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 .
Continued Examination Under 37 CFR 1.114
A request for continued examination under 37 CFR 1.114, including the fee set forth in 37 CFR 1.17(e), was filed in this application after final rejection. Since this application is eligible for continued examination under 37 CFR 1.114, and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114. Applicant's submission filed on 11/14/2025 has been entered.
Claim Status and Formal Matters
This action is in response to papers filed 11/14/2025.
Claims 25-26, 28-31, 33-34 are pending.
Claims 25, 28-30 have been amended.
Applicant's election with traverse of fragments of human chromosome 21 in the reply filed on 4/2/2020 is acknowledged. The traversal is on the ground(s) that the response asserts that searching additional chromosomes is not a search burden. This is not found persuasive because searching of art on chromosome 21 will not inherently provide art on the X chromosome or other chromosomes. Thus, searching more than one chromosome would place a serious search burned on the office.
Claims 25-26, 28-31, 33-34 are being examined.
Priority
The instant application was filed 11/14/2019 and is a continuation of 15036778, filed 05/13/2016 ,which is a national stage entry of PCT/IB2014/003062 having an international filing date: 11/26/2014 and claims foreign priority to GB1321196.6 , filed 12/02/2013.
Claim Objections
Claims 25-26, 28-31, 33-34 are objected to because of the following informalities:
Claim 25 is objected to as it recites “RCA” but does not recite the full terminology for the acronym (or abbreviation). Claims are more concise when the first time an acronym (or abbreviation) is presented the full terminology is also presented. Finally an acronym (or abbreviation) may have alternative meanings to an artisan.
Claim 25 has been amended to recite, “(i) the labelled RCA products in the first set each comprise:(x) a concatemer of both a sequence of a fragment of from blood of the pregnant woman and a first custom sequence that is not human genomic DNA.” Claim 25 later recites, “(x) a concatemer of both a sequence of a fragment of human cell-free DNA from blood of the pregnant woman and a second custom sequence that is not human genomic DNA.” The amendment is unclear if the deletion of “human” form the first recitation to encompass non-human fetus or something else. If that is the intent, it does not appear to be supported by the specification. This can be addressed by amending the claim to be consistent throughout.
Appropriate correction is required.
Response to Arguments
This is a new ground of objection necessitated by amendment.
Claim Rejections - 35 USC § 112
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claims 25-26, 28-31, 33-34 rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
Claim 25 has been amended to recite, “method for examining whether a fetus of a pregnant woman has a chromosomal abnormality” and “(e) examining whether the fetus has a chromosomal abnormality based on the results of the comparing of (d)” The claim is vague, unclear, and incomplete as to how a fetal chromosomal abnormality is determined as the active step of deposition required concatemers from the cell free DNA of a pregnant female, but does not require either concatemers to be from the fetus. Further it is unclear how a fetal abnormality is determined based on counts of concatemers from the cell free DNA from blood of a pregnant female.
Response to Arguments
This is a new ground of rejection necessitated by amendment.
Claim Rejections - 35 USC § 101
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 25-26, 28-31, 33-34 are rejected under 35 U.S.C. 101 because the claimed invention is directed to a natural correlation and mental steps without significantly more as the claims provide no active step which depend from or otherwise integrate the judicial exceptions.. The claim(s) recite(s) the abstract idea or mental step of counting, comparing and examining. Further the claim has been amended to recite, “examining whether the fetus has a chromosomal abnormality based on the results of the comparing of (d) “ which is a natural law or correlation of naturally occurring nucleic acid sequences being correlated chromosomal abnormality. This judicial exception is not integrated into a practical application because if the claims provide no active steps which depend from or otherwise integrate the judicial exception. The claim(s) does/do not include additional elements that are sufficient to amount to significantly more than the judicial exception because the claims provide no active step which provide specific limitation which would provide for significantly more.
Claim analysis
The instant claim 25 is directed towards a method for examining whether a fetus of a pregnant woman has a chromosomal abnormality, the method comprising:(a) depositing a mixture comprising at least a first set of labelled RCA products and a second set of labelled RCA products onto a support and allowing the mixture to spread across the surface of the support, wherein the first and second sets of labelled RCA products are in solution and:(i) the labelled RCA products in the first set each comprise:(x) a concatemer of both a sequence of a fragment of from blood of the pregnant woman and a first custom sequence that is not human genomic DNA, and(y) oligonucleotides that hybridize to the first custom sequence and comprise a first fluorescent label;(ii) the labelled RCA products in the second set each comprise:(x) a concatemer of both a sequence of a fragment of human cell-free DNA from blood of the pregnant woman and a second custom sequence that is not human genomic DNA, and(y) oligonucleotides that hybridize to the second custom sequence and comprise a second fluorescent label that is distinguishable from the first fluorescent label;(b) counting the number of RCA products in the first set on the support, to provide a first count;(c) counting the number of RCA products in the second set on the support, to provide a second count; (d) comparing the first and second counts, thereby determining the relative quantities of the RCA products in the first and second sets of RCA products; and (e) examining whether the fetus has a chromosomal abnormality based on the results of the comparing of (d)... The counting steps can be considered a mental step. The comparing step is a mental step or abstract idea. The examining step is a mental step and a natural correlation of correlating the detection of naturally occurring nucleic acids with chromosomal abnormality.
The depositing step is considered to be the only active step of the claim. .
Dependent claims set forth further limitations to about how the counting is done, the cell free DNA length, labeling or RCA products, and the support being planar.
According to the 2019 Patent Eligibility Guidance an initial two step analysis is required for determining statutory eligibility.
Step 1. Is the claim directed to a process, machine, manufacture, or composition of matter? In the instant case the Step 1 requirement is satisfied as the claims are directed towards a process.
Step 2A Prong one. Does the claim recite a law of nature, a natural phenomenon or an abstract idea? Yes, abstract idea and law of nature or natural phenomena.
