page 5 right col. para. 0057] to samples including blood samples [para. 0058]. Dhallan discloses “membrane stabilizers agents such has added to the sample including but not limited to aldehydes, urea formaldehyde, phenol formaldehyde, DMAE (dimethylaminoethanol), cholesterol, cholesterol derivatives, high concentrations of magnesium, Vitamin E, and Vitamin E derivatives, calcium, calcium gluconate, taurine, niacin, hydroxylamine derivatives, bimoclomol, Sucrose, astaxanthin, glucose, amitriptyline, isomer A hopane tetral phenylacetate, isomer Bhopane tetral phenylacetate, citicoline,inositol, Vitamin B, Vitamin B complex, cholesterol, hemisuccinate, Sorbitol, calcium, coenzyme Q, ubiquinone, Vitamin K, Vitamin K complex, menaquinone, zonegran, Zinc, ginkgo biloba extract, diphenylhydantoin, perftoran, polyvinylpyrrolidone, phosphatidylserine, tegretol, PABA, disodium cromglycate, nedocromil Sodium, phenyloin, Zinc citrate, mexitil, dilantin, Sodium hyaluronate, or polaxamer 188.” [disclosure page 9 left col para. 0078] [Instant specification page 11 first paragraph] (US Patent Pub 2004/0137470, Patent Pub Date: 15 July 2004).
To provide further conventionality of isolating cfRNA by adding cell-membrane stabilizing agent, Quake et al. (Quake) discloses using cell membrane stabilizers or impeding cell lysis [page 6 left col para. 0071]. Quake discloses different chemicals that can be used as cell membrane stabilizers or cell lysis impeders such as “aldehydes, urea formaldehyde, phenol formaldehyde, DMAE (dimethylaminoethanol), cholesterol, cholesterol derivatives, high concentrations of magnesium, vitamin E, and vitamin E derivatives, calcium, calcium gluconate, taurine, niacin, hydroxylamine derivatives, bimoclomol, and coenzyme Q, for example [disclosure page 6 para. 0073]. Quake discloses that “while the present description refers to DNA, fetal RNA found in maternal maybe analyzed as well [page 5 right col. para. 0063] (US Patent Pub 2007/0202525, Patent Pub Date: 30 August 2007).
To provide further conventionality of using cell membrane stabilizer and/or agents, Quake et al. (Quake 2009) discloses genetic material maybe DNA or RNA, preferably mRNA [disclosure page 2 right col para. 0026]. Quake 2009 discloses agents for stabilizing cell membrane or cell lysis inhibitors such as Vitamin E, DMAE, glucose, aldehydes, Dilantin, for example [disclosure page 6 left col para. 0065] (Quake 2009: US Patent Pub 2009/0170113, Patent Pub Date: 02 July 2009).
The recited additional element of performing DNase digestion with RNA of claim 3 step (d) does not add significantly more than the recited judicial because DNase to purify samples of RNA by removing contaminating DNA from the RNA samples of RNA is well-known and conventional. See MPEP 2106.05(d)(II)(i). To provide evidence of conventionality. Allen et al. (Allen) discloses DNA can be eliminated from a RNA samples by digesting the samples with a DNA specific DNase1 enzyme [Allen, page 8 right col para 0063] (US Patent Pub No.: US 2009/0311269, Patent Pub Date: 17 December 2009).
The recited additional element of isolating nucleic acids from a blood sample of claim 3 step (c) does not add significantly more than the recited judicial because isolating cell-free cfRNA from blood and/or tissues that is subsequently analyzed by the abstract ideas is well-known and conventional. See MPEP 2106.05(d)(II)(i). To provide evidence of conventionality, Swarup et al. (Swarup) teaches a min-review of using circulating cell-free nucleic acids for detection of human disease [title]. Swarup teaches a schematic of various pathways which cell free nucleic acids are released into the system [page 797 fig 1] (FEBS letters, 2007-03, Vol.581 (5), p.795-799). To provide further conventionality. Kurn et al. (Kurn) teaches using cell-free RNA with respect to compositions and methods for whole transcriptome analysis [claims 14, 31, and 48] (US 2011/0189679). To provide further evidence of conventionality, Kroh et al. (Kroh) teaches analysis of circulating microRNA biomarkers [abstract]. Kroh teaches analyzing microRNA in plasma and serum [page 298 right section 2.2.1]. Kroh teaches plasma and serum are acceptable types of specimens for circulating miRNA analysis [page 299 section 2.2.3] (Methods (San Diego, Calif.), 2010-04, Vol.50 (4), p.298-301).
The recited additional element of obtaining nucleic acids of claim 3 step (e) does not add significantly more than the recited judicial because reverse transcribing RNA to obtain cDNA data that is subsequently amplified is well-known and conventional. See MPEP 2106.05(d)(II) (v and viii). To provide conventionality of generating/obtaining double stranded cDNA products, Kurn teaches optionally generating double stranded cDNA products form reversed transcribed products [claims 18 and 34] [disclosure paragraphs [0028-0032]]. To provide further conventionality of transcribing RNA to obtain cDNA, Klickstein et al. (Klickstein) teach a general construction of cDNA libraries from mRNA (Klickstein: Current Protocols in Molecular Biology (1995) 5.5.1-5.5.14). To provide further conventionality of reverse transcribing RNA to cDNA, Qiagen 2011 teaches selective conversion of mature miRNAs into cDNA (i.e., double stranded DNA) [page 11 figure 2]. Qiagen 2011 teaches Simultaneous conversion of all RNA species into cDNA (i.e., double stranded DNA) in miScript HiFlex Buffer [page 12 figure 3] (Qiagen Sample and Assay Technologies: miScript PCR System Handbook 2011). To provide further conventionality of reverse transcribing RNA to cDNA, Invitrogen 2001 teaches an outline of procedures for cDNA synthesis [page 2 figure 1]. Invitrogen 2001 teaches converting mRNA to ds cDNA products [page 6 fig 3] (Invitrogen cDNA Synthesis System 2001). To provide further evidence of conventionality, Kroh teaches a protocol for reverse-transcription of miRNA using a TaqMan miRNA reverse transcription kit [page 300 left col section 2.6.1]. To provide further evidence of conventionality, Kurn teaches reverse using transcription of cell free RNA to generate double stranded cDNA products [claims 1 steps (b)-(c) and 15].
The recited additional element of amplifying nucleic acids of claim 3 step (f) does not add significantly more than the recited judicial because amplifying cDNA to produce dsDNA that is subsequently sequenced is well-known and conventional. See MPEP 2106.05(d)(II) (vii). To provide conventionality of amplifying cDNA to produce dsDNA, Kurn teaches amplifying double-stranded cDNA [claims 18 and 34]. Kurn further teaches that reversed transcribed product are amplified before being sequenced [claims 4-5]. To provide further evidence of conventionality of amplifying RNA (i.e., double stranded cDNA and in 3’ direction), Ovation teach amplifying cDNA randomly and by the 3’ end [Ovation page 1 middle col] (Ovation RNA-Seq System 2011)
The recited additional element of sequencing nucleic acids of claim 3 step (g) does not add significantly more than the recited judicial because sequencing cDNA to obtain sequencing data that is subsequently analyzed by abstract idea(s) is well-known and conventional. See MPEP 2106.05(d)(II) (v and vii). To provide evidence of conventionality, Kurn teaches sequencing of amplified reverse transcribed products [claims 4-5].
The additional element of using computer process, components, and equipment of claim 3 step (e) does not add significantly more than the recited judicial exception because using computer processes and equipment to analyze sequence data and process abstract ideas is well-known and conventional. See MPEP 2106.05(b) and 2106.05(d).
The recited additional element of sequencing nucleic acids of claim 26 does not add significantly more than the recited judicial because next generation sequencing to obtain sequencing data that is subsequently analyzed by abstract idea(s) is well-known and conventional. See MPEP 2106.05(d)(II) (ii, v, and vii).
The recited additional element of producing cDNA library of claim 29 does not add more than the recited judicial exception because producing cDNA libraries that are used to produce sequencing data that is subsequently analyzed by the abstract ideas is well-known and conventional. See MPEP 2106.05(d)(II) (ii, v, and vii).
The recited additional element of performing PCR on the cDNA of claims 30-31 does not add more than the recited judicial exception because performing on cDNA to produce nucleic acid sequences that are further sequenced to produce sequencing data that is subsequently analyzed by the abstract ideas is well-known and conventional. See MPEP 2106.05(d)(II) (ii, v, and vii).
