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 .
Priority
Receipt is acknowledged of certified copies of papers required by 37 CFR 1.55.
Information Disclosure Statement
The IDS received on September 14, 2023 is proper and is being considered by the Examiner. The NPL# C8 has been lined-through as the submission did not contain the proper document with the purported title, “Human mitochondrial mutation detecting DNA PCR primer P1 #2”. The submission relevant to the accession no. ARW56435 is directed to a GenBank accession number which was available on Jun 2017, and references a non-published document, “Genome sequence of Bacillus licheniformis strain SRCM100027”.
Drawings
The drawings received on May 11, 2023 are acceptable.
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 32-34, 37, 39, and 40 are rejected under 35 U.S.C. 101 because the claimed invention is directed to non-statutory subject matter.
Claim 32 is directed to two separate statutory subject matters which are an article of manufacture (i.e., kit) and a composition.
Claims 33-34, 37, 39, and 40 are rejected by their dependency on claim 32.
Claims 57 and 58 are rejected under 35 U.S.C. 101 because the claimed invention is directed to abstract idea (i.e., data harvesting and manipulation) without significantly more. The claims recite a method comprising the steps of determining a concentration of tRNA molecules and calculating their ratio (mutation load). This judicial exception is not integrated into a practical application because the determination of a concentration of an analyte and calculating their ratio is deemed data collection and manipulation.
The claims do not include additional elements that are sufficient to amount to significantly more than the judicial exception based on the analysis under the current Patent Eligibility Guidelines (herein, “PEG”) as discussed below.
Step 1 Inquiry under PEG
Step 1 inquiry under Patent Eligibility Guidelines (herein, “PEG”) determines whether or not the claimed invention is drawn to one of the recognized statutory classes of invention. Claims 57 and 58 satisfy the present inquiry as being drawn to a method.
Step 2A Inquiry under PEG
A recently revised PEG now performs step 2A inquiry under a 2-prong analysis, and the subject claims analyzed accordingly as follows:
Prong 1:
Prong-1 inquiry under step 2A determines whether the claims recite an abstract idea, a law of nature, or a natural phenomenon. As stated above, judicial exception of obtaining data from an analysis, that is, the amounts of tRNA by correlating the signal harvested into data and manipulating that data into a formula that calculates a ratio of a mutant amount to the entire amount of tRNA.
As stated in MPEP 2106.04(a)(2).I, steps (d) can be classified as an abstract idea belonging to the “Mathematical Concepts” grouping. Within that grouping, the claims can be classified both under “Mathematical Relationships”(A) and “Mathematical Calculations” (C):
“A. Mathematical Relationships
A mathematical relationship is a relationship between variables or numbers. A mathematical relationship may be expressed in words or using mathematical symbols. For example, pressure (p) can be described as the ratio between the magnitude of the normal force (F) and area of the surface on contact (A), or it can be set forth in the form of an equation such as p = F/A.
Examples of mathematical relationships recited in a claim include:
i. a relationship between reaction rate and temperature, which relationship can be expressed in the form of a formula called the Arrhenius equation, Diamond v. Diehr; 450 U.S. at 178 n. 2, 179 n.5, 191-92, 209 USPQ at 4-5 (1981);
ii. a conversion between binary coded decimal and pure binary, Benson, 409 U.S. at 64, 175 USPQ at 674;
iii. a mathematical relationship between enhanced directional radio activity and antenna conductor arrangement (i.e., the length of the conductors with respect to the operating wave length and the angle between the conductors), Mackay Radio & Tel. Co. v. Radio Corp. of America, 306 U.S. 86, 91, 40 USPQ 199, 201 (1939) (while the litigated claims 15 and 16 of U.S. Patent No. 1,974,387 expressed this mathematical relationship using a formula that described the angle between the conductors, other claims in the patent (e.g., claim 1) expressed the mathematical relationship in words); and
iv. organizing information and manipulating information through mathematical correlations, Digitech Image Techs., LLC v. Electronics for Imaging, Inc., 758 F.3d 1344, 1350, 111 USPQ2d 1717, 1721 (Fed. Cir. 2014). The patentee in Digitech claimed methods of generating first and second data by taking existing information, manipulating the data using mathematical functions, and organizing this information into a new form. The court explained that such claims were directed to an abstract idea because they described a process of organizing information through mathematical correlations, like Flook's method of calculating using a mathematical formula. 758 F.3d at 1350, 111 USPQ2d at 1721.