With regards to claim 25, the claim recites, “(b) counting the number of RCA products in the first set on the support, to provide a first count;(c) counting the number of RCA products in the second set on the support, to provide a second count; and(d) comparing the first and second counts, thereby determining the relative quantities of the RCA products in the first and second sets of RCA products; and (e) examining whether the fetus has a chromosomal abnormality based on the results of the comparing of (d).” The counting steps are mental steps or abstract idea. The comparing step is an abstract idea or mental step. (UNIVERSITY OF UTAH RESEARCH v. AMBRY GENETICS CORPORATION). The examining step is correlating the detection of naturally occurring nucleic acids with chromosomal abnormalities.
Step 2A prong two. Does the claim recite additional elements that integrate the judicial exception into a practical application? The answer is no as claim provides no steps which depend from or otherwise integrate the judicial exception..
Step 2B. Does the claim recite additional elements that are significantly more then the judicial exceptions? No, as the only active step of the claim is depositinga mixture, which provides no specific steps or reagents which provide for significantly more.
The art of Lizardi (US patent 6,287,824), Larsson (nature methods (2004) volume 1, pages 227-232), Chapin (Anal. Chem. 2011, 83, 7179–7185) (Weinhausel;(US20110287968), Van De Voorde( Mutation Research 751 (2012) 304–325), Dwivedi ( Critical Care 2012, 16:R151), Nillson(TRENDS in Biotechnology Vol.24 No.2 (February 2006), pages 83-88), Go ( Human Reproduction Update, Vol.17, No.3 pp. 372–382, 2011) and Sparks (Am J Obstet Gynecol 2012;206:319.e1-9.) demonstrate the step of depositing as well as the production of RCA product are routine and conventional.
Claim Rejections - 35 USC § 103
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
Claim 25-26, 28-31, 33-34 is/are rejected under 35 U.S.C. 103 as being unpatentable over, Lizardi (US patent 6,287,824), Larsson (nature methods (2004) volume 1, pages 227-232), Chapin (Anal. Chem. 2011, 83, 7179–7185) (Weinhausel;(US20110287968), Van De Voorde( Mutation Research 751 (2012) 304–325), Dwivedi ( Critical Care 2012, 16:R151), Nillson(TRENDS in Biotechnology Vol.24 No.2 (February 2006), pages 83-88), Go ( Human Reproduction Update, Vol.17, No.3 pp. 372–382, 2011) and Sparks (Am J Obstet Gynecol 2012;206:319.e1-9.)
The prior art as detailed below demonstrates that cell free DNA or circulating DNA was known at the time of filing. The prior art demonstrates cloning or placing DNA in padlock probes, molecular inversion probes, or other circular vectors was on slides and allowing to spread by sandwiching with a coverslip was known at the time of filing. Further the analysis of cell free DNA from pregnant females for examination of aneuploidy was known.
Lizardi provides a patent on rolling circle amplification. (title, abstract). Lizardi teaches, “In the method, the ligation reaction can be divided by spreading the ligation reaction onto a surface to form a spread, where the separate amplification reactions are the locations of circular vectors on the surface after spreading. In this case, a replica of the amplification reactions can be made by contacting the spread with a second surface to which nucleic acids can bind. Any number or all of the amplification reactions can be ordered as an array of reaction droplets or in an array of reaction vessels. In this case, following amplification, all or part of the contents of any number or all the individual reaction droplets or reaction vessels are transferred by one to one mapping to a new set of reaction droplets or reaction vessels. “(column 6, 3rd and 4th paragraph).
Larsson teaches the use of two different padlock probes to produce rolling circle amplification products that are spread across a slide (figure 4, in situ hybridization of mitochondrial genome (230-231)). Larsson teaches the use of padlock probes that have regions complementary to Lin16 or Lin33 fluorescent probes (Lin16 and lin33 are not present in human genome). Larson teaches counting of the red and green fluorescence. Larson teaches determining heteroplasmy based on counts (page 231, 1st column, top).
Chapin teaches:
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Chapin teaches after particles comprising miRNA specific probes have been incubated with the biotinylated reporter sequences which bind to the probe, then washed and the particle solution was “placed on a cover glass…, sandwiched with another smaller cover glass…., and imaged with a CCD camera…. Attached to an inverted microscope.(7181 right column. Because the target and ligated adapter with the primer site is hybridized to the target/adapter probe that is in turn bound to the particle, placing particle solution on the cover glass deposits the labelled RCA products on the surface of a planar substrate and allow them to spread out.
Thus Chapin teaches the use of custom sequences hybridized to concatemers of target sequences and custom sequences for the detection of rolling circle amplification products which are deposited on a support and allowed to spread.
While, Lizardi, Larrson, Chapin teach they do not specifically teach the use of cell free DNA as part of the concatemer or cell free DNA from pregnant females to examin aneuploidy of a fetus..
Van De Voorde provides a review of examination of methylation in breast cancer. Van De Voorde teaches, “The targeting of specific regions of interest in the genome is accomplished by array capture or padlock capture before sequencing. Padlock probes are designed such that a region of interest is covered by overlapping probes on both the sense and antisense DNA strands. Each probe consists of two target specific arms connected by a universal backbone sequence. After hybridization to the DNA in two places, leaving the backbone sequence unbound, the gap between the two arms is polymerized and then ligated to form circular DNA. The backbone sequence is used for universal PCR, allowing for the amplification of tens of thousands of probes within a single reaction.” (page 309, 2nd column bottom).Van De Voorde teaches, “The discovery that cfDNA can be shed into the bloodstream and the presence of abnormally high DNA concentrations in serum/ plasma of patients with breast cancer have opened a new research area, indicating that plasma DNA might be a suitable target for the development of non-invasive diagnostic, prognostic and follow-up tests for cancer [8]. Serum and plasma are more readily accessible body fluids and the collection of a sample is patient-friendly and does not require a specialist [95,99]. The analysis can be repeated any time throughout the follow-up period. According to the origin of cfDNA, it appears that serum tends to contain more DNA than plasma.”(page 310, 1st column bottom).
