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 .
Claim Status and Formal Matters
This action is in response to papers filed 7/24/2025.
Claims 1, 4-8 have been amended.
Claims 10-11 have been added by amendment.
Applicant’s election of group I in the reply filed on 2/26/2025 is acknowledged. Because applicant did not distinctly and specifically point out the supposed errors in the restriction requirement, the election has been treated as an election without traverse (MPEP § 818.01(a)).
Claims 9 withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected invention, there being no allowable generic or linking claim. Election was made without traverse in the reply filed on2/26/2025.
Applicant's election with traverse of a first and second signal in the reply filed on 2/19/2025 is acknowledged. The traversal is on the ground(s) that the response traverses the rejection asserting there is no serious search burden. This is not found persuasive because searching a method which requires detecting a first signal in one well and a second signal in a different well has a different design, does not overlap in scope and is not an obvious variant of methods requiring both signals in a single well.
Claim 9 withdrawn from further consideration pursuant to 37 CFR 1.142(b), as being drawn to a nonelected species, there being no allowable generic or linking claim. Applicant timely traversed the restriction (election) requirement in the reply filed on 2/26/2025.
Claims 1, 4-8, 10-11 are being examined.
The prior art rejections have been withdrawn.
Priority
The instant application was filed 06/09/2022 and is a continuation of PCT/JP2020/045438 , filed 12/07/2020 and claims foreign priority to JP2019-222153, filed 12/09/2019. The foreign priority document is not in English.
Information Disclosure Statement
The response request reconsideration of the IDS of 8/20/2024 as the response asserts there is an English translation of the abstracts of the Chinese applications. However, review of the applications did not reveal any English translation.
Claim Objections
Claims 1, 4-8, 10-11 objected to because of the following informalities:
Claim 1 has been amended to recite, “A method for detecting a target nucleic acid in a biological sample, the method comprising: bringing liquid comprising a target nucleic acid into contact with a well array having a plurality of wells such that at most one molecule of the target nucleic acid is included per well; wherein the target nucleic acid comprises a first single-stranded nucleic acid, a second single-stranded nucleic acid, a double stranded nucleic acid resulting from complementary binding of the first single-stranded nucleic acid and the second single-stranded nucleic acid, or a combination thereof: sealing the wells such that the target nucleic acid remains in the [[well]] wells; amplifying, in the wells,signals derived from the target nucleic acid; wherein the signals derived from the target nucleic acid in the amplifying comprises a first signal emitted by binding of a first specific binding substance to the first single-stranded nucleic acid, and the second signal different from the first signal and emitted by binding of a second specific binding substance to a single-stranded second nucleic acid, wherein the first signal and the second signal are luminescence signals having different wavelengths: wherein the first specific binding substance comprises a nucleic acid sequence complementary to the first single-stranded nucleic acid, and the second specific binding substance comprises a nucleic acid sequence complementary to the second single-stranded nucleic acid: detecting the signals emitted from the wells; and detecting, based on the signal comprisesthe first single-stranded nucleic acid, the second single-stranded nucleic acid that is complementary to the first single-stranded nucleic acid, or a double-stranded nucleic acid resulting from complementary binding of the first single- stranded nucleic acid and the second single-stranded nucleic acid, wherein wells comprising the first single-stranded nucleic acid, wells comprising the second single-stranded second nucleic acid, and wells comprising the double-stranded nucleic acid, in which the first nucleic acid and the second nucleic acid are complementary bound, are individually detected; wherein the double-stranded nucleic acid has a double-strand when sealed within a well, wherein the amplifying the signals comprises conducting an invasive cleavage assay (ICA) reaction, and wherein the liquid comprises reagents for the ICA reaction, the method further comprises delivering the reagents for the ICA reaction contained in the liquid to the wells on a plate and sealing the wells with oil, or delivering the reagents and oil contained in the liquid to wells on a plate from a delivery port and filling the wells.” The claim is wordy confusing and awkward. For example referencing first binding substance is wordy when the claim also requires it is a nucleic acid.
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 1, 4-8, 10-11 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 has been amended to require, “ wherein the signals derived from the target nucleic acid in the amplifying comprises a first signal emitted by binding of a first specific binding substance to the first single-stranded nucleic acid, and the second signal different from the first signal and emitted by binding of a second specific binding substance to a single-stranded second nucleic acid, wherein the first signal and the second signal are luminescence signals having different wavelengths: wherein the first specific binding substance comprises a nucleic acid sequence complementary to the first single-stranded nucleic acid, and the second specific binding substance comprises a nucleic acid sequence complementary to the second single-stranded nucleic acid”. The claim later recites, “wherein the amplifying the signals comprises conducting an invasive cleavage assay (ICA) reaction, and wherein the liquid comprises reagents for the ICA reaction, the method further comprises delivering the reagents for the ICA reaction contained in the liquid to the wells on a plate and sealing the wells with oil, or delivering the reagents and oil contained in the liquid to wells on a plate from a delivery port and filling the wells.” Thus the claim is confusing and unclear as the claim appears to require the binding substance produces a signal and later requires invasive cleavage assay. Thus it is unclear how a signal is derived or generated.