“C. Mathematical calculations
A claim that recites a mathematical calculation, when the claim is given its broadest reasonable interpretation in light of the specification, will be considered as falling within the "mathematical concepts" grouping. A mathematical calculation is a mathematical operation (such as multiplication) or an act of calculating using mathematical methods to determine a variable or number, e.g., performing an arithmetic operation such as exponentiation. There is no particular word or set of words that indicates a claim recites a mathematical calculation. That is, a claim does not have to recite the word "calculating" in order to be considered a mathematical calculation. For example, a step of "determining" a variable or number using mathematical methods or "performing" a mathematical operation may also be considered mathematical calculations when the broadest reasonable interpretation of the claim in light of the specification encompasses a mathematical calculation.
Examples of mathematical calculations recited in a claim include:
i. performing a resampled statistical analysis to generate a resampled distribution, SAP America, Inc. v. Investpic, LLC, 898 F.3d 1161, 1163-65, 127 USPQ2d 1597, 1598-1600 (Fed. Cir. 2018), modifying SAP America, Inc. v. Investpic, LLC, 890 F.3d 1016, 126 USPQ2d 1638 (Fed. Cir. 2018);
ii. calculating a number representing an alarm limit value using the mathematical formula ‘‘B1=B0 (1.0–F) + PVL(F)’’, Parker v. Flook, 437 U.S. 584, 585, 198 USPQ 193, 195 (1978);
iii. using a formula to convert geospatial coordinates into natural numbers, Burnett v. Panasonic Corp., 741 Fed. Appx. 777, 780 (Fed. Cir. 2018) (non-precedential);
iv. managing a stable value protected life insurance policy via performing calculations, Bancorp Servs., LLC v. Sun Life Assur. Co. of Canada (U.S.), 687 F.3d 1266, 1280, 103 USPQ2d 1425, 1434 (Fed. Cir. 2012);
v. using an algorithm for determining the optimal number of visits by a business representative to a client, In re Maucorps, 609 F.2d 481, 482, 203 USPQ 812, 813 (CCPA 1979); and
vi. calculating the difference between local and average data values, In re Abele, 684 F.2d 902, 903, 214 USPQ 682, 683-84 (CCPA 1982).
Therefore, the steps of procuring signals which correlate to an amount of target analyte that already exist in nature, and applying a mathematical formula in the form of taking their ratio in generating a subsequent data tantamount to a data harvesting and manipulation of data without significant application.
Prong 2:
Prong-2 inquiry under step 2A determines whether or not the claims recite additional elements that integrate the judicial exception into a practical application in a manner that imposes a meaningful limit on the judicial exception.
The dependent claim 58 further recite that the analyte is mt-tRNA, but this does not negate the fact that the analyte is naturally existing molecule and the data harvested from such an analyte is data and its manipulation falls under the same analysis as discussed above, railing to pose a meaningful limit on the judicial exception.
As explained by the Supreme Court, in order to transform a judicial exception into a patent-eligible application, the additional element or combination of elements must do ‘more than simply stat[e] the [judicial exception] while adding the words ‘apply it’”. Alice Corp. v. CLS Bank, 573 U.S. __, 134 S. Ct. 2347, 2357, 110 USPQ2d 1976, 1982-83 (2014) (quoting Mayo Collaborative Servs. V. Prometheus Labs., Inc., 566 U.S. 66, 72, 101 USPQ2d 1961, 1965). Thus, for example, claims that amount to nothing more than an instruction to apply the abstract idea using a generic computer do not render an abstract idea eligible. Alice Corp., 134 S. Ct. at 2358, 110 USPQ2d at 1983. See also 134 S. Ct. at 2389, 110 USPQ2d at 1984 (warning against a § 101 analysis that turns on “the draftsman’s art”) (MPEP 2106.05(f))
Step 2B Inquiry under PEG
Step 2B inquiry of the PEG determines whether or not additional elements are provided and whether such elements amount to significantly more than the judicial exception in the claims.
No additional steps are recited in the claims.
In conclusion, the claims are not patent eligible under 35 U.S.C. 101.