Step 2B: Consideration of Combination of Additional Elements
With respect to the above additional elements being considered as a whole and/or in combination, the combination additional elements are as follows:
(a) performing centrifugation on a test blood sample of a test subject to obtain a plasma sample,
(b) adding a cell membrane stabilizing agent to the plasma sample,
(c) isolating cell-free ribonucleic acid (cfRNA) from the plasma sample wherein the cfRNA comprises cell-free messenger RNA,
(d) performing DNAse digestion on the isolated cfRNA.
(e) reverse transcribing the cfRNA to obtain cDNA,
(f) amplifying the cDNA to produce double-stranded cDNA amplification wherein the amplifying is initiated at a 3’ end of the cDNA and randomly throughout a whole transcriptome of the test blood sample, and
(g) performing a sequencing assay on the double-stranded cDNA amplification to obtain sequencing data.
Here, El-Hefnaway et al. (2004) teach centrifuging (page 567m col. 2, par. 2) for cf RNA extracted from maternal blood plasma required treatment to DNase (page 566, col. 1, par. 1, col. 2, par. 2; page 567, col. 2, par. 1and 568, col. 1 par. 1) to remove contaminating DNA; El-Hefnaway et al. teach mRNA detection from plasma (page 567, col. 2, par. 1) and amplification by PCR, so El-Hefnawy et al. teach (a), (c ), (d ) and (f). (Clinical Chemistry, 2004-03, Vol.50 (3), p.564-573).
Huang et al. (2009) teach (page 1968, col. 2, par. 2-4) isolating cf RNA from seminal plasma by centrifugal force followed by DNase to remove contaminating DNA. Then cfRNA is amplified by PCR and reverse transcribed into cDNA (page 1969, col. 1, par. 3), so Huang teach (a), (c), (d), (e) and (f). (Clinical chemistry (Baltimore, Md.), 2009-11, Vol.55 (11), p.1967-1976)
Prenatal Diagnosing (2008) teaches protocols for nucleic acid extraction from plasma (page 271), including centrifuge and DNase (page 271, section 3.2 #1 and 5); collection of blood plasma, and DNase for RNA extraction is taught (page 277-278, sections 2.1 and 2.2 #4 and 5) followed by cDNA synthesis (page 278, section 2.3). Extraction of cf RNA, DNase, centrifuge and DNase are taught (page 280 #1, 4, 6). Synthesis of cDNA from RNA (page 281, 3.3) is taught. Prenatal Diagnosis also evidence (a), (c), (d), and (e) as common protocols. (Prenatal Diagnosing: Hahn, Sinuhe, editor.; Jackson, Laird G., editor.2008).
Ovation teach amplifying cDNA randomly and by the 3’ end [Ovation page 1 middle col] (Ovation RNA-Seq System 2011)
As evidenced by El-Hefnaway et al., Huang et al., Prenatal Diagnosing (2008), and Ovation, the prior art teaches that in order to obtain cell free nucleic acid, more specifically ribonucleic acids, from a blood sample, centrifugation is a required step to separate blood elements such plasma, red blood cells, white blood cells, and platelet. Thus, in order to obtain cell-free RNA, centrifugation must be performed. Extraction of biomarkers from plasma necessarily requires centrifugation.
Furthermore, regarding the utilization of DNase, El-Hefnaway et al. (page 566), Huang et al. (1968), and Prenatal Diagnosing (2008) provide evidence, that in order to purify a sample of RNA, DNase is routinely used to remove DNA which is considered a contaminant when obtaining cell free RNA (mRNA) from a blood sample. Next in the sequence of steps, reverse transcription converts unstable RNA molecules into stable complementary DNA (cDNA) which is an essential processing step so that the cfRNA can be converted to cDNA for subsequent amplification, sequencing, or reliably quantified. Lastly, regarding (f), amplifying cDNA to produce double stranded cDNA for subsequent analysis is also a required step such that cfRNA can be converted to cDNA for subsequent amplification, sequencing, or reliably quantified. Therefore, the combination of additional elements (a), (c), (d), (e) and (f) is conventional.
Dov-Drnovsek et al. (2008) teach extracting cell free mRNA from plasma, creating cDNA from mRNA (page 126, col. 1, last sentence) and cDNA is amplified by PCR and sequencing data is determined (page 126, col. 2, par. 1); a centrifugation of plasma is also taught (Abstract and page 128, col. 1, par. 2); Dov-Drnovsek et al. therefore evidence (a), (c ), (e) and (f). (Annals of the New York Academy of Sciences, 2008-08, Vol.1137 (1), p.125-129).
Poon et al. (2000) evidence cell free RNA from maternal blood plasma (page 1833) which is converted to cDNA and cDNA is used in the PCR (Fig 1, caption) to amplify and sequence the DNA (page 1833, col. 2, par. 1); Poon et al. therefore evidence (c), (e), (f) and (g). (Clinical chemistry (Baltimore, Md.), 2000-11, Vol.46 (11), p.1832-1834).
In addition to El-Hefnaway et al. (2004) and Huang et al. (2009), the prior art of Dov-Drnovsek et al. (2008) and Poon et al. (2000) further evidence that isolating cell free RNA from plasma followed by transcription to cDNA, which is amplified and sequenced is routine, conventional and well understood. While the use of DNase is not necessarily taught in all references concerning RNA extraction from plasma and subsequence transcription to cDNA, El-Hefnaway et al. and Huang et al., provide sufficient evidence that DNase is routinely used for removal of DNA where cell free RNA sample purification is concerned.
Regarding (b) of claim 1, reciting the addition of a cell membrane stabilizer prior to cfRNA isolation, it is well known, routine and conventional that cell membrane stabilization would aid enrichment of the cfRNA sample.
Dhallan et al. (2004) evidence centrifuging to isolate free nucleic acid (par. 0083) before which a cell membrane stabilizer to is added to prevent lysis of cells (par. 0044, 0057, 0064-0065); cDNA amplification is also taught (par. 0064 and 0265); reverse transcription of DNA from RNA is also suggested as routine (par. 0189); Dhallan et al. evidences the same list of cell membrane stabilizers (par. 004) as listed in Applicant’s specification (page 11, par. 1). Dhallan et al. evidences (a), (b), (e) and (f).
Puzycks (2009) is a review teaching formaldehyde as a cell membrane stabilizer for cell free nucleic acid extraction prior to centrifugation. Puzycks teach that formaldehyde prevents cell lysis (page 2, col. 1, par. 3). Puzycks also teach extract of cfRNA from plasma (page 3, col. 1, last par.) therefore evidence (a), (b) in the context of extracting techniques including extracting cfRNA. It is noted that Applicants instant specification (page 11, par. 1-2) teach adding the stabilizer to maternal blood to reduce cell lysis and not necessarily after centrifugation which would produce a separation of plasma and platelets. (Prenatal diagnosis, 2008-01, Vol.28 (1), p.1-6).
Maron et al. (2007) is a review teaching formaldehyde is a known cell stabilizer to impede cell lysis and is used with centrifugation (page 6, middle col., par. 2). Maron et al. teach cell membrane stabilization in the context of mRNA extraction (Abstract). (American Journal of Medical Genetics Part C: Seminars in Medical Genetics, 2007-02, Vol.145C (1), p.5-17).
Quake et al. (US 2007/0202525, par. 0073-0076) and (US 2009/0170113, par. 0065-0068) teach cell membrane stabilizers in combination with plasma centrifugation prior to cell free nucleic acid extraction including RNA enrichment (par. 0064 in Quake 2007 and par. 0056 in Quake 2009). Quake et al. also teach that mRNA is the preferred genetic material (par. 0026 in Quake 2007 and 2009), as listed in Applicant’s instant specification in par. 0044.
The prior art of Dhallan et al. (2004), Puzycks (2009), Maron et al. (2007), and Quake et al. therefore evidence preventing cell lysis in the field of mRNA extraction from plasma was well known, routine and conventional. Here, adding cell membrane stabilizer is an integral step to ensure that RNA and/or other nucleic contamination can be prevented by stabilizing cell membrane and/or preventing cell lysis or cell leakage of nucleic acid into the plasma sample. For example, adding cell membrane stabilizer to prevent capture of lymphocyte DNA/RNA contamination from the buffy coat of the blood elements of the centrifuged sample(s) would be an essential and required step for purifying cfRNA from plasma such that the encompassed cfRNA/mRNA can be reverse transcribed, amplified, and sequenced for subsequent data processing. Therefore, using cell stabilizer to prevent nucleic acid contamination if conventional.
