DETAILED ACTION
Notice of Pre-AIA or AIA Status
1. The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Election/Restrictions
2. Applicant’s election without traverse of Group I and the species of 5' (N)n-HG-(N)m-HG-(N)p-HG-(N)q-3’ in the reply filed on 11 November 2025 is acknowledged.
Upon further consideration, the election of species requirement is withdrawn. The species of 5'(N)n-CGH-(N)m-CGH-(N)p-3' has been rejoined with the elected species.
Claim Status
3. Claims 1, 2, 4-6, 8, 13, 16, 20-21, 26, 27, 37, 40, 43, 47, 51, 53, 59, 72 and 73 are pending and have been examined herein.
Claim Objections
4. Claims 1, 2, 4-6, 8, 13, 16, 20-21, 26, 27, 37, 40, 43, 47, 51, 53, 59, 72 and 73 are objected to because of the following informalities:
Claims 1, 2, 4-6, 8, 13, 16, 20-21, 26, 27, 37, 40, 43, 47, 51, 53, 59, 72 and 73 are objected to because the claims recite a capital letter at each step (e.g., “a. Providing”, “b. Incubating”, “c. Detecting”). See claims 1, 37, and 47. MPEP 608.01(m) states: “Each claim begins with a capital letter and ends with a period. Periods may not be used elsewhere in the claims except for abbreviations.” Capitalizing the first word suggests that the step or clause is a new sentence, whereas claims consist of a single sentence.
Claim 37 is objected to because the claim recites steps “d.” and “e.” and then recites steps “a.” and “b.” Since claim 37 depends from claim 13, which depends from claim 1 and which already recites a step “a.” and “b.”, claim 37 should recite “f. melting nucleic acids” and “g. annealing and extension under general conditions.”
Appropriate correction is required.
Claim Rejections - 35 USC § 112(b) - Indefiniteness
5. 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 6, 51, 53 and 59 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 6 is indefinite because it is unclear as to how this claim is intended to further limit the method of claim 1 and it is unclear as to what is encompassed by this claim. Claim 6 recites “wherein the difference between A….; and B. of at least 0.50° C…that is methylated instead of unmethylated in said target nucleic acid sequence of interest, wherein the difference is A-B and wherein the difference is a positive difference.” It is unclear as to whether the claim intends to recite “wherein the difference…is at least 0.50°C” rather than “wherein the difference …of at least 0.50° C.” It is also unclear as to what is meant by “however which does not comprise said hydrophobic nucleotide(s), when hybridized to said target nucleic acid sequence of interest” and “however which does not comprise said hydrophobic nucleotide(s), when hybridized to said target nucleic acid sequence of interest. “
Claim 6 is further indefinite over the recitation of “such as at least 0.75 preferably at least 1.0°C per NOI, such as per cytosine, that is methylated instead of unmethylated in said target nucleic acid sequence of interest. It is unclear as to what the “per cytosine” and “that is methylated instead of unmethylated” are referring back to. Further, the phrases "such as" and “preferably” render the claim indefinite because it is unclear whether the limitations following the phrases are part of the claimed invention. See MPEP § 2173.05(d).
Claim 51, and thereby dependent claims 53 and 59, are indefinite because claim 51 recites (N)q but does not define what constitutes “q.” Claim 53 is further indefinite because the claim defines “p” as an integer > 1. However, claim 51, from which claim 53 depends defines “p” as an integer > 0. It is confusing for claim 53 to define “p” in a manner that is not consistent with claim 51, from which it depends, while relying on claim 51 to define “n” and “m”, as well as “N”, “H” and “G”.
Claim Rejections - 35 USC § 112(d) / Fourth paragraph
6. The following is a quotation of 35 U.S.C. 112(d):
(d) REFERENCE IN DEPENDENT FORMS.—Subject to subsection (e), a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.
The following is a quotation of pre-AIA 35 U.S.C. 112, fourth paragraph:
Subject to the following paragraph [i.e., the fifth paragraph of pre-AIA 35 U.S.C. 112], a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.
Claims 26 and 27 are rejected under 35 U.S.C. 112(d) or pre-AIA 35 U.S.C. 112, 4th paragraph, as being of improper dependent form for failing to further limit the subject matter of the claim upon which it depends, or for failing to include all the limitations of the claim upon which it depends.
Claims 26 and 27 depend from subsequent claim 72. As set forth in MPEP 608.01(n), the test for proper dependency requires that a dependent claim references a claim previously set forth:
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Herein, claims 26 and 27 depend from a subsequent / latter presented claim rather than “a claim previously set forth.”
Accordingly, claims 26 and 27 are not in proper dependent form.
Applicant may cancel the claim(s), amend the claim(s) to place the claim(s) in proper dependent form, rewrite the claim(s) in independent form, or present a sufficient showing that the dependent claim(s) complies with the statutory requirements.