Claim Rejections - 35 USC § 112
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claims 1, 2, 3, 6, 18, 25, 29, 31-34, 37, 39, 40, 42, 43, 50-55, 57, 58, and 64 are rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
Claim 1 is indefinite for reciting the phrase, “terminal region is complementary to a region of a tRNA that is associated with a pathogenic mutation” because it is unclear what a region that is “associated” with the mutation means. For example, it is unclear whether the terminal region actually contains the site of mutation or is simply complementary to a region adjacent (“associated”) to the mutation, or whether the tRNA does not even contain any mutation but is “associated” with mutations in other target sequences they bind, etc.
Claims 2, 40, and 51 are indefinite for reciting the trademark, Splint R® ligase. Usage of a trademark to define a product in a claim renders the claim indefinite because a trademark does not identify the actual product, but the source of the product. Because the formulation of a product defined by a trademark can change over a period of time while retaining the same tradename, its usage renders the claim indefinite in its metes and bounds. For example, the product, Coke® has retained its tradename while changing its formulation over a period of time.
Claim 25 is indefinite because it is unclear what is meant by a terminal region that is adjacent and non-adjacent regions of the tRNA. The term, “adjacent” necessarily encompass any region that is found on the tRNA which the terminal regions of the padlock probe anneals because their “adjacency” does not necessarily limit them to any particular location or gaps (or no gaps) therebetween. In other words, two terminal ends which are separated by a gap of any nucleotides are still considered “adjacent” to each other. For the purpose of prosecution, the phrase, “adjacent regions” has been construed to mean that they are immediately adjacent to each other (i.e., one end of the terminal region abuts the other terminal region), and the “non-adjacent” region has been construed to mean as recited by the claim.
Claim 33 is indefinite for the following reasons. Claim 33 recites the phrase, “one or more” padlock probes, but the actual claim recites that at least two padlocks probes are required (i.e., first padlock probe of (i) and a second padlock probe of (ii)). Therefore, the subject-phase is incongruent with the actual requirement in the claim.
Claim 64 is an improper multiple dependent claim because the claim depends from independent claim 57 and its dependency from method of usage of claim 42 that draws its dependency from independent claim 1.
Claims 2, 3, 6, 18, 25, 29, 31-34, 37, 39, 40, 42, 43, 50-55, 57, 58, and 64 are also indefinite by way of their dependency on claim 1.
The following is a quotation of the first paragraph of 35 U.S.C. 112(a):
(a) IN GENERAL.—The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor or joint inventor of carrying out the invention.
The following is a quotation of the first paragraph of pre-AIA 35 U.S.C. 112:
The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor of carrying out his invention.
Claims 1-3, 18, 25, 29, 31-34, 37, 39, 40, 42, 43, 50-55, 57, 58, and 64 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, as failing to comply with the written description requirement. The claim(s) contains subject matter which was not described in the specification in such a way as to reasonably convey to one skilled in the relevant art that the inventor or a joint inventor, or for applications subject to pre-AIA 35 U.S.C. 112, the inventor(s), at the time the application was filed, had possession of the claimed invention. This is a Written Description Rejection.
The Federal Circuit reiterated that mere use of the same words in the specification and the claim (an in ipsis verbis test) is not sufficien to establish written description.
Rather, the written description requirement ensures that, “an applicant invented the subject matter which is claimed. Further, the written description requirement for a claimed genus may be satisfied through a sufficient description of a representative number of species by 1) reduction to practice; 2) reduction to drawing; or 3) disclosure of relevant identifying characteristics (i.e., structure of other physical and/or chemical properties, functional characteristics coupled with a known or disclosed correlation between function and structure) (MPEP 2163 at II(A)(3)(a)(ii)).
Reduction to Practice
I – Products (Padlock probe, a kit/composition comprising said padlock probe):
Claims 1 and 32 are directed to a product (a padlock probe and a kit/composition comprising one or more of said padlock probe(s)).
Claim 1 embraces a broad genus of any padlock probe which comprises a pair of terminal regions, wherein a terminal region (of said probe) is complementary to a region of tRNA that is associated with a pathogenic mutation.
To this end, the specification does not provide a reasonable number of species so as to show possession of a padlock probe that comprises a terminal sequence complementary to a region of a tRNA that is associated with all types of mutations presently known and unknown for any and all kind of diseases known and unknown, where the padlock probe has not yet been optimized nor fabricated to work.
The specification discloses a specific target for such a mutation found on a specific type of tRNA, for a mitochondrial DNA, having two different regions defined by SEQ ID numbers 55 and 56; and SEQ ID numbers 57 and 58). These two pair of terminal regions in no way is demonstrative of all mutations or associated with all mutations found on the tRNA.