Thus, in combination and as a whole, these additional elements (a-g) are conventional and routine for extracting ribonucleic acids (i.e., cfRNA/mRNA) from blood samples, processing plasma from said blood samples (a), stabilizing cellular membranes to prevent cellular DNA/RNA contamination to obtain high concentrate of RNA (b), isolating cfRNA (c), digesting DNA contaminants (d), and reverse transcribing cDNA to produce double stranded DNA (e) for subsequent amplification and sequencing of targeted cDNA to obtain sequencing data. Therefore, and as evidenced by El-Hefnaway et al., Huang et al., Prenatal Diagnosing (Hanh), Drnovsek et al., Poon et al., Puzycks et al. Dhallen et al., Maron et al., and Quake et al., the ordered combination of data gathering elements for obtaining sequencing data from cfRNA/cfmRNA obtained from a blood samples are routine, well-known, and conventional. See MPEP 2106.05(a)(II)(ii), 2106.05(d)(II) (i-iii, v, vii).
In conclusion, and when viewed as a whole, these additional claim element(s) do not provide meaningful limitation(s) to transform the abstract idea recited in the instantly presented claims into a patent eligible application of the abstract idea such that the claim(s) amounts to significantly more than the abstract idea itself. Therefore, the claim(s) are rejected under 35 U.S.C. 101 as being directed to non-statutory subject matter.
Response to Arguments
Applicant’s arguments, filed 24 March 2026, have been fully considered but the rejection is maintained. Furthermore, upon further consideration, a new ground(s) of rejection is made in view of amendments received 24 March 2026.
The Applicant states the claims are patent eligible under Step 2B. The Applicant states the claims as a whole provide significantly more than the recited judicial exception. The Applicant points to claim 3 steps (a-g) for guidance [remarks, pages 7-8]. The Applicant points to the MPEP 2106.05 for further guidance. The Applicant states molecular assessment of tissue types represented in transcripts of a plasma sample, it was not well-understood, routine, or conventional as of the effective filing date of the Application for one of ordinary skill in the art to perform the following ordered combination of seven additional elements beyond the alleged judicial exception. The Applicant points to claim 3 steps (a-g) for further guidance. The Applicant states claim 3 recites an ordered combination of additional elements that amounts to significantly more than the recited judicial exception and therefore provides an inventive concept [remarks, pages 8-9].
In response, and as noted above in steps 2B, the claims do not contain additional elements that provide significantly more because the claimed steps are routine and conventional steps for obtaining total RNA. The MPEP 2106.05d(II)(2) directs the utilization of what courts have recognized, or those in the art would recognize, as elements that are well-understood, routine, conventional activity in the relevant field when making the required determination. Here, the MPEP 2106.05d(II)(2) and 2106.305(d)(II)(i-iii, v, vii-viii) provide both the cases and their conventional activity and describe techniques recognize by the courts as laboratory techniques that are considered well-understood, routine, and conventional activity (i.e., PCR , amplifying). As such, the claims do not provide significantly more because the claimed steps utilize routine and conventional additional elements (i.e., centrifugation on a test blood sample of a test subject to obtain a plasma sample, adding a cell membrane stabilizing agent to the plasma sample, isolating cell-free ribonucleic acids (cfRNA) from a blood test sample com, using DNase on the isolated samples, reverse transcribing the cfRNA to obtain complementary DNA (cDNA), amplifying the cDNA to produce double stranded cDNA amplification products, and performing sequencing analysis for deconvolving tissue types).
Furthermore, and importantly, the MPEP 2106.05d(II)(2) states ”the specification of the application may indicate that additional elements are well-known or conventional.” Here, the specification discloses [7] “Second, total RNA in plasma from a sample is determined using methods known in the art.”, [10] “Methods of the invention involve isolating total RNA from a biological sample. Total RNA can be isolated from the biological sample using any methods known in the art.”, [10] “The RNA is extracted from this gel slice and eluted using methods known in the art. Alternatively, fetal specific RNA may be concentrated by known methods, including centrifugation and various enzyme inhibitors.”, [11] “While any known sequencing method can be used to sequence the amplified cDNA mixture, single molecule sequencing methods are preferred.” As such, the Applicants’ disclosure discloses that the methods/method steps used for obtaining total RNA (i.e., centrifugation on a test blood sample of a test subject to obtain a plasma sample, adding a cell membrane stabilizing agent to the plasma sample, isolating cell-free ribonucleic acids (cfRNA) from a blood test sample com, using DNase on the isolated samples, reverse transcribing the cfRNA to obtain complementary DNA (cDNA), amplifying the cDNA to produce double stranded cDNA amplification products, and performing sequencing analysis for deconvolving tissue types) are methods known in the art. Here, the claimed steps/methods are contained either explicitly or implicitly within the known methods described by the specification and MPEP 2106.05(d)(II)(i-iii, v, vii-viii), 2106.05(g)(i, vi)).
Additionally, and regarding the ordered combination of additional elements, the combination is either explicitly or implicitly evidenced by El-Hefnaway et al., Huang et al., Prenatal Diagnosing (Hanh), Drnovsek et al., Poon et al., Puzycks et al. Dhallen et al., Maron et al., and Quake et al. as noted in Step 2B of the 101 analyses above. These references evidence that the ordered steps are the required steps for isolating and processing cfRNA from a blood samples. As further evidence by teachings of El-Hefnaway et al., Huang et al., Prenatal Diagnosing (Hanh), Drnovsek et al., Poon et al., Puzycks et al. Dhallen et al., Maron et al., and Quake et al., there also is significant overlap between the methods of the references with respect to processing blood sample and obtaining cfRNA from said sample for subsequently nucleic acid sequencing method to yield sequencing data. Here, because of the overlap of the methods (i.e., centrifuging, adding membrane stabilizer, isolating cfRNA/mRNA, DNAse digestion, reverse transcribing, amplifying, and sequencing) of El-Hefnaway et al., Huang et al., Prenatal Diagnosing (Hanh), Drnovsek et al., Poon et al., Puzycks et al. Dhallen et al., Maron et al., and Quake et al., one would recognize that these steps of the instant claims are not unique or unconventional but are in fact conventional ordered steps for obtaining cfRNA from a plasma sample.
Moreover, it is also noted that the mRNA extraction from plasma prior to reverse transcription to cDNA that is subsequently amplified before sequencing is well known. Extraction of mRNA subsequent cDNA sequencing does not necessarily require the use of DNase or a cell membrane stabilizing agent, although both DNase and cell membrane stabilizers are elements that are routinely taught in the context of mRNA analysis. Even the instant specification teaches that the cell stabilizer may be added to reduce cell lysis (page 11, par. 1-2). The specification does not require DNase or the cell stabilizer as an element necessary for deriving the cDNA sequencing data to be used by the deconvolution algorithm. Therefore, one would recognize that adding DNase, and cell stabilizers are extra-solution activities which are part of the pre-solution activities of claim 1, step (a) of (f).
MPEP 2106.05(g) sets forth examples of pre-solution activities of data gathering to include:
vi. Determining the level of a biomarker in blood, Mayo, 566 U.S. at 79, 101 USPQ2d at 1968. See also PerkinElmer, Inc. v. Intema Ltd., 496 Fed. App'x 65, 73, 105 USPQ2d 1960, 1966 (Fed. Cir. 2012) (assessing or measuring data derived from an ultrasound scan, to be used in a diagnosis).
Similarly, limitations (a) to (f) are pre-solution activities for gathering sequence data for the deconvolution algorithm of claim 3. Furthermore, MPEP 2106.05(d)(II) states, the courts have recognized that routine, conventional and well understood elements include:
i. Determining the level of a biomarker in blood by any means, Mayo, 566 U.S. at 79, 101 USPQ2d at 1968; Cleveland Clinic Foundation v. True Health Diagnostics, LLC, 859 F.3d 1352, 1362, 123 USPQ2d 1081, 1088 (Fed. Cir. 2017);
ii. Using polymerase chain reaction to amplify and detect DNA, Genetic Techs. Ltd. v. Merial LLC, 818 F.3d 1369, 1376, 118 USPQ2d 1541, 1546 (Fed. Cir. 2016); Ariosa Diagnostics, Inc. v. Sequenom, Inc., 788 F.3d 1371, 1377, 115 USPQ2d 1152, 1157 (Fed. Cir. 2015);
vii. Amplifying and sequencing nucleic acid sequences, University of Utah Research Foundation v. Ambry Genetics, 774 F.3d 755, 764, 113 USPQ2d 1241, 1247 (Fed. Cir. 2014).
These elements are additionally analogous to elements (a), (c), (f) and (g) of claim 3. Therefore, because the combinations of steps (a) to (g) are routine and taught in the prior in the art in context of cfRNA extraction, the combination of additional elements of claim 3, steps (a) to (g) do not add significantly more to the abstract idea so as to render the claims as a whole patent eligible.