Dwivedi teaches cloning of cell free DNA into vectors (page 3, 1st column, DNA sequence analyses of cfDNA from severe sepsis patients.”
Nilsson teaches a review article on analysis of genes using closing and replicating circles (title). Nilsson teaches, “Padlock probes are equipped with several features that make them useful tools for gene analysis. The circularization reaction is inherently specific [13] because correct hybridization of both end-sequences is required before the ligase can join the ends. The ligase reaction further provides robust discrimination between allelic variants under standard reaction conditions [14]. A unique property is that the two target-recognition sequences required for specific detection are present in the same molecule; therefore, padlock probes are less likely to crossreact when used in multiplexed reactions. Any linear probes remaining can be removed from the circularized ligation products by exonucleolytic degradation, thereby reducing opportunities for cross-reactive amplification products to form. The combination of these properties makes padlock probes particularly suitable for multiplexed genetic analyses.” (page 83, 2nd column, bottom). Nillsson teaches, “The RCA produces a long, tandem-repeated single-stranded DNA molecule that can be visualized by hybridizing short fluorescence-labeled oligonucleotides to the hundreds of sequence repeats that are typically present in the product” (page 87, 1st column, top) Nillsson teaches, “Detailed understanding of multicellular organisms and many human diseases will ultimately require analyses of individual cells in their natural tissue environment. In analyses of cancers, for example, tumor cells of different stages and grades are mixed with non-transformed cells that support the tumor cells with nutrients and oxygen. Improved monitoring of tumor progression could be obtained through analysis of the genetic and epigenetic status of individual transformed cells in tissue samples from the patient. Moreover, some fundamental biological phenomena, such as random monoallelic expression, asynchronous replication [45] or stochastic gene expression [46], can only be studied at the level of single cells. If the in situ genotyping approach with probe circularization can be extended to also enable analyses of single-copy nuclear genes and transcripts in low-abundance, this will open a new and exciting arena for single-cell analyses” (page 87, 2nd column)
Weinhausel teaches isolation of cell free DNA from serum of breast cancer patients and performing rolling circle amplification (0207)
However, it would have been prima facie obvious to one of ordinary skill in the art prior to the effective filing date of the claims to analyze in cell free DNA in padlock, MIP or other circular probes with non-target sequence or genome sequence with cell free DNA, distribute the RCA product over a surface, detect by hybridization of probes to non-human sequences in RCA product and compare levels of RCA probes to determine the amount of two sequences being assayed. The artisan would be motivated to apply known padlock/ circular probes and rolling circle amplification to human cell free DNA as the art teaches cell free DNA was a non-invasive method of assaying a biological system. The artisan would be motivated to use the circular probes such as padlock probes as Nillsson teaches the probes are inherently specific and non-reacted probes can be easily degraded. The artisan would have a reasonable expectation of success as the artisan is merely using known method in known ways.
While, Lizardi, Larsson, Chapin, Weinhausel, Van De Voorde, Dwivedi, Nillson suggest the use of RCA to analyze cell free nucleic acids, but do not suggest examination of fetal chromosomal abnormalities based on cell-free fetal DNA.
However, Go provides a review of cell free fetal DNA and RNA in maternal plasma ((abstract, throughout). Go suggests the detection of fetal aneuploidy using cell free DNA ((abstract, conclusion).
Sparks teaches, “We assayed cell-free DNA from a training set and a blinded validation set of pregnant women, comprising 250 disomy, 72 T21, and 16 T18 pregnancies” (abstract). Sparks teaches, “The chromosomal dosage resulting from fetal aneuploidy is directly related to the fraction of fetal cfDNA. For example, a cfDNA sample containing 4% DNA from a T21 fetus should exhibit a 2% increase in the proportion of reads from chromosome 21 (chr21) as compared to a normal fetus. Distinguishing these 2 scenarios with high confidence requires a large number (_93,000) of chr21 observations.24 Because MPSS is indiscriminate with respect to chromosomal origin, and because chr21 represents _1.5% of the human genome, _6.3 million uniquely mapped reads are required to ensure sufficient chr21 counts. Given typical MPSS mapping yields of _25%, this translates to 25 million raw sequencing reads per sample. This requirement constrains the throughput, cost efficiency, and clinical utility of MPSS for aneuploidy detection.” (page 1, bottom 2nd column-top 3rd column).
Therefore it would have been prima facie obvious to one of ordinary skill in the art prior to the effective filing date of the claims to use the methods of Lizardi, Larsson, Chapin, Weinhausel, Van De Voorde, Dwivedi, Nillson for analysis of fetal aneuploidy via cell free DNA in maternal blood. The artisan would be motivated as Go and Sparks teach screening of fetal aneuploidy by cell free DNA analysis eliminates the need for invasive fetal testing. Thea artisan would have a reasonable expectation of success as the artisan is merely using known cell free fetal DNA in a maternal blood sample via known rolling circle amplification assays.
With regards to claim 26 Lizardi, Larrson and Chapin teach counting labels for analysis of one or more images.
With regards to claim 28, Larsson teaches fragments at each end of the padlock probes that are 10 to 30 nucleotides (table 1).
Therefore, it would have been prima facie obvious to one of ordinary skill in the art prior to the effective filing date of the claims to design padlock probes with the ends of the probe being complementary to the 10-30 nucleotides of the human genome. The artisan would be motivated as Larsson demonstrates probes with 3’ and 5’ sequence complementary to approximately 15 nucleotides of the human mitochondrial genome work. The artisan would have a reasonable expectation of success as the artisan is merely substituting genomic sequences for mitochondrial sequences.