Claim Rejections - 35 USC § 112 - Enablement
6. 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, 2, 4-6, 8, 13, 16, 20-21, 26-27, 37, 40, 43, 47, 51, 53, 59, 72 and 73
are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, because the specification, while being enabling for methods wherein the epigenetic modification is methylation and wherein in step “b” the temperature is higher than that between the oligonucleotide and the target nucleic acid of interest when the NOI is not methylated does not reasonably provide enablement for methods which determine any epigenetic modification status or methods wherein in step “b” the temperature is higher than that between the oligonucleotide and the target nucleic acid of interest when the NOI is “unmodified” in any manner. The specification does not enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the invention commensurate in scope with these claims.
The following factors have been considered in formulating this rejection (In re Wands, 858F.2d 731, 8 USPQ2d 1400 (Fed. Cir. 1988): the breadth of the claims, the nature of the invention, the state of the prior art, the relative skill of those in the art, the predictability or unpredictability of the art, the amount of direction or guidance presented, the presence or absence of working examples of the invention and the quantity of experimentation necessary.
The claims are drawn to a method for determining the epigenetic modification status of at least one nucleotide of interest (NOI) in a non-modified target nucleic acid sequence of interest and includes a step of “Incubating said oligonucleotide with said target nucleic acid of interest at a temperature which is higher than the melting temperature between said oligonucleotide and the target nucleic acid sequence of interest when said NOI is unmodified.”
The specification (para [0103]; para numbering herein is with respect to the published application) defines the term “non-modified” as follows:
“The term “non-modified” as used herein refers to that a nucleic acid of interest has not undergone a step of modification or pre-treatment to convert unmethylated nucleotides to detectable moieties and/or to convert methylated nucleotides to detectable moieties. Examples of such modifications or pre-treatments include bisulfite conversion, restriction enzyme digestion or TET enzymatic conversion. Non-modified as defined herein thus only refers to the absence of nucleic acid treatments or modifications that act selectively on either methylated or unmethylated nucleic acids.”
However, the specification does not provide a limiting definition in the specification for “unmodified” as recited at step “b.” of claim 1.
Accordingly, claim 1 encompasses methods wherein the temperature used in step “b.” is higher than the melting temperature for the oligonucleotide annealed to the target nucleic acid when the NOI is unmodified in any unspecified manner. Such unmodified NOIs can include NOIs in which the nucleotide is not different or the nucleotide does not have a fluorescent label attached to it or the nucleotide does not have a radioactive label attached to it.
Further, the specification (para [0102] teaches that the epigenetic modification can be any “covalent modification of a nucleobase, which typically is inherited to daughter cells.” The specification teaches that while the epigenetic modification is preferably a methylation, “it could also be alkylation, acetylation, hydroxylation, methoxylation or other modifications of the nucleobases” (para [0102]).
However, all the examples and guidance provided in the specification are limited to methods wherein the epigenetic modification is methylation and the “unmodified” NOI in step “b” is an unmethylated NOI.
The specification exemplifies methods wherein the presence of the intercalator in the anchor sequence of the oligonucleotide increases the difference in melting temperature between the methylated and non-methylated target nucleic acid bound to the oligonucleotide.
The specification (para [0155]) teaches:
In particular, the invention is based on the finding that the oligonucleotides described herein have a larger difference in affinity to methylated versus unmethylated target nucleic acid compared similar oligonucleotides not comprising hydrophobic nucleotides. This larger difference in affinity allows for successful detection of methylation by simple methods, such as PCR. The difference in melting temperature between the oligonucleotide of the invention and the methylated target nucleic acid sequence compared to an unmethylated target nucleic acid sequence of interest is preferably at least 1° C. higher than the difference in melting temperature between an oligonucleotide of identical sequence except lacking the hydrophobic nucleotides and the methylated target nucleic acid sequence compared to an unmethylated target nucleic acid sequence.
The specification (para [0334]) also teaches:
“The BasePrimer approach is based on a selective amplification of methylated DNA using hydrophobic nucleotides. The discrimination can be realized by a single primer, herein named a BasePrimer. A BasePrimer consists of three parts: an anchor, a loop, and a starter sequence. The anchor sequence contains the hydrophobic nucleotides. The anchor sequence has higher thermal stability when bound to methylated DNA compared to unmethylated DNA. Thus, the anchor sequence is used to create stability for the starter sequence that is placed in the 3′-end of the primer. The starter sequence should have low thermal stability, and therefore not bind to the DNA template if it does not get support from the anchor sequence (FIG. 4B). The PCR will only start if the anchor sequence binds with high thermal stability, thus allowing the starter sequence to bind (FIG. 3A, “Cycle 1”). Note that the amplicons created during the PCR will not contain any methylated nucleotides as generally the polymerase adds dNTPs with non-methylated nucleotides in PCR reactions.”