Therefore, because the Applicants’ disclosure acknowledges/discloses that the steps (i.e., physical steps (a-g)) as known methods in the art and the claims encompass additional elements the courts have recognized (MPEP 2106.05(d)(II)(i-iii, v, vii-viii), 2106.05(g)(i, vi)), or those in the art would recognize (i.e., conventionality references), as elements that are well-understood, routine, and conventional under Step 2B of the 101 analyses, the Applicant’s argument the claimed method provides an inventive, unconventional concept is not persuasive.
Here, the Applicant is invited to provide as to which steps or limitations of the ordered combination are unconventional because, as noted above, the specification discloses that total RNA from a sample is determined using methods known in the art, that total RNA can be isolated from the biological sample using any methods known in the art, that RNA is extracted from this gel slice and eluted using methods known in the art and alternatively fetal specific RNA may be concentrated by known methods, including centrifugation and various enzyme inhibitors, that while any known sequencing method can be used to sequence the amplified cDNA mixture, single molecule sequencing methods are preferred, and that fetal RNA can be purified using standard techniques in the art. As such, the specification discloses that the steps (a-g) are conventional, so Applicant is encouraged to clarify which of these steps of the ordered combination are required so that to construct an unconventional ordered combination of data gathering elements needed to obtain sequencing data from mRNA extracted from a blood samples.
As such, the Applicants’ disclosure discloses that the combination of methods/method steps used for obtaining total RNA (i.e., centrifugation on a test blood sample of a test subject to obtain a plasma sample, adding a cell membrane stabilizing agent to the plasma sample, isolating cell-free ribonucleic acids (cfRNA) from a blood test sample com, using DNase on the isolated samples, reverse transcribing the cfRNA to obtain complementary DNA (cDNA), amplifying the cDNA to produce double stranded cDNA amplification products, and performing sequencing analysis for deconvolving tissue types) are, in fact, methods known in the art.
Furthermore, the claims do not provide significantly more because the claims are drawn to mere gathering and analyzing information (i.e., sequencing data) using conventional techniques (i.e., isolating of cell-free RNA (cfRNA), reverse-transcribing of cfRNA to obtain cDNA, amplifying the cDNA, and sequencing the cDNA)/clinical/laboratory methods for obtaining input for quantifying data (i.e., total RNA) and displaying the result (i.e., deconvolved tissue types). See MPEP 2106.05(a)(II)(iii) and 2106.05(g).
Therefore, the instant claimed invention is not patent eligible subject matter under 35 U.S.C § 101.
Claim Rejections - 35 USC § 103
The instant rejection is maintained for reason for record in the Office Action mailed 05 November 2025 and modified in view of the amendments filed 24 March 2026. It is noted the amendments received 24 March 2026 necessitated new ground(s) of rejection.
The rejection of claims 3, 26, and 28-30 under pre-AIA 35 U.S.C. 103(a) as being unpatentable over Lo et al. (Patent Pub: US 2004/0203037, Patent Pub Date: 14 October 2004) in view of Dhallen et al. (US Patent No.: 2004/0137470, Patent Pub Date 15 July 2004) in view of Kurn et al. (US2011/0189679 Pub: 04 August 2011, Cited in the Office Action mailed 06 June 2025) in view of Gong et al. (Cited in the Office Action mailed 10 October 2024) (PloS one, 2011-11, Vol.6 (11), p.e27156-e27156) in view of Li et al. (Journal of clinical oncology, 2006-04, Vol.24 (11), p.1754-1760) in the Office Action mailed 05 November 2025 is withdrawn in view of the amendments received 24 March 2026.
The rejection of claim 12-13 and 16 under pre-AIA 35 U.S.C. 103(a) as being unpatentable over Lo in view of Dhallen in view of Kurn in view of Gong in view of Li, as applied to claims 3, 26, and 28-30, and in further view of McGill (Tutorial: RMA Analysis using the Microarray Platform Website) (Cited in the Office Action mailed 06 June 2025) in the Office Action mailed 05 November 2025 is withdrawn in view of the amendments received 24 March 2026.
The rejection of claims 7-8 under pre-AIA 35 U.S.C. 103(a) as being unpatentable over Lo in view of Dhallen in view of Kurn in view of Gong in view of Li, as applied to claims 3, 26, and 28-30, and in further view of Bruder et al. (BMC genomics, 2010-04, Vol.11 (1), p.251-251) in the Office Action mailed 05 November 2025 is withdrawn in view of the amendments received 24 March 2026
The rejection of claims 21 under pre-AIA 35 U.S.C. 103(a) as being unpatentable over Lo in view of Dhallen in view of Kurn in view of Gong in view of Li as applied to claims 3, 26, and 28-30, and in further view of Shiroguchi et al. (Proceedings of the National Academy of Sciences - PNAS, 2012-01, Vol.109 (4), p.1347-1352) in the Office Action mailed 05 November 2025 is withdrawn in view of the amendments received 24 March 2026.
The rejection of claims 22-25 under pre-AIA 35 U.S.C. 103(a) as being unpatentable over Lo in view of Dhallen in view of Kurn in view of Gong in view of Li, as applied to claims 3, 26, and 28-30, and in further view of Xie et al. (BMC systems biology, 2011-12, Vol.5 (Suppl 3), p.S4-S4, Article S4) in the Office Action mailed 05 November 2025 is withdrawn in view of the amendments received 24 March 2026.
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 3, 26, and 28-33 are rejected under pre-AIA 35 U.S.C. 103(a) as being unpatentable over Lo et al. (Patent Pub: US 2004/0203037, Patent Pub Date: 14 October 2004) in view of Dhallen et al. (US Patent No.: 2004/0137470, Patent Pub Date 15 July 2004) (Cited in the Office Action mailed 05 November 2025) in view of Kurn et al. (US2011/0189679 Pub: 04 August 2011, Cited in the Office Action mailed 06 June 2025) in view of Lao et al. (US Patent Pub No.: US 2010/0124765, Patent Pub Date: 02 May 2010) in view of Gong et al. (Cited in the Office Action mailed 10 October 2024) (PloS one, 2011-11, Vol.6 (11), p.e27156-e27156) in view of Li et al. (Journal of clinical oncology, 2006-04, Vol.24 (11), p.1754-1760).
Claim 3 step (a) recites performing centrifugation on a test blood sample of a test subject to obtain a plasma sample.
Claim 3 step (b) adding a cell membrane stabilizing agent to the plasma sample.
Claim 3 step (c) recites isolating cell-free ribonucleic acids (cfRNA) from a blood test sample com, wherein the cfRNA is messenger RNA, and wherein isolating the cfRNA comprises adding a cell-membrane stabilizing agent to the test blood sample.
Claim 3 step (d) performing DNase digestion on the isolated.
Claim 3 step (e) recites reverse transcribing the cfRNA to obtain complementary DNA (cDNA). Claim 3 step (f) recites amplifying the cDNA to product double stranded cDNA amplification products, wherein the amplifying is initiated at a 3' end of the cDNA and randomly throughout a whole transcriptome of the test blood sample.
Claim 3 step (g) recites performing sequencing assay on the double stranded cDNA a0mplification products to obtain collection of sequencing data, wherein collection of sequence data comprises test fractional contributions of tissue types.
Claim 3 step (h) recites computer processing the collection of sequencing data, wherein the computer processing comprises deconvolving the test fractional contributions of tissue types from the plasma derived from the test blood sample of the test subject against a reference cell-free transcriptome in plasma derived from a reference blood sample of a reference subject. Claim 3 step (e) recites wherein deconvolving the test fractional contributions of tissue types further comprises (1) identifying a panel of tissue-associated transcripts, (2) determining total ribonucleic acid (RNA) in plasma derived from the test blood sample of the test subject, and (3) assessing the total RNA of (2) against the panel of tissue-associated transcripts of (1), wherein the total RNA is considered a summation of the tissue-associated transcripts.
Lo et al. (Lo) discloses centrifuging between 1,500-3,000g and centrifugation can be done before transferring to a fresh tube for RNA extraction [Lo, page 5 left col para 0050]. Lo discloses centrifuging tubes at 1,600g [Lo, page 7 left col para 0080], as in instant claim 3 step (a).
Lo discloses a method for diagnosing, monitoring, and predicting preeclampsia in pregnant women by determining the amount one or more mRNA species (i.e., total mRNA) in the pregnant women’s blood sample [claim 1 step (i)] and comparing the amount of mRNA (i.e., total mRNA) of step (i) to a standard control [par. 0044 and Lo claim 1]. Lo discloses the pregnant women’s sample as acellular [Lo, claim 7], as in instant claim 3 step (c) isolating cell-free ribonucleic acids (cfRNA) from a blood test sample com, wherein the cfRNA is messenger RNA (mRNA).