II – Methods of use:
The presently claimed method employs the padlock probe or the kit/composition comprising one or more of the padlock prob(s) which have been discussed above as lacking written description. Therefore, the method also lacks description as lacking possession of a reasonable number of species of padlock probes having terminal regions which are complementary to regions on tRNA which comprise any mutation or regions which are associated with any pathogenic mutation.
Reduction to Drawing
The specification only provides a generalized depiction of using padlock probes for the identification and quantification of target nucleic acids.
Disclosure of Relevant Identifying Characteristics
While one could argue that a skilled artisan would be able to identify the “representative number of species” of the remaining mutations on the regions of a tRNA or regions associated with pathogenic mutations and derive such padlock probes, such method would not satisfy the written description for the genus claims when, “the claims require an essential or critical feature which is not adequately described in the specification and which is not conventional in the art or known to one of ordinary skill in the art” (MPEP 2163(I)(A)). For the claims at issue, such essential or critical feature is the actual mutations and the padlock probes which can operate to detect a genus of such mutations. Applicants have not disclosed enough number of species within the claimed genus.
As stated in University of California v. Eli Lilly and Co. at page 1404:
An adequate written description of a DNA ... "requires a precise definition, such as by structure, formula, chemical name, or physical properties," not a mere wish or plan for obtaining the claimed chemical invention. Fiers v. Revel, 984 F.2d 1164, 1171, 25 USPQ2d 1601, 1606 (Fed. Cir. 1993). Accordingly, "an adequate written description of a DNA requires more than a mere statement that it is part of the invention and reference to a potential method for isolating it; what is required is a description of the DNA itself." Id. at 1170, 25 USPQ2d at 1606.
Therefore, for the foregoing reasons, the genus embraced by the claims is not sufficiently described by the number of species disclosed in the specification, and therefore, the specification lacks written description of the claims.
Claim Rejections - 35 USC § 103
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
Claims 1-3, 6, 18, 25, 29, 32, 33, 37, 39, 40, 42, 43, 50-55, 57, 58, and 64 are rejected under 35 U.S.C. 103 as being unpatentable over Bockelmann et al. (US 2012/0264630 A1, published October 18, 2012) in view of Ueki et al. (Mitochondrion, 2006, pages 29-36) and Jin et al. (Nucleic Acids Research, 2016, vol. 44, no. 13, e116, pages 1-14).
With regard to claim 1, Bockelmann et al. teach a padlock probe comprising terminal regions complementary to a target nucleic acid, wherein a terminal region is complementary to a region of the target nucleic acid that is associated with a mutation (see “Circularizable Mutant Probe” on section [0123]; also principle of the assay in Fig. 21).
With regard to claim 2, Bocklemann et al. explicitly teach that their method requires the incubation period of 25 to 35 minutes (“the incubation time for step iv) is 25 to 90 minutes, more preferably 25 to 35 minutes”, section [0085]) at temperature of 37oC (“THRCA is carried out a temperature in the range of 37oC.”, section [0040]).
With regard to claim 3, the terminal regions are 5-13 nucleotides in length (see below alignment between Mut DNA target and Mut circularizable probe with underlined regions):
Mut DNA target2:
5’- GCT ACT CGC TGA AAT TAA TAC GAa TCA CTA GGT GCC ACG G- 3’
Mut Circular probe:
5’ - TCG TAT TAA TTT CAG CGA GTT TCT GAC TCG TCA TGT CTC AGC TCT AGT ACG CTG ATC TTA GTG TCA GGA TAC GGT GTA GAC CTA GTG AT – 3’
With regard to claim 25, the terminal regions are complementary to adjacent regions of the target nucleic acid (see Fig. 2 and above alignment).
With regard to claim 29, the padlock probe comprises from 5’ to 3’, a first terminal region; a first primer binding site; a second primer binding site; and a second terminal region, wherein the first primer binding site is a forward primer binding site and the second primer binding site is a reverse primer binding site or visa versa (any region found on the circular probe of Bockelmann et al. can be utilized for forward or reverse primer region; also see Mutant forward primer (MutFP) and Mutant reverse primer (MutRP) on sections [0128] and [0129]).
With regard to claim 32, Bockelmann et al. explicitly teach a kit of reagents for performing the disclosed method.
With regard to claim 33, the artisans teach the use of both wildtype and mutant circularization probe (therefore, a first padlock that comprises the mutant specific nucleotide and the second padlock that does not comprise the mutant nucleotide).