The claim recites, “signals derived from the target nucleic acid in the amplifying comprises a first signal emitted by binding of first specific binding substance to the first single-stranded nucleic acid, and the second signal different from the first signal and emitted by binding of second specific binding substance to a single-stranded second nucleic acid, wherein the first signal and the second signal are luminescence signals having different wavelengths: wherein the first specific binding substance comprises a nucleic acid sequence complementary to the first single-stranded nucleic acid, and the second specific binding substance comprises a nucleic acid sequence complementary to the second single-stranded nucleic acid.” The recitation of “signals derived from the target nucleic acid in the amplifying” is vague, unclear, and incomplete as it is unclear how the signals is derived by binding.
The recitation of invasive cleavage assay suggests there are non-invasive cleavage assays. The specification provides no definition or standard to differentiate invasive relative to non-invasive cleavage assays. Thus the metes and bounds are unclear what is required.
The claim recites “a first specific binding substance to the first nucleic acid.” The recitation of “specific binding substance ” suggests there is “non-specific binding substances.” The specification provides no standard or definition on how to differentiation a specific binding substance from a non-specific binding substance.
The claim recites “a second specific binding substance to the second nucleic acid.” The recitation of “specific binding substance ” suggests there is “non-specific binding substances.” The specification provides no standard or definition on how to differentiation a specific binding substance from a non-specific binding substance.
The claim recites, “wherein the double-stranded nucleic acid has a double-strand when sealed within a well.” The metes and bounds are unclear how the artisan determines which double stranded double stranded nucleic acid is in the well before to allow selective sealing of only double stranded target nucleic acids or it the claim encompasses sealing all wells.
Claim 1 recites, “sealing the [[well]] wells such that the target nucleic acid remains in the [[well]] wells.” Claim 1 later recites, “wherein the liquid comprises reagents for the ICA reaction, the method further comprises delivering the reagents for the ICA reaction contained in the liquid to the wells on a plate and sealing the wells with oil, or delivering the reagents and oil contained in the liquid to wells on a plate from a delivery port and filling the wells.” The metes and bounds are unclear how or when the wherein clause relates to the first sealing step. Thus the claim is vague unclear and incomplete.
Response to Arguments
The response traverses the rejection in view of the amendments. This argument has been thoroughly reviewed but is not considered persuasive as the response has not addressed the issues and raised new issues.
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(s) 1, 4-8, 10-11 is/are rejected under 35 U.S.C. 103 as being unpatentable over White (White et al. Mobile DNA (2014) 5:30), , Diffenbach (PCR methods and Applications (1993) volume 3, pages S30-S37), Roux et al(PCR Methods and Applications (1995) volume 4, pages s185-s194) Wittwer (METHODS 25, 430–442 (2001)), McCoy (US20140274786)
With regards to claim 1, White teaches, “Droplet digital PCR (ddPCR) has recently emerged as a robust tool to provide precise measurements of nucleic acid target concentrations [14,15]. In ddPCR, input DNA is partitioned, along with PCR reagents, into approximately 20,000 droplets as a water-in-oil emulsion within a single thermocycled reaction well [16]. Detection of target DNA relies on fluorogenic probes in a 5’-nuclease assay (TaqMan™) [17,18]. Briefly, an oligonucleotide probe, which anneals specifically to a target DNA within the primer binding sites, is included in the PCR with the primers. The probe is modified at the 5’ end with a fluorescent moiety, which is quenched in the intact probe by a modification at the 3’ end with a quencher moiety. The probe anneals to the target DNA during the annealing/extension step of the PCR. During extension of the primer that anneals to the same DNA strand as the probe, the 5’ to 3’ nuclease activity of Taq polymerase cleaves the probe, which separates the 5’-fluorescent nucleotide of the probe from the 3’ quencher, generating a fluorescent signal. Sequestration of template DNA occurs in ddPCR, such that some droplets contain no copies and others one or more copies of the template target DNA [14,16]. Identification of template target DNA-containing droplets is achieved through fluorescence analysis of the droplets according to the 5’-fluorogenic probes used in the ddPCR. Droplets containing one or more target templates generate increased fluorescence compared to droplets containing non-target DNA. Thus, the quantification comes from the ability to essentially detect a single DNA template sequestered into a droplet through PCR amplification of the templates followed by counting of fluorescent droplets. The concentration of the input target DNA is calculated according to a Poisson distribution of template DNA molecules partitioned into the fluorescence-positive droplets [16].”
White teaches
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White does not specifically teach detection of the first strand and second strand in the same droplet or well.