With regards to claim 29, Dwivedi teaches a fragment of chromosome 21 (table 5, 150-5).
With regards to claim 30, Nillsson teaches, “the target DNA is fragmented using restriction enzymes (figure 2 legend).
With regards to claim 31, Larsson teaches two RCA products are labeled with CDy3 or FITC.
With regards to claim 33, Nilsson teaches, “When using the efficient F29 DNA polymerase, ~1000 monomers are produced during a one-hour RCA, which means that a billion-fold amplification is achieved after three cycles of C2CA.” (page 85, 2nd column).
With regards to claim 34, Larsson teaches a slide.
Response to Arguments
The response the rejection in view of the amendment. This argument is not persuasive as the teachings of Sparks and Go address the new limitations to the claims.
Double Patenting
The nonstatutory double patenting rejection is based on a judicially created doctrine grounded in public policy (a policy reflected in the statute) so as to prevent the unjustified or improper timewise extension of the “right to exclude” granted by a patent and to prevent possible harassment by multiple assignees. A nonstatutory double patenting rejection is appropriate where the conflicting claims are not identical, but at least one examined application claim is not patentably distinct from the reference claim(s) because the examined application claim is either anticipated by, or would have been obvious over, the reference claim(s). See, e.g., In re Berg, 140 F.3d 1428, 46 USPQ2d 1226 (Fed. Cir. 1998); In re Goodman, 11 F.3d 1046, 29 USPQ2d 2010 (Fed. Cir. 1993); In re Longi, 759 F.2d 887, 225 USPQ 645 (Fed. Cir. 1985); In re Van Ornum, 686 F.2d 937, 214 USPQ 761 (CCPA 1982); In re Vogel, 422 F.2d 438, 164 USPQ 619 (CCPA 1970); In re Thorington, 418 F.2d 528, 163 USPQ 644 (CCPA 1969).
A timely filed terminal disclaimer in compliance with 37 CFR 1.321(c) or 1.321(d) may be used to overcome an actual or provisional rejection based on nonstatutory double patenting provided the reference application or patent either is shown to be commonly owned with the examined application, or claims an invention made as a result of activities undertaken within the scope of a joint research agreement. See MPEP § 717.02 for applications subject to examination under the first inventor to file provisions of the AIA as explained in MPEP § 2159. See MPEP §§ 706.02(l)(1) - 706.02(l)(3) for applications not subject to examination under the first inventor to file provisions of the AIA . A terminal disclaimer must be signed in compliance with 37 CFR 1.321(b).
The USPTO Internet website contains terminal disclaimer forms which may be used. Please visit www.uspto.gov/patent/patents-forms. The filing date of the application in which the form is filed determines what form (e.g., PTO/SB/25, PTO/SB/26, PTO/AIA /25, or PTO/AIA /26) should be used. A web-based eTerminal Disclaimer may be filled out completely online using web-screens. An eTerminal Disclaimer that meets all requirements is auto-processed and approved immediately upon submission. For more information about eTerminal Disclaimers, refer to www.uspto.gov/patents/process/file/efs/guidance/eTD-info-I.jsp.
Claims 25-26, 28-31, 33-34 rejected on the ground of nonstatutory double patenting as being unpatentable over claims1-20 of U.S. Patent No. 10,208,336 and ), Van De Voorde( Mutation Research 751 (2012) 304–325), Dwivedi ( Critical Care 2012, 16:R151), Nillson(TRENDS in Biotechnology Vol.24 No.2 (February 2006), pages 83-88), Go ( Human Reproduction Update, Vol.17, No.3 pp. 372–382, 2011) and Sparks (Am J Obstet Gynecol 2012;206:. Although the claims at issue are not identical, they are not patentably distinct from each other because they are coextensive in scope.
The instant claims are drawn to method for examining whether a fetus of a pregnant woman has a chromosomal abnormality, the method comprising:(a) depositing a mixture comprising at least a first set of labelled RCA products and a second set of labelled RCA products onto a support and allowing the mixture to spread across the surface of the support, wherein the first and second sets of labelled RCA products are in solution and:(i) the labelled RCA products in the first set each comprise:(x) a concatemer of both a sequence of a fragment of cell-free DNA from blood of the pregnant woman and a first custom sequence that is not human genomic DNA, and(y) oligonucleotides that hybridize to the first custom sequence and comprise a first fluorescent label;(ii) the labelled RCA products in the second set each comprise:(x) a concatemer of both a sequence of a fragment of human cell-free DNA from blood of the pregnant woman and a second custom sequence that is not human genomic DNA, and(y) oligonucleotides that hybridize to the second custom sequence and comprise a second fluorescent label that is distinguishable from the first fluorescent label;(b) counting the number of RCA products in the first set on the support, to provide a first count;(c) counting the number of RCA products in the second set on the support, to provide a second count; products; and (e) examining whether the fetus has a chromosomal abnormality based on the results of the comparing of (d)..
Claims of ‘336 are drawn to A method of sample analysis, comprising: (a) producing a liquid sample comprising at least a first population of rolling circle amplification (RCA) products and a second population of RCA products, wherein the first and second populations of labeled RCA products are distinguishably labeled and the RCA products are not produced or labeled on or in a porous capillary membrane; (b) filtering the liquid sample through a porous capillary membrane, thereby producing an array of the RCA products on the membrane; and (c) determining the amount of the first labeled population of RCA products and the amount of the second labeled population of RCA products in an area of the membrane. Claim 5 draws the invention to wherein the RCA products are distinguishably labeled by hybridizing fluorescently labeled oligonucleotides to the RCA products.
The claims of ‘336 do not specifically teach a non-human target sequence or cell free DNA or determining aneuploidy.