With regard to claim 37, the artisans already teach that the reagents used in the method comprise first and second primers (see above).
With regard to claims 42 and 53, the artisans teach the method of providing a sample, hybridizing the padlock probe, circularizing the one or more padlock probes, amplifying the one or more padlock probes via THRCA (Tagged Hyperbranched Rolling Circle Amplification) and detecting the amplified padlock probes, see claim 8 for example).
With regard to claim 54, the detection is via fluorescence detection (see section [0022]).
Bockelmann et al., while explicitly teaching that their method is for the detection of mutations, such as single nucleotide polymorphisms (“THRCA may be used to detect a genetic polymorphism of a single or a plurality of base pair(s) (mutations).”, section [0077]), do not explicitly teach all types of target nucleic acids from which the mutation can be detected.
Consequently, Bockelmann et al. do not explicitly teach that their padlock probe is designed to target a mutation non tRNA comprising a region that is associated with a pathogenic mutation, or that the tRNA is a mitochondrial tRNA (claim 1, in-part, claims 18, 43).
Bockelmann et al. do not explicitly teach that the method comprises a capture probe and thus do not teach a kit comprising a capture probe (claim 39).
While Bockelmann et al. explicitly teach ligation of the padlock probes with a ligase, the artisans do not use SplintR® ligase (claims 40 and 51), or isolating from urine, muscle, or blood (claim 50), or purifying using magnetic beads (claim 52).
Bockelman et al. do not teach that the amount of tRNAs (or target nucleic acid) is quantified (claim 55).
Bockelmann et al. do not explicitly teach that their method is employed for detecting and/or diagnosing/assessing the severity of a tRNA-associated disease in a subject by determining the concentration of a mutant tRNA in a percent of total tRNA of the sample that includes a wildtype tRNA (claim 57), said tRNA being mitochondrial tRNA (claims 58 and 64).
Ueki et al. teach that mutations found in mitochondrial tRNA is correlated with patients having lactic acidosis:
“we have designed … to detect the mutation in all mitochondrial tRNA genes in 44 patients who had severe lactic acidosis using single strand conformation polymorphism (SSCP), automated DNA sequence and PCR-RFLP methods, to see [whether] mitochondrial tRNA mutations are one of the common causes of patients having lactic acidosis” (page 30)
Ueki et al. teach that in patients with Liegh disease, patient one had 92% and patient two had 78% of an A3243G mutation in the mitochondrial tRNALeu(UUR) gene, patient 21 had 97% of an A8296G mutation in the mitochondrial tRNALys gene, and patient 24 had 67% of a C3303T mutation in the mitochondrial tRNALeu(UUR) gene (page 32, 2nd column).
Ueki et al. teach the extracting the sample from blood (see page 30, 2nd column, section 2.2.1).
Jin et al. teach a well-known usage of T4 DNA ligase in reactions that involve ligation, such as padlock probe RCA:
“[l]igation of DNA probes followed by DNA amplification offers …” (page 2, 1st column, bottom paragraph)
“One embodiment of this approach is the use of a ‘padlock’ probe followed by rolling circle amplification” (page 2, 2nd column, 1st paragraph)
“previously described miRNA ligation-based detection methods use T4 DNA Ligase, despite its highly inefficient ligation of DNA in an RNA:DNA hybrid (page 2, 2nd column, 2nd paragraph)
Jin et al. teach that SplintR Ligase is superior in its efficiency over the conventionally employed T4 DNA ligase:
“A superior enzyme for this type of ligation is Chlorella virus DNA ligase (sold commercially as SplintR Ligase) … Compared to T4 DNA Ligase, Chlorella virus DNA ligase has a faster maximum turnover rate … relatively poor binding of the T4 enzyme to the RNA:DNA hybrid often results in abortive ligation with the accumulation of the adenylated DNA oligonucleotides in preference to productive ligation” (page 2, 2nd column, 2nd paragraph)
It would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to combine the teachings of Bockelmanne et al. with the teachings of Ueki et al. and Jin et al., thereby arriving at the invention as claimed for the following reasons.
With regard to the usage of a ligase commercially sold and available in the art for the same purpose of ligating the two adjacent regions of the padlock probes, doing so would have been an obvious application based on the teachings provided by Jin et al. who teach that SplintR Ligase was more efficient at its ligation reaction over the traditionally employed T4 DNA ligase (which Bockelmann et al. used3).