However, Diffenbach teaches parameters and principles of promoter design include primer length, terminal nucleotide, GC content, melting temperature, PCR product length, and placement of target sequence (s30-s34). Diffenbach teaches PCR software was known (s35).
Roux teaches optimization of PCR by the presence of enhancing agents, Mg2+, annealing temperature, primer design, cycle number, hot start PCR (s185-s194).
Designing oligonucleotides to hybridize to specific targets, which are equivalents to those taught in the art is routine experimentation. The prior art teaches the parameters and objectives involved in the selection of oligonucleotides that function as primers and/or probes, see Diffenbach and Roux. The prior art is replete with guidance and information necessary to permit the ordinary artisan in the field of nucleic acid detection to design primers and probes. As discussed above, the ordinary artisan would be motivated to have designed and tested new primers to obtain additional oligonucleotides that function to detect both strands or nucleic acids as taught by White and identify oligonucleotides with improved properties. Thus, for the reasons provided above, the ordinary artisan would have designed additional oligonucleotides using the teachings in the art at the time the invention was made. The claimed mutations are obvious over the cited prior art, absent secondary considerations
Therefore it would have been prima facie obvious to one of skill in the art prior to the effective filing date of the claims to provide a single digital PCR reaction to detect both strands of the target nucleic acid. The artisan would be motivated to design primers and probes to detect each strand to eliminate the time and expense of requiring a PCR reaction to detect each strand individually. The artisan would have a reasonable expectation of success as the artisan is merely using known methods of primer and probe design.
With regards to claim 4, 6, 11 White teaches, “Because the two loci are not linked, each droplet has a probability of being positive for either one of the loci, and some droplets will be either negative or positive for both.”
With regards to claim 5, White teaches, “For each experiment, a threshold of fluorescence is set relative to negative controls to measure the quantity of droplets that do or do not contain template target DNA.” (page 3, 1st column, top).
While White teaches “Because the two loci are not linked, each droplet has a probability of being positive for either one of the loci, and some droplets will be either negative or positive for both.” White does not teach providing ratios.
However, McCoy teaches, “[0312] The method of any preceding paragraph, further comprising a step of processing the data such that a given partition is counted as positive for both targets if the data indicates the given partition contains both targets and is counted as negative for both targets if the data indicates the given partition contains only one or neither of the targets, wherein the step of determining a level is based on the processed data.”
With regards to claim to claim 7, McCoy teaches the amount of single-stranded template or the ratio of single-stranded to double- stranded template is inherently informative in many situations [0162].
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 provide analysis including rations of single stranded to double stranded nucleic acid as taught by McCoy. The artisan would be motivated to provide additional mathematical or statistical analysis to examine the target nucleic acids. The artisan would have a reasonable expectation of success as the artisan is using known methods.
McCoy does not specifically teach having melting temperatures within 10oC.
However, McCoy teaches, “0148] The discussion above assumes equal cleavage rates for a given number of extension events, but the melting temperature (T.sub.m) of the probe (bound to product) may cause that to differ. Distinguishable intensities may result by having a higher T.sub.m for one of the probes so the probability of cleavage with each extension step is higher. Alternatively, if the T.sub.m is lower, then less of the probe would be cleaved. Accordingly, different signal intensities may be produced and/or signal intensities may be adjusted by selecting probes having distinct melting temperatures.”
Further Wittwer provides a review of real time multiplex PCR. Wittwer teaches the second probe should be 5-10 degrees higher than the other probe. (page 433, 1st column, last paragraph).
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 the second probe to have a melting temperature of 5-10 degrees Celsius from the first probe. The artisan would be motivated as both McCoy and Witwer suggest having probes of different melting temperatures, while Witwer specifically suggests 5-10 degrees Celsius. The artisan would have a reasonable expectation of success as the artisan is merely designing probes as suggested by the art.
With regards to claim 10, White teaches, “. The 3’ end of the 3’ L1Hs primer makes use of an AC dinucleotide at position 5926 of L1Hs, which gives the primer specificity to only these youngest L1 elements. Thus, although the primer can probably anneal to a significant portion of genomic L1 elements, it will only be able to prime DNA synthesis from these actively mobile, and therefore most interesting, L1 elements. Additionally, the L1 3’-end probe makes use of a G nucleotide at position 6011 of L1Hs, making it also specific for only the youngest L1 elements.” Thus the target nucleic is between 10 to 1000 bases.
Summary
No claims are allowed.
Conclusion
Applicant's amendment necessitated the new ground(s) of rejection presented in this Office action. Accordingly, THIS ACTION IS MADE FINAL. See MPEP § 706.07(a). Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any nonprovisional extension fee (37 CFR 1.17(a)) pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to STEVEN C POHNERT PhD whose telephone number is (571)272-3803. The examiner can normally be reached Monday- Friday about 6:00 AM-5:00 PM, every second Friday off.
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/Steven Pohnert/Primary Examiner, Art Unit 1683