Van De Voorde provides a review of examination of methylation in breast cancer. Van De Voorde teaches, “The targeting of specific regions of interest in the genome is accomplished by array capture or padlock capture before sequencing. Padlock probes are designed such that a region of interest is covered by overlapping probes on both the sense and antisense DNA strands. Each probe consists of two target specific arms connected by a universal backbone sequence. After hybridization to the DNA in two places, leaving the backbone sequence unbound, the gap between the two arms is polymerized and then ligated to form circular DNA. The backbone sequence is used for universal PCR, allowing for the amplification of tens of thousands of probes within a single reaction.” (page 309, 2nd column bottom).Van De Voorde teaches, “The discovery that cfDNA can be shed into the bloodstream and the presence of abnormally high DNA concentrations in serum/ plasma of patients with breast cancer have opened a new research area, indicating that plasma DNA might be a suitable target for the development of non-invasive diagnostic, prognostic and follow-up tests for cancer [8]. Serum and plasma are more readily accessible body fluids and the collection of a sample is patient-friendly and does not require a specialist [95,99]. The analysis can be repeated any time throughout the follow-up period. According to the origin of cfDNA, it appears that serum tends to contain more DNA than plasma.”(page 310, 1st column bottom).
Dwivedi teaches cloning of cell free DNA into vectors (page 3, 1st column, DNA sequence analyses of cfDNA from severe sepsis patients.”
Nilsson teaches a review article on analysis of genes using closing and replicating circles (title). Nilsson teaches, “Padlock probes are equipped with several features that make them useful tools for gene analysis. The circularization reaction is inherently specific [13] because correct hybridization of both end-sequences is required before the ligase can join the ends. The ligase reaction further provides robust discrimination between allelic variants under standard reaction conditions [14]. A unique property is that the two target-recognition sequences required for specific detection are present in the same molecule; therefore, padlock probes are less likely to crossreact when used in multiplexed reactions. Any linear probes remaining can be removed from the circularized ligation products by exonucleolytic degradation, thereby reducing opportunities for cross-reactive amplification products to form. The combination of these properties makes padlock probes particularly suitable for multiplexed genetic analyses.” (page 83, 2nd column, bottom). Nillsson teaches, “The RCA produces a long, tandem-repeated single-stranded DNA molecule that can be visualized by hybridizing short fluorescence-labeled oligonucleotides to the hundreds of sequence repeats that are typically present in the product” (page 87, 1st column, top) Nillsson teaches, “Detailed understanding of multicellular organisms and many human diseases will ultimately require analyses of individual cells in their natural tissue environment. In analyses of cancers, for example, tumor cells of different stages and grades are mixed with non-transformed cells that support the tumor cells with nutrients and oxygen. Improved monitoring of tumor progression could be obtained through analysis of the genetic and epigenetic status of individual transformed cells in tissue samples from the patient. Moreover, some fundamental biological phenomena, such as random monoallelic expression, asynchronous replication [45] or stochastic gene expression [46], can only be studied at the level of single cells. If the in situ genotyping approach with probe circularization can be extended to also enable analyses of single-copy nuclear genes and transcripts in low-abundance, this will open a new and exciting arena for single-cell analyses” (page 87, 2nd column)
Weinhausel teaches isolation of cell free DNA from serum of breast cancer patients and performing rolling circle amplification (0207)
However, it would have been prima facie obvious to one of ordinary skill in the art prior to the effective filing date of the claims to analyze in cell free DNA in padlock, MIP or other circular probes with non-target sequence or genome sequence with cell free DNA, distribute the RCA product over a surface, detect by hybridization of probes to non-human sequences in RCA product and compare levels of RCA probes to determine the amount of two sequences being assayed. The artisan would be motivated to apply known padlock/ circular probes and rolling circle amplification to human cell free DNA as the art teaches cell free DNA was a non-invasive method of assaying a biological system. The artisan would be motivated to use the circular probes such as padlock probes as Nillsson teaches the probes are inherently specific and non-reacted probes can be easily degraded. The artisan would have a reasonable expectation of success as the artisan is merely using known method in known ways.
While the claims and Weinhausel, Van De Voorde, Dwivedi, Nillson suggest the use of RCA to analyze cell free nucleic acids, but do not suggest examination of fetal chromosomal abnormalities based on cell-free fetal DNA.
However, Go provides a review of cell free fetal DNA and RNA in maternal plasma ((abstract, throughout). Go suggests the detection of fetal aneuploidy using cell free DNA ((abstract, conclusion).
Sparks teaches, “We assayed cell-free DNA from a training set and a blinded validation set of pregnant women, comprising 250 disomy, 72 T21, and 16 T18 pregnancies” (abstract). Sparks teaches, “The chromosomal dosage resulting from fetal aneuploidy is directly related to the fraction of fetal cfDNA. For example, a cfDNA sample containing 4% DNA from a T21 fetus should exhibit a 2% increase in the proportion of reads from chromosome 21 (chr21) as compared to a normal fetus. Distinguishing these 2 scenarios with high confidence requires a large number (_93,000) of chr21 observations.24 Because MPSS is indiscriminate with respect to chromosomal origin, and because chr21 represents _1.5% of the human genome, _6.3 million uniquely mapped reads are required to ensure sufficient chr21 counts. Given typical MPSS mapping yields of _25%, this translates to 25 million raw sequencing reads per sample. This requirement constrains the throughput, cost efficiency, and clinical utility of MPSS for aneuploidy detection.” (page 1, bottom 2nd column-top 3rd column).
Therefore it would have been prima facie obvious to one of ordinary skill in the art prior to the effective filing date of the claims to use the methods of the claims and prior art of record for analysis of fetal aneuploidy via cell free DNA in maternal blood. The artisan would be motivated as Go and Sparks teach screening of fetal aneuploidy by cell free DNA analysis eliminates the need for invasive fetal testing. Thea artisan would have a reasonable expectation of success as the artisan is merely using known cell free fetal DNA in a maternal blood sample via known rolling circle amplification assays.