Lo discloses prior to the amplification step, a DNA (cDNA) copy of the mRNA of interest must be synthesized by reverse transcription [disclosure 0057], as in claim 3 step (f).
Lo discloses teaches quantitatively determining the amount of one or more mRNA species in the pregnant women blood such as those encoding hCRH, GAPDH [par. 0043, Lo claims 1 step (i) and claim 7], as in instant claim 3 step (h) sub step (1).
Lo discloses determining the amount of mRNA species (i.e., hCRH, GAPDH) [Lo, claim 1 step (i)], as in instant claim 3 step (h) sub step (2) determining total ribonucleic acid (RNA) in plasma derived from the test blood sample of the test subject. Here, determining the amount of mRNA species (i.e., hCRH, GAPDH) reads on determining the total amount of RNA because claim 1 of Lo teaches determining the “amount of mRNA species (i.e., hCRH, GAPDH) in the pregnant woman’s blood” which is the total amount of that mRNA species in the pregnant woman’s blood.
Dependent claim(s): 30-33
Lo discloses “Prior to the amplification step, a DNA copy (cDNA) of the mRNA of interest must be synthesized. This is achieved by reverse transcription, which can be carried out as a separate step, or in a homogeneous reverse transcription-polymerase chain reaction (RT-PCR), a modification of the polymerase chain reaction for amplifying RNA.” [disclosure page 5 right col para. 0057], as in instant claims 30-31. Here, the synthesize cDNA copies can be reverse transcribed, in a separate step, or in a homogeneous reverse transcription-polymerase chain reaction (RT-PCR) which reads on performing PCR on cDNA libraries.
Lo discloses using centrifugation force at 1,600xg and 11, 900g [Lo, page 7 left col para 0078, 0080] and recentrifuged at 16,000g [Lo, page 11 right col para 0143], as in instant claim 32.
Lo discloses normalizing (i.e., rescaling) transcripts (such as KISS1, nβhCG, nGAPDH, and β-globin transcripts [Lo, page 3 right col para 0029]. Lo discloses normalizing for each transcript was calculated by dividing the transcript level in an individual placenta or plasma Sample to the corresponding hPL mRNA level in the same sample [Lo, page 13 left col para 0158], as in instant claim 33.
Lo does not teach wherein isolating the cfRNA comprises adding a cell-membrane stabilizing agent to the test blood sample claim 3 step (b). Lo does not explicitly teach amplifying the cDNA to product double stranded cDNA amplification products of claim 3 step (f). Lo does not teach wherein the amplifying is initiated at a 3' end of the cDNA and randomly throughout a whole transcriptome of the test blood sample of claim 3 step (f). Therefore, it is obvious that the amplification can be initiated a the 3’ regions of the linear region, target region. Lo does not teach performing sequencing assay on the double stranded cDNA amplification products to obtain collection of sequencing data of claim 3 step (g). Lo does not teach the collection of sequence data comprises test fractional contributions of tissue types of claim 3 step (g). Lo does not teach computer processing the collection of sequencing data, wherein the computer processing comprises deconvolving the test fractional contributions of tissue types from the plasma derived from the test blood sample of the test subject against a reference cell-free transcriptome in plasma derived from a reference blood sample of a reference subject of claim 3 step (g). Lo does not teach wherein deconvolving the test fractional contributions of tissue types further comprises of claim 3 step (h). Lo does not teach assessing the total RNA of (2) against the panel of tissue-associated transcripts of (1) of claim 3 step (h) sub step (3). Lo does not teach wherein the total RNA is considered a summation of the tissue-associated transcripts of claim 3 step (h) sub step (3). Lo does not teach claims 26 and 28-29.
Dhallan
Dhallan et al. (Dhallan) discloses that agents were added to sample to inhibit cell lysis such as membrane stabilizers, a cross-linker, or a cell lysis inhibitor [disclosure page 5 right col. para. 0057]. Dhallen discloses “membrane stabilizers agents such as may be added to the Sample including but not limited to aldehydes, urea formaldehyde, phenol formaldehyde, DMAE (dimethylaminoethanol), cholesterol, cholesterol derivatives, high concentrations of magnesium, Vitamin E, and Vitamin E derivatives, calcium, calcium gluconate, taurine, niacin, hydroxylamine derivatives, bimoclomol, Sucrose, astaxanthin, glucose, amitriptyline, isomer A hopane tetral phenylacetate, isomer Bhopane tetral phenylacetate, citicoline,inositol, Vitamin B, Vitamin B complex, cholesterol, hemisuccinate, Sorbitol, calcium, coenzyme Q, ubiquinone, Vitamin K, Vitamin K complex, menaquinone, zonegran, Zinc, ginkgo biloba extract, diphenylhydantoin, perftoran, polyvinylpyrrolidone, phosphatidylserine, tegretol, PABA, disodium cromglycate, nedocromil Sodium, phenyloin, Zinc citrate, mexitil, dilantin, Sodium hyaluronate, or polaxamer 188.” [disclosure page 9 left col para. 0078] [Instant specification page 11 first paragraph]. Dhallan discloses “DNA that has been reverse transcribed from an RNA sample, such as cDNA. The Sequence of RNA can be determined according to the invention if it is capable of being made into a double stranded DNA form to be used as template DNA” [disclosure page 18 right col para. 0189], as in instant claim 3 step (b) adding a cell-membrane stabilizing agent to the test blood sample.
It would be obvious to one of ordinary skill in the art by the effective filing date of the claimed invention to modify Lo in view of Dhallan because Dhallen discloses methods analyzing nucleic acids (i.e., RNA) from cells. One of ordinary skill in the art would be motivated to combine Lo in view Dhallan because Dhallan discloses methods for using cell-membrane inhibitors and/or cell lysis inhibitors for quantifying free fetal DNA and discloses specific cell-membrane inhibitor agents (i.e., cell membrane stabilizers).Thus, there is a reasonable expectation of success using an acellular sample from a pregnant women of Lo and adding the cell lysis inhibitor of Dhallan to said acellular sample would a construct a claimed step that can isolate RNA from other cells or cells not previously removed in a sample and/or prevent intracellular mRNA contamination in order to yield a purified total RNA (i.e., mRNA) that can be utilized for deconvolving test fractional contributions of tissue types from a blood sample.
Kurn
Kurn et al. (Kurn) discloses RNA can comprise cell-free RNA [claims 15, 32, and 48]. Kurn teaches reverse transcribed cDNA products [claim 18 and 34], as in instant claim 3 step (e).
Kurn discloses amplifying double stranded cDNA [claim 18 and 34], as in instant claim 3 step (f).
Kurn discloses sequencing the reversed transcribed products [claim 4-5], as in instant claim 3 step (g) performing sequencing assay on the double stranded cDNA amplification products to obtain collection of sequencing data.
Dependent claim(s): 26 and 29
Kurn discloses using amplified cDNA can be from massively parallel sequencing enable by next generation sequencing technologies and platform, as representing by the RNA-seq data using Illumina’s Genome Analyzer [disclosure paragraph [0097]]. Kurn discloses “In one aspect, the invention provides for a method for whole transcriptome sequencing comprising providing a RNA sample, reverse transcribing the sample, amplifying the RNA sample using one or more primers to produce amplified products, and performing sequencing on the products [disclosure page 1 left col para. 0004], as in claim 26.
Kurn discloses generating double stranded cDNA product [claim 18 and 34], as in instant claim 29.
It would have been obvious to one of ordinary skill in the art by the effective filing date of the claimed invention to modify Lo in view of Dhallan in view of Kurn because Kurn discloses methods for compositions and whole transcriptome analysis [title]. One of ordinary skill in the art would be motivated to combine Lo in view of Dhallan in view of Kurn because Kurn discloses steps for generating double-stranded cDNA, and amplifying said double-stranded cDNA which can be utilized to process the mRNA species of Lo to determine total RNA species of a particular gene. Thus, there is a reasonable expectation of success to combine Lo in view of Dhallan in view of Kurn to produce RNA/mRNA data (i.e., total RNA) that can be subsequently summed and utilized for deconvolving test fractional contribution of tissues type and/or tissue types from a blood sample because the combination of Lo in view of Dhallan in view of Kurn which would yield a predictable method for generating double stranded cDNA via reverse transcription of cell-free RNA to produce test fractional contributions from the cell-free RNA data which could be further deconvoluted for determining tissue and/or cell type.