Thus, the specification teaches that methylation of the NOI, and particularly cytosine, increases the thermal stability of the oligonucleotide containing the intercalator bound to the target nucleic acid sequence comprising the methylated NOI as compared to the target nucleic acid comprising the unmethylated NOI. The specification does not teach that the presence of other types of epigenetic modification, including alkylation, acetylation, hydroxylation, methoxylation or other modifications of the nucleobases that are inherited by daughter cells, also confer a change in thermal stability that permit the oligonucleotide to anneal to the target nucleic acid of interest at a higher temperature than the melting temperature between the oligonucleotide and the target nucleic acid of interest in which the NOI is unmodified in an unspecified manner.
The specification does not provide any guidance as to how to perform the claimed methods to detect other types of epigenetic modifications in target nucleic acids other than the epigenetic modification of methylation. Further, the specification does not provide any guidance as to how to perform step “b” of claim 1 wherein the temperature is higher in comparison to a melting temperature that melts the oligonucleotide when bound to a target nucleic acid of interest when the NOI is unmodified in a manner that is not methylation (i.e., when the NOI is not unmethylated).
Absent any information in the specification regarding the effect of other types of epigenetic modification, including alkylation, acetylation, hydroxylation, methoxylation or other inherited modifications on the thermal stability of the hybridization products, it is unpredictable as to whether the claimed method can be performed to detect any type of epigenetic modification in a target nucleic acid of interest.
Case law has established that “(t)o be enabling, the specification of a patent must teach those skilled in the art how to make and use the full scope of the claimed invention without ‘undue experimentation.’” In re Wright 990 F.2d 1557, 1561. In re Fisher, 427 F.2d 833, 839, 166 USPQ 18, 24 (CCPA 1970) it was determined that “(t)he scope of the claims must bear a reasonable correlation to the scope of enablement provided by the specification to persons of ordinary skill in the art”. The amount of guidance needed to enable the invention is related to the amount of knowledge in the art as well as the predictability in the art. Furthermore, the Court in Genetech Inc. v Novo Nordisk 42 USPQ2d 1001 held that “(I)t is the specification, not the knowledge of one skilled in the art that must supply the novel aspects of the invention in order to constitute adequate enablement”.
In the instant situation, given the lack of specific teachings in the specification and the lack of guidance provided in the specification and prior art, it would require undue experimentation for one of skill in the art to make and use the broadly claimed invention.
7. The prior art made of record and not relied upon is considered pertinent to applicant's disclosure.
Christensen, U.B. (U.S. 20100068704) discloses methods for detecting the methylation status (i.e., an epigenetic modification status) of a target nucleic acid, the methods comprising providing an oligonucleotide that consists of a sequence that is complementary to the target nucleic acid and includes a nucleotide complementary to a nucleotide of interest (NOI), as well as a hydrophobic nucleotide that is positioned between the nucleotide complementary to the NOI and the nucleotide immediately 5’ thereof; incubating the oligonucleotide with a target nucleic acid at a temperature that is higher than the melting temperature of a complex formed between the oligonucleotide and the target nucleic acid having an unmethylated NOI; and detecting hybridization of the oligonucleotide to the target nucleic acid as indicative of methylation of the NOI, wherein the hydrophobic nucleotide has the structure of X-Y-Q, wherein X is a nucleotide or an analog of a nucleotide, Y is a linker and Q is an intercalator (e.g., para [0040-0041], [0062], [0143], [0605] In the method of Christensen, the target nucleic acid is treated with sodium bisulfite to convert non-methylated CpGs to dUpG (para [0107], [0137], and [0143]). Further, Christensen teaches that the oligonucleotides are “Stemless Beacons” (e.g., para [0604] and discusses the advantages of using oligonucleotides that do not form a protruding stem-like structure (e.g, para [0036], and [0104]). Christensen does not teach or suggest the presently claimed methods which require that the target nucleic acid is “non-modified” - i.e., not treated to modify methylated or unmethylated nucleotides (and particularly not treated with sodium bisulfite) and which require that the oligonucleotide further includes, 3’ to the above sequence comprising the hydrophobic nucleotide (herein “An”), a loop sequence (Lp), which is not complementary to the target nucleic acid sequence and which consists of a single nucleic acid sequence capable of forming a protruding structure, or consists of two or more nucleic acid sequences capable of hybridizing at least partly to one another to form a complex which is capable of forming a protruding structure, and a starter sequence (St), capable of hybridizing to a target starter sequence, wherein the target starter sequence is a sequence of the target nucleic acid sequence positioned 5' to the target anchor sequence. As disclosed in the present specification, the claimed method provides increased discrimination between methylated and unmethylated nucleotides and the ability to distinguish between methylated and unmethylated nucleotides using the oligonucleotide as a primer in a PCR assay.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to CARLA J MYERS whose telephone number is (571)272-0747. The examiner can normally be reached M-Th 6:30-5:00 EST.
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If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, Wu-Cheng Winston Shen can be reached on 571-272-3157. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
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/CARLA J MYERS/Primary Examiner, Art Unit 1682