Dependent claims of 336 render the instant claims obvious.
Response to Arguments
The response traverses the rejection in view of the amendment. This argument is not persuasive in view of the teachings of Go and Sparks,
Claims 25-26, 28-31, 33-34 rejected on the ground of nonstatutory double patenting as being unpatentable over claims1-15 of U.S. Patent No. 10,526,643 and ), Van De Voorde( Mutation Research 751 (2012) 304–325), Dwivedi ( Critical Care 2012, 16:R151), Nillson(TRENDS in Biotechnology Vol.24 No.2 (February 2006), pages 83-88), Go ( Human Reproduction Update, Vol.17, No.3 pp. 372–382, 2011) and Sparks (Am J Obstet Gynecol 2012;206: Although the claims at issue are not identical, they are not patentably distinct from each other because they are coextensive in scope.
The instant claims are drawn to quantifying a first and second set of RCA products labeled by hybridization by spreading a mixture on a support and counting.
Claims of ‘643 are drawn to A method of quantifying a first chromosome or chromosomal locus relative to a second chromosome or chromosomal locus in a sample comprising fragments of human genomic DNA, comprising: contacting the sample with a first set of probes and a second set of probes, wherein the probes each comprise: (a) a targeting oligonucleotide comprising: (i) an internal target-complementary sequence that is in the range of 10 to 30 nucleotides in length and complementary to a fragment of the human genomic DNA, (ii) an upstream flanking sequence that is in the range of 10 to 30 nucleotides in length and not complementary to human genomic DNA, and (iii) a downstream flanking sequence that is in the range of 10 to 30 nucleotides in length and not complementary to human genomic DNA, and (b) a second oligonucleotide comprising a head sequence and a tail sequence having free 5' and 3' ends respectively, wherein the head sequence and the tail sequence are complementary to the upstream flanking sequence and the downstream flanking sequence, respectively, and wherein the probes of the first set each specifically recognize a distinct target sequence within the first chromosome or chromosomal locus and wherein the probes of the second set each specifically recognize a distinct target sequence within the second chromosome or chromosomal locus, providing conditions under which the target sequences in the first and second chromosome or chromosomal locus are at least partially single stranded, providing conditions for annealing and ligation, under which conditions the probes hybridize to their target sequences and generate ligation products, each ligation product being a circle of nucleic acid comprising the second oligonucleotide and a fragment of human genomic DNA that is in the range of 10 to 30 nucleotides in length, providing conditions for rolling circle amplification of the circles of nucleic acid, labelling the rolling circle amplification products in solution by hybridizing them to labelled oligonucleotides to produce hybridized labelled rolling circle amplification products, distributing the hybridized labelled rolling circle amplification products on the surface of a support; counting the number of first rolling circle amplification products on the support, wherein the first rolling circle amplification products are amplified from the ligation products generated by probes of the first set and labelled by a first label, to provide a first count, counting the number of second rolling circle amplification products on the support, wherein the second rolling circle amplification products are amplified from the ligation products generated by probes of the second set and labelled by a second label, to provide a second count, and comparing the first and second counts, thereby determining the relative quantities of the first chromosome or chromosomal locus relative to a second chromosome or chromosomal locus in the sample.
The claims do not specifically teach the use of cell free human DNA.
Van De Voorde provides a review of examination of methylation in breast cancer. Van De Voorde teaches, “The targeting of specific regions of interest in the genome is accomplished by array capture or padlock capture before sequencing. Padlock probes are designed such that a region of interest is covered by overlapping probes on both the sense and antisense DNA strands. Each probe consists of two target specific arms connected by a universal backbone sequence. After hybridization to the DNA in two places, leaving the backbone sequence unbound, the gap between the two arms is polymerized and then ligated to form circular DNA. The backbone sequence is used for universal PCR, allowing for the amplification of tens of thousands of probes within a single reaction.” (page 309, 2nd column bottom).Van De Voorde teaches, “The discovery that cfDNA can be shed into the bloodstream and the presence of abnormally high DNA concentrations in serum/ plasma of patients with breast cancer have opened a new research area, indicating that plasma DNA might be a suitable target for the development of non-invasive diagnostic, prognostic and follow-up tests for cancer [8]. Serum and plasma are more readily accessible body fluids and the collection of a sample is patient-friendly and does not require a specialist [95,99]. The analysis can be repeated any time throughout the follow-up period. According to the origin of cfDNA, it appears that serum tends to contain more DNA than plasma.”(page 310, 1st column bottom).
Dwivedi teaches cloning of cell free DNA into vectors (page 3, 1st column, DNA sequence analyses of cfDNA from severe sepsis patients.”