Lao
Lao et al. (Lao) discloses method for collecting and detecting oligonucleotides [Lao, title]. Lao discloses methods for amplification of cDNA [Lao, claim 30]. Lao discloses a method for producing double stranded extended primers [Lao, claim 6]. Lao discloses the target sequence can be double stranded [Lao, page 13 left col para 0097]. Lao discloses the linear primer can be used to initiate priming as desired [Lao, page 13 right col para 0103]. Leo discloses the first primer is a linear primer [Lao, claim 11]. Lao discloses the 3’ target regions (i.e., first and second target region) can comprise random regions [Lao, claims 37-38]. Lao discloses the linear primer can be used to initiate priming as desired and using the linear primer to amplify sections of the target sequence [Lao, page 13 right col para 0103], as in claim 3 step (f) wherein the amplifying is initiated at a 3' end of the cDNA and randomly throughout a whole transcriptome of the test blood sample. Therefore, it is obvious that the amplification can be initiated a the 3’ regions of the linear region, target region.
It would have been obvious to one of ordinary skill in the art by the effective filing date of the claimed invention to modify Lo in view of Dhallan in view of Kurn in view of Lao because Lao discloses sequence amplification with target nucleic acid that can be mRNA, miRNA, siRNA [Lao, page 2 right col para 0030] and discloses that target nucleic acids can be single or double stranded [Lao, page 2 right col para 0031]. One of ordinary skill in the art would be motivated to combine the methods of Lo in view of Dhallan in view of Kurn in view of Lao because Lao discloses analyzing RNA molecules form different sources (i.e., lysed cells, whole blood, perspiration, buccal swabs [Lao, page 2 right col para 0030]) which discloses the methods of Lao can be utilized for processing cell-free RNA. One of ordinary skill in the art would have a reasonable expectation of successes combining methods of centrifuging, and isolating cfRNA, amplification of Lo, membrane stabilizers of Dhallan, and amplifying and sequencing of double stranded of cDNA of Kurn with initial amplification of 3’ end of cDNA and randomly of Lao because the initial amplification methods, primers, and kits of Lao can be utilized to add primers to the RNA molecules of Lo and Kurn such that amplification can be initiated at the 3’ end and/or randomly. Therefore, thus combining Lo, Dhallen, Kurn, and Lao would yield a series of clinical/laboratory step or construct a protocol for centrifuging a blood sample, adding cell membrane stabilizer to the to prevent cellular leakage of RNA or DNA molecules, using DNase to remove contaminating DNA molecules, reverse transcribing cfRNA, amplifying the cfRNA, and sequencing the on the RNA molecules to provide nucleic acid data such that tissue type can deconvolved.
Gong
Gong also teaches deconvolving transcriptional profiling data [abstract]. Gong teaches deconvolution of transcriptional profiling data using quadratic programming with application to complex clinical blood samples [abstract]. Gong teaches method to various existing platforms to estimate proportions of different pure cell or tissue types and gene expression profiling of distinct phenotypes, with a focus on complex samples collected in clinical trials [abstract]. Gong teaches tissue types [page 2 table 1]. Gong teaches blood is a complex tissue type [page 3 right col deconvolution of circulating cells from whole blood sample second para.], as in claim 3 step (h) wherein the computer processing comprises deconvolving the test fractional contributions of tissue types.
Dependent claim 28
Gong also teaches deconvolving transcriptional profiling data [abstract]. Gong teaches deconvolution of transcriptional profiling data using quadratic programming with application to complex clinical blood samples [abstract], as in claim 28.
It would be obvious to one of ordinary skill in the art by the effective filing date of the claim invention to modify Lo in view of Dhallan in view of Kurn in view in view of Lao in view of Gong because Gong teaches methods for deconvolution of cell types. One of ordinary skill in the art would be motivated to combine Lo in view of Dhallan in view of Kurn in view in view of Lao in view of Gong because Gong teaches methods for deconvolving cell and tissues type from RNA data but teaches deconvolving using quadratic programming for deconvolving RNA data for estimating proportions of different pure cell or tissue types and gene expression profiling of distinct phenotypes mixing fractions for more than ten species of circulating cells and to provide accurate estimates for relatively rare cell types [abstract]. Here, the would be a reasonable expectation of success combining Lo in view of Dhallan in view of Kurn in view Lao in view of Gong because Gong teaches using quadratic programming for deconvolution of cell and tissue types. As such, combining Lo in view of Dhallan in view of Kurn in view of Lao in view of Gong would yield a predictable method using quadratic programming for deconvolution of tissue types.
Li
Li teaches using a HGU133A microarrays for profiling and identifying the differences in serum mRNA transcriptomes between cancer patients and healthy controls. Li teaches analyzing 14,268 genes with the microarray [page 1756 right col second para.]. Li teaches using 35 control subjects [page 1756 left col results] as in claim 3 step (h) test blood sample of the test subject against a reference cell-free transcriptome in plasma derived from a reference blood sample of a reference subject. Here, using the microarray teaches a reference cell-free transcriptome because the microarray, HGU133A, contains mRNA transcripts (i.e., 14,268 genes) for profiling and identifying transcript differences in serum mRNA transcriptomes between cancer and healthy patients. Furthermore, the use of healthy controls teaches the samples included reference sample (i.e., controls).
Li teaches “Ten significant upregulated candidates were selected from the list of 62 based on their reported cancer association: H3F3A, TPTl, FTHl, NCOA4, ARCR, THSMB, PRKCBl, FTLl, COX4Il, and SERPl (i.e., panel of identified tissue-associated transcripts)” which also teaches identifying a panel of tissue-associated transcripts of claim 3 step (e) sub step (1). Li teaches “five transcripts (H3F3A, TPTl , FTHl, NCOA4, and ARCR) were confirmed to be significantly elevated in OSCC sera” [page 1756 right col third para] which also teaches identifying a panel of tissue-associated transcripts of claim 3 step (h) sub step (1).
Li teaches regular qPCR was performed to quantify a subset of differently expressed transcripts in serum of OSCC patients (n = 32) versus controls (n = 35). Li teaches “regular PCR was performed on total RNA to obtain specific amplicons of the aforementioned subset of genes/transcripts. These amplicons underwent a serial dilution with the starting amount determined by a spectrophotometer. Those diluted amplicons, also used as positive controls, were run in parallel with the samples under identical qPCR conditions and amplified with the same set of primers.” [page 1756 Quantitative PCR], which also teaches determining a total RNA of claim 3 step (h) sub step (2).
Li teach detecting oral squamous cell carcinoma (OSCC) in blood samples [abstract]. Li teaches serum circulating human mRNA profiling and its utility for oral cancer detection [abstract]. Li teaches microarrays were used to profile and identify the differences in serum mRNA transcriptomes between cancer and healthy patients [page 1756 right col second paragraph]. Li teaches “we identified 62 transcripts from the differently expressed 335 genes such that these 62 genes/transcripts (i.e., summed total mRNA) [Spec page 8 top of page] are all upregulated in OSCC serum.” [page 1756 right col second para]. Li teaches “Ten significant upregulated candidates were selected from the list of 62 based on their reported cancer association: H3F3A, TPTl, FTHl, NCOA4, ARCR, THSMB, PRKCBl, FTLl, COX4Il, and SERPl (i.e., panel of identified tissue-associated transcripts)” which also teaches identifying a panel of tissue-associated transcripts of claim 3 step (e) sub step (1). Li teaches “five transcripts (H3F3A, TPTl , FTHl, NCOA4, and ARCR) were confirmed to be significantly elevated in OSCC sera” [page 1756 right col third para], as in claim 3 step (h) sub step (3) assessing the total RNA of (2) against the panel of tissue-associated transcripts of (1) and the total RNA is considered a summation of the tissue-associated transcripts of claim 3 step (e) sub step (3).
Here, Li teaches that the obtained amplicons of subset of genes/transcripts were run in parallel with the samples under identical qPCR conditions and amplified with the same set of primers which makes obvious that the total RNA (i.e., summed total mRNA transcripts) was assessed against the panel of tissue-associated transcripts because the total RNA and samples were run in parallel. Moreover, Li al teaches assessing a set of 62 transcripts (i.e., total summed mRNA of tissue-associated transcripts) against a set of 10 candidate transcripts (i.e., panel of tissue-associated transcripts). Therefore, it is obvious that Li teaches a method for deconvolving of tissue types.