As well, employing a reagent that yields the same outcome of ligating two ends of adjacent nucleic acids would have been also obvious as such rationale is within that which was provided in KSR, wherein the Supreme Court particularly emphasized “the need for caution in granting a patent based on the combination of elements found in the prior art,” Id. at 415, 82 USPQ2d at 1395, and discussed circumstances in which a patent might be determined to be obvious. Importantly, the Supreme Court reaffirmed principles based on its precedent that “[t]he combination of familiar elements according to known methods is likely to be obvious when it does no more than yield predictable results.” Id. at 415-16, 82 USPQ2d at 1395. The Supreme Court stated that there are “[t]hree cases decided after Graham [that] illustrate this doctrine.” Id. at 416, 82 USPQ2d at 1395. (1) “In United States v. Adams, . . . [t]he Court recognized that when a patent claims a structure already known in the prior art that is altered by the mere substitution of one element for another known in the field, the combination must do more than yield a predictable result.”
With regard to the application of the teachings of Bockelmann et al. for targeting mutations found on mitochondrial tRNA or tRNA encoding gene, doing so would have been obvious for the following reasons.
While Bockelmann et al. did not explicitly teach that their method should be directed for the detection of mutations found on mitochondrial tRNA or tRNA encoding gene, one of ordinary skill in the art of molecular diagnostics would have recognized that such detection means would have been applicable for detection of mutations, including single base mutations found on any target nucleic acid, so long as there was a reason to do so.
Indeed, as evidenced by Ueki et al., tRNA mutations have been known in the art to be implicated with diseases, such as lactic acidosis, stroke-like episodes (MELAS), maternal inherited Leigh syndrome (MILS), etc. (see page 30, 1st column, 1st paragraph).
As well, Ueki et al. teach specific mutations found on mitochondrial tRNA sequences which are implicated diseases (see above).
Therefore, one of ordinary skill in the art would have been motivated to apply the teachings of Bockelmann et al. to assay for the mutation from target nucleic acids which are known to be implicated with diseases. One of ordinary skill in the art would have also had a reasonable expectation of success because the method disclosed by Bockelmann et al. was capable of detecting as little as a single base nucleotide change (i.e., SNP), and such a capability would have been useful in the detection of single base mutations found on mitochondrial tRNA that were implicated with diseases.
As to the quantification of the amount of mutant tRNAs in the sample and the calculation of the ratio of the amount of mutant tRNA to the total population, doing so would have been an obvious application as it is conventional in the art to determine the quantity of marker analytes and calculate their amount in a ratio of analytes to total samples (i.e., ratio of tRNAmut/tRNAmut+wildtype). Quantification of analytes based on fluorescence has been well-established in the art of molecular diagnostics and applying that to the quantification of Bocklemann et al. would have been well-within the purview of the artisan.
Lasty, one of ordinary skill in the art would have been motivated to combine the teachings of Bockelmann et al. with conventionally practiced method of capturing the resulting amplification product. This is because Bocklemann et al. already teach that the primer employed in generating the RCA product from the circularized probe comprises a tag, such as biotin (see claim 6). Because biotin has been conventionally utilized to concentrate, isolate and purify based on its binding to bead-bound streptavidin, one of ordinary skill in the art would have been motivated to isolate the amplified products from the circularized padlock probes by their binding to a solid-substrate prior to detecting their presence/amount for the well-known desire to reduce background signal.
Therefore, the invention as claimed is deemed prima facie obvious over the cited references.
Claims 31 and 34 are rejected under 35 U.S.C. 103 as being unpatentable over Bockelmann et al. (US 2012/0264630 A1, published October 18, 2012) in view of Ueki et al. (Mitochondrion, 2006, pages 29-36) and Jin et al. (Nucleic Acids Research, 2016, vol. 44, no. 13, e116, pages 1-14), as applied to claims 1-3, 6, 18, 25, 29, 32, 33, 37, 39, 40, 42, 43, 50-55, 57, 58, and 64 above, and further in view of Fan et al. (US 2015/0368704 A1, published December 24, 2015).
The teachings of Bockelmann et al., Ueki et al., and Jin et al. have already been discussed above.