Nilsson teaches a review article on analysis of genes using closing and replicating circles (title). Nilsson teaches, “Padlock probes are equipped with several features that make them useful tools for gene analysis. The circularization reaction is inherently specific [13] because correct hybridization of both end-sequences is required before the ligase can join the ends. The ligase reaction further provides robust discrimination between allelic variants under standard reaction conditions [14]. A unique property is that the two target-recognition sequences required for specific detection are present in the same molecule; therefore, padlock probes are less likely to crossreact when used in multiplexed reactions. Any linear probes remaining can be removed from the circularized ligation products by exonucleolytic degradation, thereby reducing opportunities for cross-reactive amplification products to form. The combination of these properties makes padlock probes particularly suitable for multiplexed genetic analyses.” (page 83, 2nd column, bottom). Nillsson teaches, “The RCA produces a long, tandem-repeated single-stranded DNA molecule that can be visualized by hybridizing short fluorescence-labeled oligonucleotides to the hundreds of sequence repeats that are typically present in the product” (page 87, 1st column, top) Nillsson teaches, “Detailed understanding of multicellular organisms and many human diseases will ultimately require analyses of individual cells in their natural tissue environment. In analyses of cancers, for example, tumor cells of different stages and grades are mixed with non-transformed cells that support the tumor cells with nutrients and oxygen. Improved monitoring of tumor progression could be obtained through analysis of the genetic and epigenetic status of individual transformed cells in tissue samples from the patient. Moreover, some fundamental biological phenomena, such as random monoallelic expression, asynchronous replication [45] or stochastic gene expression [46], can only be studied at the level of single cells. If the in situ genotyping approach with probe circularization can be extended to also enable analyses of single-copy nuclear genes and transcripts in low-abundance, this will open a new and exciting arena for single-cell analyses” (page 87, 2nd column)
Weinhausel teaches isolation of cell free DNA from serum of breast cancer patients and performing rolling circle amplification (0207)
However, it would have been prima facie obvious to one of ordinary skill in the art prior to the effective filing date of the claims to analyze in cell free DNA in padlock, MIP or other circular probes with non-target sequence or genome sequence with cell free DNA, distribute the RCA product over a surface, detect by hybridization of probes to non-human sequences in RCA product and compare levels of RCA probes to determine the amount of two sequences being assayed. The artisan would be motivated to apply known padlock/ circular probes and rolling circle amplification to human cell free DNA as the art teaches cell free DNA was a non-invasive method of assaying a biological system. The artisan would be motivated to use the circular probes such as padlock probes as Nillsson teaches the probes are inherently specific and non-reacted probes can be easily degraded. The artisan would have a reasonable expectation of success as the artisan is merely using known method in known ways.
While the claims and Weinhausel, Van De Voorde, Dwivedi, Nillson suggest the use of RCA to analyze cell free nucleic acids, but do not suggest examination of fetal chromosomal abnormalities based on cell-free fetal DNA.
However, Go provides a review of cell free fetal DNA and RNA in maternal plasma ((abstract, throughout). Go suggests the detection of fetal aneuploidy using cell free DNA ((abstract, conclusion).
Sparks teaches, “We assayed cell-free DNA from a training set and a blinded validation set of pregnant women, comprising 250 disomy, 72 T21, and 16 T18 pregnancies” (abstract). Sparks teaches, “The chromosomal dosage resulting from fetal aneuploidy is directly related to the fraction of fetal cfDNA. For example, a cfDNA sample containing 4% DNA from a T21 fetus should exhibit a 2% increase in the proportion of reads from chromosome 21 (chr21) as compared to a normal fetus. Distinguishing these 2 scenarios with high confidence requires a large number (_93,000) of chr21 observations.24 Because MPSS is indiscriminate with respect to chromosomal origin, and because chr21 represents _1.5% of the human genome, _6.3 million uniquely mapped reads are required to ensure sufficient chr21 counts. Given typical MPSS mapping yields of _25%, this translates to 25 million raw sequencing reads per sample. This requirement constrains the throughput, cost efficiency, and clinical utility of MPSS for aneuploidy detection.” (page 1, bottom 2nd column-top 3rd column).
Therefore it would have been prima facie obvious to one of ordinary skill in the art prior to the effective filing date of the claims to use the methods of the claims and prior art of record for analysis of fetal aneuploidy via cell free DNA in maternal blood. The artisan would be motivated as Go and Sparks teach screening of fetal aneuploidy by cell free DNA analysis eliminates the need for invasive fetal testing. Thea artisan would have a reasonable expectation of success as the artisan is merely using known cell free fetal DNA in a maternal blood sample via known rolling circle amplification assays.
Dependent claims of 643 render the instant claims obvious.
Response to Arguments
The response traverses the rejection in view of the amendment. This argument is not persuasive in view of the teachings of Go and Sparks,
Claims 25-26, 28-31, 33-34 rejected on the ground of nonstatutory double patenting as being unpatentable over claims1-20 of U.S. Patent No. 10,934,579 and ), Van De Voorde( Mutation Research 751 (2012) 304–325), Dwivedi ( Critical Care 2012, 16:R151), Nillson(TRENDS in Biotechnology Vol.24 No.2 (February 2006), pages 83-88), Go ( Human Reproduction Update, Vol.17, No.3 pp. 372–382, 2011) and Sparks (Am J Obstet Gynecol 2012;206 . Although the claims at issue are not identical, they are not patentably distinct from each other because they are coextensive in scope.
The instant claims are drawn to quantifying a first and second set of RCA products labeled by hybridization by spreading a mixture on a support and counting.
Claims of ‘579 are drawn to A method of sample analysis, comprising: (a) producing a liquid sample comprising at least a first population of rolling circle amplification (RCA) products and a second population of RCA products, wherein the first and second populations of labeled RCA products are distinguishably labeled and the RCA products are not produced or labeled on or in a porous capillary membrane; (b) filtering the liquid sample through a porous capillary membrane, thereby depositing the RCA products on the membrane; and (c) determining the amount of the first labeled population of RCA products and the amount of the second labeled population of RCA products in an area of the membrane.. Claim 5 draws the invention to wherein the RCA products are distinguishably labeled by hybridizing fluorescently labeled oligonucleotides to the RCA products.
The claims of ‘579 do not specifically teach a non-human target sequence or human cell free DNA.