It would be obvious to one of ordinary skill in the art by the effective filing date of the claim invention to modify Lo in view of Dhallan in view of Kurn in view of Lao in view of Gong in view of Li because Li teaches methods for analyzing serum circulating human mRNA detecting OSCC [abstract] and identifying mRNA transcripts associated with OSCC [page 1756 right col third para]. One of ordinary skill in the art would be motivated to combine Lo in view of Dhallan in view of Kurn in view of Lao in view of Gong in view of Li because Li teaches methods using microarrays to profile and identify differences in serum mRNA transcriptomes, revealing five mRNA transcripts upregulated in OSCC, and using qPCR to quantify the transcripts. Here, there is a reasonable expectation of success combining Lo in view of Dhallan in view of Kurn in view in view of Lao of Gong in view of Li because the reference cell-free transcriptome in plasma, the identified mRNA transcripts data, the assessing of total RNA and panel of tissue-associated transcripts of Li could be incorporated into the methods of Lo, Dhallan, Kurn, Lao, and Gong for deconvolving tissue types. As such, combining Lo in view of Dhallan in view of Kurn in view of Lao in view of Gong in view of Li would yield a predictable method using a reference cell-free transcriptome in plasma derived from a reference blood sample of a reference subject and assessing total RNA against a panel of tissue-associated transcripts for deconvolving tissue type of a sample(s).
Claim 12-13 and 16 is rejected under pre-AIA 35 U.S.C. 103(a) as being unpatentable over Lo in view of Dhallen in view of Kurn in view of Lao in view of Gong in view of Li, as applied to claims 3, 26, and 28-33, and in further view of McGill (Tutorial: RMA Analysis using the Microarray Platform Website) (Cited in the Office Action mailed 06 June 2025).
Lo in view of Dhallen in view of Kurn in view of Lao in view of Gong in view of Li teach claims 3, 26, and 28-33.
Lo in view of Dhallen in view of Kurn in view of Lao in view of Gong in view of Li teach a method for performing centrifugation on a test blood sample of a test subject to obtain a plasma sample, adding a cell membrane stabilizing agent to the plasma sample, isolating cell-free ribonucleic acids (cfRNA) from a blood test sample com, using DNase on the isolated samples, reverse transcribing the cfRNA to obtain complementary DNA (cDNA), amplifying the cDNA to product double stranded cDNA amplification products, and performing sequencing analysis for deconvolving tissue types.
Lo in view of Dhallen in view of Kurn in view of Lao in view of Gong in view of Li do not teach claims 12-13 and 16.
With respect to claim instant claim 12, the claim is rendered obvious because McGill teaches calcualting signal intensity of probes [page 3 Affymetrix arrays]. McGill teaches using RMA which that uses intensity values [page 4 RMA]. Lo discloses that oligonucleotide probes specific to mRNA binding (i.e., hCRH, hPL, KISS1, GAPDH) can be used to detect the presence of mRNA species and indicate the amount of mRNA species in comparison to the standard control, based on the intensity of the signal imparted by the probe [disclosure page 5 right col para. 0062]. Kurn discloses sequencing [Kurn, claims 4-5].
McGill et al. (McGill) teaches RMA analysis using microarray platform [title]. McGill teaches using Affymetrix GeneChip and calculating probe intensity for the expression of a targeted gene [page 3]. McGill teaches RMA properties that has probe intensities that are in log (base 2) scale [page 4], as in claim 13.
With respect to claim 16, the claim is rendered obvious because McGill teaches calcualting signal intensity of probes [page 3 Affymetrix arrays]. McGill teaches using RMA which that uses intensity values [page 4 RMA]. Kurn discloses using RNA-seq and quantification PCR [disclosure page 9 para 0093 example 3].
It would be obvious to one ordinary skill in the art by the effective filing date of the claimed invention to modify Lo in view of Dhallen in view of Kurn in view of Lao in view of Gong in view of Li and in further view of McGill because McGill teaches analyzing probe intensities of microarray data using raw microarray algorithm (RMA). One of ordinary skill in the art would recognize that the probe signal data from the microarrays of Lo, in combination with the raw microarray algorithm (RMA) of McGill, could be utilized for deconvolving samples/cells into different cells/tissues. Here, the methods of Lo in view of Dhallen in view of Kurn in view of Lao in view of Gong in view of Li and in further view of McGill would yield a predictable method that uses an RMA to convert microarray probe intensity data to log scales that can be utilized for deconvolving test fractional contributions for deconvolving tissue types of a test blood sample.
Claims 7-8 are rejected under pre-AIA 35 U.S.C. 103(a) as being unpatentable over Lo in view of Dhallen in view of Kurn in view of Lao in view of Gong in view of Li, as applied to claims 3, 26, and 28-33, and in further view of Bruder et al. (BMC genomics, 2010-04, Vol.11 (1), p.251-251).
Lo in view of Dhallen in view of Kurn in view of Lao in view of Gong in view of Li teach claims 3, 26, and 28-33.
Lo in view of Dhallen in view of Kurn in view of Lao in view of Gong in view of Li teach a method for performing centrifugation on a test blood sample of a test subject to obtain a plasma sample, adding a cell membrane stabilizing agent to the plasma sample, isolating cell-free ribonucleic acids (cfRNA) from a blood test sample com, using DNase on the isolated samples, reverse transcribing the cfRNA to obtain complementary DNA (cDNA), amplifying the cDNA to product double stranded cDNA amplification products, and performing sequencing analysis for deconvolving tissue types.
Lo in view of Dhallen in view of Kurn in view of Lao in view of Gong in view of Li do not teach claims 7-8.
Bruder et al. (Bruder) teach using Pavlidis template matching (PTM) pattern recognition algorithm to isolate transcripts only expressed by one of five tissue groups. Bruder teaches a number of these transcripts were compared to the gene atlas expression sets though the BioGPS portal [page 4 left col second para.]. Bruder teaches transcripts identified by NOVA followed by PTM analysis [page 5 figure 3]. Bruder teaches RNA from brain, liver, lung, spleen, and blood [page 3 right col microarray analysis and TaqMan development], as in claim 7.
Bruder teaches “The 609 probes on the ferret array corresponding to these genes were analyzed by ANOVA to filter out the genes that varied over the analyzed tissues despite being classified as housekeeping genes.” Bruder teaches “remaining transcripts were clustered using K-means clustering to distinguish between the genes that varied within each group from those that were truly non variable between the groups [page 4 right col first paragraph], as in claim 8.
It would be obvious to one ordinary skill in the art by the effective filing date of the claimed invention to modify Lo in view of Dhallen in view of Kurn in view of Lao in view of Gong in view of Li and in further view of Bruder because Bruder teaches analyzing transcriptome sequencing and development of microarray platform [abstract]. One of ordinary skill in the art would be motivated to combine Lo in view of Dhallen in view of Kurn in view of Lao in view of Gong in view of Li and in further view of Bruder because Bruder teaches parametric testing to isolate transcripts differentially expressed between five tissue and using Pavlidis Template Matching (PTM) pattern recognition algorithm to isolate transcripts only expressed by one of the five analyzed tissues [page 4 left col. second para.]. Here, although Bruder teaches analyzing RNA from a domestic ferret, the PTM algorithm can be applied to analyze human RNA because Bruder teaches using array data set containing human housekeeping genes [page 4 left col last para.]. Thus, there is a reasonable expectation of success to combine Lo in view of Dhallen in view of Kurn in view of Lao in view of Gong in view of Li and in further view of template matching algorithm of Bruder because the Pavlidis template matching (PTM) pattern recognition algorithm of Bruder could be utilized to recognize patterns within RNA and deconvolved data to identify tissue-associated transcripts. Therefore, combining Lo in view of Dhallen in view of Kurn in view of Lao in view of Gong in view of Li with the Pavlidis template matching (PTM) pattern recognition algorithm of Bruder would yield a predictable method for using a template matching algorithm to identify a panel of tissue-associated transcripts and applying a quality control function for filtering the tissue-associated transcripts for deconvolving tissue types.
Claims 21 are rejected under pre-AIA 35 U.S.C. 103(a) as being unpatentable over Lo in view of Dhallen in view of Kurn in view of Lao in view of Gong in view of Li as applied to claims 3, 26, and 28-33, and in further view of Shiroguchi et al. (Proceedings of the National Academy of Sciences - PNAS, 2012-01, Vol.109 (4), p.1347-1352).
Lo in view of Dhallen in view of Kurn in view of Lao in view of Gong in view of Li teach claims 3, 26, and 28-33.
Lo in view of Dhallen in view of Kurn in view of Lao in view of Gong in view of Li teach a method for performing centrifugation on a test blood sample of a test subject to obtain a plasma sample, adding a cell membrane stabilizing agent to the plasma sample, isolating cell-free ribonucleic acids (cfRNA) from a blood test sample com, using DNase on the isolated samples, reverse transcribing the cfRNA to obtain complementary DNA (cDNA), amplifying the cDNA to product double stranded cDNA amplification products, and performing sequencing analysis for deconvolving tissue types.