While Bocklemann et al. explicitly suggest that multiplex assay can be performed (“detection of a genetic polymorphism by multiplexing”, section [0077]; “THRCA may be used to detect a genetic polymorphism of a single or a plurality of base pair(s) (mutations)”, section [0077]), the artisans do not explicitly teach a kit comprising a third and a fourth padlock probe, said third padlock comprising a terminal region complementary to a region of a gene encoding tRNA that is associated with a pathogenic mutation but does not comprise the pathogenic mutation, and said fourth padlock comprising a terminal region complementary to the gene encoding tRNA that is associated with a pathogenic mutation and comprises the mutation.
Bocklemann et al. explicitly teach that their method produces RCA products which comprises a barcode which is associated with particular mutation or with a particular patient (“THRCA … the case of detection of a genetic polymorphism by multiplexing … the barcode sequence may be associated with a particular mutation or with a particular patient”, section [0077]), but utilizes primer sequences to produce RCA products having a barcode thereon (“inventors … modified the HRCA technique … whereby the forward primer is constituted, from its 5’ end to 3’ end, by a barcode nucleotide sequence, a linear spacer …”, section [0018]), and therefore, their padlock probe does not comprise a tag or a barcode sequence.
Fan et al. teach a method of producing barcode containing RCA products from a padlock probe, wherein the padlock itself comprises a barcode sequence therein (“[b]arcode probes used in the methods presented herein can include padlock probes”, section [0034]; “one or more unique padlock probes with unique molecular barcodes”, section [0041]).
It would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to combine the teachings of Bockelmann et al., Ueki et al., and Jing et al., with the teachings of Fan et al., thereby arriving at the invention as claimed for the following reasons.
As discussed above, Bocklemann et al. already teach that their method could be used in a multiplex detection utilizing padlock probes which are specific for different single base mutations as well as using them as a pair for a wildtype and a mutant counterpart detection.
Therefore, one of ordinary skill in the art would have been reasonably motivated to arrive at a kit comprising a multiple pairs of such padlock probes, each pair directed to a mutant and a wildtype counterpart of the known mitochondrial tRNA mutations disclosed in Ueki et al.’s teachings, rendering the invention as claimed prima facie obvious.
As to providing a molecular barcode sequence on the padlock probe itself over the use of primer comprising a molecular barcode (as done by Bockelmann et al.), one of ordinary skill in the art would have recognized that the two different means would have resulted in a similar product, that is, an RCA product comprising a unique region (i.e., molecular barcode sequence) which can later be assayed to decode the RCA product in various way, such as identifying the mutation itself, or the sample source/origin, etc., such ways which have already been known in the art of molecular diagnostics, as well as evidenced by Bockelmann et al.
“THRCA … the case of detection of a genetic polymorphism by multiplexing … the barcode sequence may be associated with a particular mutation or with a particular patient” (section [0077], Bockelmann et al.)
Therefore, utilizing an alternative means of producing an RCA product comprising a molecular barcode would have been an obvious mean of substituting one well-known means with the another, yielding a predictable outcome, the rationale which had been expressed by the Supreme Court in KSR as being obvious (see above for citations).
Therefore, the invention as claimed is deemed prima facie obvious over the cited references.
Conclusion
No claims are allowed.
Claim 6 is free of prior art as the prior art does not provide a sufficient motivation to arrive at a padlock probe comprising two terminal regions that consists of the specific sequences identified by SEQ ID Numbers 55 and 56; or SEQ ID Numbers 57 and 58.
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Any inquiry concerning this communication or earlier communications from the Examiner should be directed to Young J. Kim whose telephone number is (571) 272-0785. The Examiner can best be reached from 7:30 a.m. to 4:00 p.m (M-F). The Examiner can also be reached via e-mail to Young.Kim@uspto.gov. However, the office cannot guarantee security through the e-mail system nor should official papers be transmitted through this route.
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/YOUNG J KIM/Primary Examiner
Art Unit 1637 September 20, 2025
/YJK/
1 Fig. 2 shows an exemplary embodiment of a circularizable probe (i.e., padlock probe) directed to a wild-type target nucleic acid, but the invention is also directed to a circularizable probe to a mutant target nucleic acid (i.e., SNP) that operate on the same principle.
2 Lowercase “a” is the single base mutation.
3 see, “if the target nucleic acid contains said polymorphic base or bases, obtaining the hybridized and circularized single-stranded DNA by ligation of said hybridized single-stranded DNA with the target nucleic acid in the presence of a DNA ligase (for example T4 DNA ligase) …” (section [0081].