Van De Voorde provides a review of examination of methylation in breast cancer. Van De Voorde teaches, “The targeting of specific regions of interest in the genome is accomplished by array capture or padlock capture before sequencing. Padlock probes are designed such that a region of interest is covered by overlapping probes on both the sense and antisense DNA strands. Each probe consists of two target specific arms connected by a universal backbone sequence. After hybridization to the DNA in two places, leaving the backbone sequence unbound, the gap between the two arms is polymerized and then ligated to form circular DNA. The backbone sequence is used for universal PCR, allowing for the amplification of tens of thousands of probes within a single reaction.” (page 309, 2nd column bottom).Van De Voorde teaches, “The discovery that cfDNA can be shed into the bloodstream and the presence of abnormally high DNA concentrations in serum/ plasma of patients with breast cancer have opened a new research area, indicating that plasma DNA might be a suitable target for the development of non-invasive diagnostic, prognostic and follow-up tests for cancer [8]. Serum and plasma are more readily accessible body fluids and the collection of a sample is patient-friendly and does not require a specialist [95,99]. The analysis can be repeated any time throughout the follow-up period. According to the origin of cfDNA, it appears that serum tends to contain more DNA than plasma.”(page 310, 1st column bottom).
Dwivedi teaches cloning of cell free DNA into vectors (page 3, 1st column, DNA sequence analyses of cfDNA from severe sepsis patients.”
Nilsson teaches a review article on analysis of genes using closing and replicating circles (title). Nilsson teaches, “Padlock probes are equipped with several features that make them useful tools for gene analysis. The circularization reaction is inherently specific [13] because correct hybridization of both end-sequences is required before the ligase can join the ends. The ligase reaction further provides robust discrimination between allelic variants under standard reaction conditions [14]. A unique property is that the two target-recognition sequences required for specific detection are present in the same molecule; therefore, padlock probes are less likely to crossreact when used in multiplexed reactions. Any linear probes remaining can be removed from the circularized ligation products by exonucleolytic degradation, thereby reducing opportunities for cross-reactive amplification products to form. The combination of these properties makes padlock probes particularly suitable for multiplexed genetic analyses.” (page 83, 2nd column, bottom). Nillsson teaches, “The RCA produces a long, tandem-repeated single-stranded DNA molecule that can be visualized by hybridizing short fluorescence-labeled oligonucleotides to the hundreds of sequence repeats that are typically present in the product” (page 87, 1st column, top) Nillsson teaches, “Detailed understanding of multicellular organisms and many human diseases will ultimately require analyses of individual cells in their natural tissue environment. In analyses of cancers, for example, tumor cells of different stages and grades are mixed with non-transformed cells that support the tumor cells with nutrients and oxygen. Improved monitoring of tumor progression could be obtained through analysis of the genetic and epigenetic status of individual transformed cells in tissue samples from the patient. Moreover, some fundamental biological phenomena, such as random monoallelic expression, asynchronous replication [45] or stochastic gene expression [46], can only be studied at the level of single cells. If the in situ genotyping approach with probe circularization can be extended to also enable analyses of single-copy nuclear genes and transcripts in low-abundance, this will open a new and exciting arena for single-cell analyses” (page 87, 2nd column)
Weinhausel teaches isolation of cell free DNA from serum of breast cancer patients and performing rolling circle amplification (0207)
However, it would have been prima facie obvious to one of ordinary skill in the art prior to the effective filing date of the claims to analyze in cell free DNA in padlock, MIP or other circular probes with non-target sequence or genome sequence with cell free DNA, distribute the RCA product over a surface, detect by hybridization of probes to non-human sequences in RCA product and compare levels of RCA probes to determine the amount of two sequences being assayed. The artisan would be motivated to apply known padlock/ circular probes and rolling circle amplification to human cell free DNA as the art teaches cell free DNA was a non-invasive method of assaying a biological system. The artisan would be motivated to use the circular probes such as padlock probes as Nillsson teaches the probes are inherently specific and non-reacted probes can be easily degraded. The artisan would have a reasonable expectation of success as the artisan is merely using known method in known ways.
While the claims and Weinhausel, Van De Voorde, Dwivedi, Nillson suggest the use of RCA to analyze cell free nucleic acids, but do not suggest examination of fetal chromosomal abnormalities based on cell-free fetal DNA.
However, Go provides a review of cell free fetal DNA and RNA in maternal plasma ((abstract, throughout). Go suggests the detection of fetal aneuploidy using cell free DNA ((abstract, conclusion).
Sparks teaches, “We assayed cell-free DNA from a training set and a blinded validation set of pregnant women, comprising 250 disomy, 72 T21, and 16 T18 pregnancies” (abstract). Sparks teaches, “The chromosomal dosage resulting from fetal aneuploidy is directly related to the fraction of fetal cfDNA. For example, a cfDNA sample containing 4% DNA from a T21 fetus should exhibit a 2% increase in the proportion of reads from chromosome 21 (chr21) as compared to a normal fetus. Distinguishing these 2 scenarios with high confidence requires a large number (_93,000) of chr21 observations.24 Because MPSS is indiscriminate with respect to chromosomal origin, and because chr21 represents _1.5% of the human genome, _6.3 million uniquely mapped reads are required to ensure sufficient chr21 counts. Given typical MPSS mapping yields of _25%, this translates to 25 million raw sequencing reads per sample. This requirement constrains the throughput, cost efficiency, and clinical utility of MPSS for aneuploidy detection.” (page 1, bottom 2nd column-top 3rd column).
Therefore it would have been prima facie obvious to one of ordinary skill in the art prior to the effective filing date of the claims to use the methods of the claims and prior art of record for analysis of fetal aneuploidy via cell free DNA in maternal blood. The artisan would be motivated as Go and Sparks teach screening of fetal aneuploidy by cell free DNA analysis eliminates the need for invasive fetal testing. Thea artisan would have a reasonable expectation of success as the artisan is merely using known cell free fetal DNA in a maternal blood sample via known rolling circle amplification assays.
Dependent claims of 579 render the instant claims obvious.
Response to Arguments
The response traverses the rejection in view of the amendment. This argument is not persuasive in view of the teachings of Go and Sparks,
Summary
No claims are allowed.
Conclusion
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/Steven Pohnert/Primary Examiner, Art Unit 1683
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