Lo in view of Dhallen in view of Kurn in view of Lao in view of Gong in view of Li do not teach claims 21.
Shiroguchi et al. (Shiroguchi) teach “(A) Correlation between the number of spike-in molecules for five different spike-in sequences as measured by digital PCR and digital counting of unique barcodes [page 1349 fig 2], as in claim 21.
It would be obvious to one ordinary skill in the art by the effective filing date of the claimed invention to modify Lo in view of Dhallen in view of Kurn in view of Lao in view of Gong in view of Li and in further view of Shiroguchi because Shiroguchi teaches method for digitally counting/recalibrating RNA-seq sequencing data. One of ordinary skill in the art would be motivated to combine Lo in view of Dhallen in view of Kurn in view of Lao in view of Gong in view of Li and in further view of Shiroguchi because Shiroguchi teaches digital quantification of the E. coli transcriptome, (A) conventional and digital counting results for the fumA transcription unit (TU) as a function of genome position [page 1350 fig 3]. Here, although Shiroguchi teaches applying the digital counting to E. coli transcriptome analysis, Shiroguchi teaches the technique can be applied to eukaryotic system without substantial modification and the performance of digital and conventional counting in a simulation of differential expression analysis is key to application of RNA-seq data [page 1351 left col first para.]. Therefore, one of ordinary skill in the art would expect a reasonable success that combining Lo in view of Dhallen in view of Kurn in view of Lao in view of Gong in view of Li and in further view of Shiroguchi would yield a predictable method using digital counting analysis for analyzing/digitally counting RNA/mRNA transcripts for deconvolving tissue types.
Claims 22-25 are rejected under pre-AIA 35 U.S.C. 103(a) as being unpatentable over Lo in view of Dhallen in view of Kurn in view of Lao in view of Gong in view of Li, as applied to claims 3, 26, and 28-33, and in further view of Xie et al. (BMC systems biology, 2011-12, Vol.5 (Suppl 3), p.S4-S4, Article S4).
Lo in view of Dhallen in view of Kurn in view of Lao in view of Gong in view of Li teach claims 3, 26, and 28-33.
Lo in view of Dhallen in view of Kurn in view of Lao in view of Gong in view of Li teach a method for performing centrifugation on a test blood sample of a test subject to obtain a plasma sample, adding a cell membrane stabilizing agent to the plasma sample, isolating cell-free ribonucleic acids (cfRNA) from a blood test sample com, using DNase on the isolated samples, reverse transcribing the cfRNA to obtain complementary DNA (cDNA), amplifying the cDNA to product double stranded cDNA amplification products, and performing sequencing analysis for deconvolving tissue types.
Lo in view of Dhallen in view of Kurn in view of Lao in view of Gong in view of Li do not teach claims 22-25.
With respect to claim 22, the claim is rendered obvious because Li teaches “We used three criteria to determine differentially expressed genes between OSCCs and controls. First, we excluded genes that were assigned as absent call in all samples. Second, a two-tailed t test was used to compare the average gene expression levels between the two groups. The critical a level of .05 was defined for statistical significance. Third, fold changes were calculated for those genes that showed statistically significant differences (P < .05). Only those that exhibited at least two-fold change were included for additional analysis.” [page 1755-1756 right col bottom of 1755]. Gong also teaches deconvolving transcriptional profiling data [abstract]. Gong teaches deconvolution of transcriptional profiling data using quadratic programming with application to complex clinical blood samples [abstract]. Gong teaches estimated fractions for several circulating cell populations [page 5 figure 3]. Xie teaches “Totally 1296 genes were differentially expressed between each pair of the three tissues. The numbers of genes that have significant methylation difference between tissues are given in this Venn diagram” [page 6 fig 3]. Xie teaches integrating DNA methylation profiles and RNA-seq data of the human heart, kidney, and liver [page 2 right col. results]. Xie teaches differences in sample clustering of 18 tissue sample using all 27,578 DNA methylation markers [page 4 fig 2]. Xie teaches correlations of gene expression and DNA methylation [page 7 figures 4-5]. Li teaches using a HGU133A microarrays for profiling and identifying the differences in serum mRNA transcriptomes between cancer patients and healthy controls. Li teaches analyzing 14,268 genes with the microarray [page 1756 right col second para.]. Li teaches using 35 control subjects [page 1756 left col results].
Xie et al. (Xie) teach “PCA is that it disassociates the correlation between markers when methylation data are transformed into principal components. We plotted the 18 samples in the first three principal components, and the samples are grouped into their tissue labels with the second and third principal components [page 5 left col third para.]. Xie teaches “Totally 1296 genes were differentially expressed between each pair of the three tissues. The numbers of genes that have significant methylation difference between tissues are given in this Venn diagram” [page 6 fig 3]. Xie teaches integrating DNA methylation profiles and RNA-seq data of the human heart, kidney, and liver [page 2 right col. results]. Xie teaches differences in sample clustering of 18 tissue sample using all 27,578 DNA methylation markers [page 4 fig 2]. Xie teaches correlations of gene expression and DNA methylation [page 7 figures 4-5], as in claim 23.
With respect to claim 24, the claim is rendered obvious because Dhallan discloses methods for to screen prostate cancer and to monitor the severity of the disease [disclosure page 64 right col para. 0900]. Xie teaches using PCA [page 5 left col third para.]. Xie teaches “there are dramatic differences between tissues. Heart has a number of genes with significantly lower expression than both kidney and liver, and liver has more low-expression genes than kidney.” [page 4 left col second para and fig 1]. Xie teaches “Unpaired t test was used to detect the methylation differences among tissues and the results are summarized in Figure 3. From the 1296 significantly expressed genes, only about one third of them (483 genes) were not shown to have significant changes in methylation between the three tissues. In total, 610 genes were shown to have significant methylation difference between heart and liver, 599 genes were significant between kidney and liver, and 418 genes were significant between heart and kidney.” [page 5 right col first para.]. Xie teaches “Furthermore, as many disease states such as cancer may present cells with altered differentiation patterns (i.e. evidence for stem cells in cancer) or characteristics of mixed lineages (epithelial to mesenchymal transition) we need to understand the patterns of DNA methylation between different types of normal cells in order to properly gauge the significance of any aberrant findings.” [page 9 left col middle para]. Here, Dhallan, and Xie make obvious a determination whether cancer of a tissue differs or does not differ from a known reference cancer state. It is obvious that the healthy state will be compared against cancer states and/or healthy/control state/profile references to determine if genetic alterations differ or do not differ from a known reference state.
Xie et al. (Xie) teach “PCA is that it disassociates the correlation between markers when methylation data are transformed into principal components.” [page 5 left col third para.]. Xie teaches integrating DNA methylation profiles and RNA-seq data of the human heart, kidney, and liver [page 2 right col. results]. Xie teaches differences in sample clustering of 18 tissue sample in the first three principle component. Xie teaches the samples are grouped into their tissue labels with the second and third principal components (Figure 2C) [page 5 left col third paragraph]. As in claim 25.
It would be obvious to one ordinary skill in the art by the effective filing date of the claimed invention to modify Lo in view of Dhallen in view of Kurn in view of Lao in view of Gong in view of Li and in further view of Xie because Xie teaches using PCA analysis for investigating the relationship between sample in RNA-seq data [page 9 right col RNA-seq data quantification and DNA methylation sample clustering]. One of ordinary skill in the art would be motivated to combine Lo in view of Dhallen in view of Kurn in view of Lao in view of Gong in view of Li and in further view of Xie because Xie teaches correlating RNA-seq with DNA methylation data for identifying unique signatures of global DNA for correctly classifying various tissue [abstract]. Therefore, there is a reasonable expectation of success to combine Lo in view of Dhallen in view of Kurn in view of Lao in view of Gong in view of Li and in further view of Xie to yield a predictable for integrating and performing principal component analysis (PCA) for calcualting differences between test fractional contribution of tissue types for deconvolving tissue types.
Response to Arguments
Applicant’s arguments, filed 24 March 2026, have been fully considered but the rejection is maintained. Furthermore, upon further consideration, a new ground(s) of rejection is made in view of amendments received 24 March 2026. Here, Lo, Dhallen, Kurn, Lao, Gong, Li, McGill, Bruder, Shiroguchi, and Xie address the amendments filed 24 March 2026.
Conclusion
Claims 3, 6-8, 12-13, 16, 19, and 21-26, and 28-30 are rejected.
No claims are allowed.
Finality
This Office action is a Non-Final action. A shortened statutory period for reply to this action is set to expire THREE MONTHS from the mailing date of this action.
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/J.C.P./Examiner, Art Unit 1687
/Anna Skibinsky/
Primary Examiner, AU 1635
Prosecution Insights