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and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114.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 5/4/2026 has been entered.
Applicant’s arguments have been thoroughly reviewed and considered. Claim 8 has been canceled. Claims 1-2, 4-5, 7, 12, 15-16, 20, 28-36, and 38 are pending and are examined on their merits herein.
Information Disclosure Statement
The information disclosure statement (IDS) submitted on 5/8/2026 is in compliance with the provisions of 37 CFR 1.97. Accordingly, the information disclosure statement is being considered by the examiner.
Response to Applicant’s Amendments
Claim Objections
Claims 1, 8, and 32 were objected to for minor informalities. Claim 8 has been canceled, so this rejection has been rendered moot. In light of Applicant’s amendments to the claims submitted 5/4/2026, the objections for claims 1 and 32 have been withdrawn.
35 USC 112(b) Rejections
Claims 1-2, 4-5, 7-8, 12, 15-16, 20, 28-36, and 38 were rejected for various indefiniteness issues. In light of Applicant’s amendments to the claims submitted 5/4/2026, these rejections have been withdrawn for all currently pending claims. Claim 8 has been canceled, so this rejection has been rendered moot. See also new grounds of rejection below.
35 USC 103 Rejections
Claims 1-2, 4-5, 7-8, 12, 15-16, 20, 28-30, and 33-34 were rejected under 35 U.S.C. 103 as being unpatentable over Christians et al. (US 2017/0275691 A1), in view of Nodine (WO 2018/138334 A1), and in view of Mercer (US 2018/0148778 A1).
Claims 31-32, 35-36, and 38 were rejected under 35 U.S.C. 103 as being unpatentable over Christians et al. (US 2017/0275691 A1), in view of Nodine (WO 2018/138334 A1), in view of Mercer (US 2018/0148778 A1), and further in view of Wu et al. (US 10,465,232 B1).
In light of Applicant’s amendments to the claims submitted 5/4/2026, these rejections have been withdrawn for all currently pending claims. Claim 8 has been canceled, so this rejection has been rendered moot. See new grounds of rejection and “Response to Applicant’s Arguments” below.
Response to Applicant’s Arguments
Applicant has provided arguments against the 35 USC 103 Rejections presented in the Final Rejection mailed 1/15/2026, namely that the cited references do not teach steps (h)-(j) of newly amended claim 1 (Remarks, pages 9-10).
While Christians, the primary reference recited, does teach the measurement of disease levels over time (e.g. para. 282), this does not appear to be related to minimal residual disease. Mercer, also cited in the Final Rejection, does mention minimal residual disease (e.g. para. 333), but does not appear to actively measure this value. Thus, the previously presented 35 USC 103 Rejections have been withdrawn for all currently pending claims, and new grounds of rejection are provided below.
Claim Rejections - 35 USC § 112(b)
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, 4-5, 7, 12, 15-16, 20, 28-36, and 38 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 recites the limitations "the target sample" and “the first target sequence” in lines 1 and 4 of step (a). There is insufficient antecedent basis for these limitations in the claim, as “a target sample” and “a first target sequence” are not recited earlier in the claim.
Additionally, in line 1 of step (j), the phrase “the subject” also lacks antecedent basis, as “a subject” is not recited previously in the claim.
Additionally, the claim is rejected because steps (g) and (h) are unclear with regard to the sample used. Step (g) requires repeating steps (a) through (e) of the method, but does not recite that this occurs in a second nucleic acid sample, meaning that this repetition may occur in the same, initial sample. This is then supported by the use of “the nucleic acid sample” in step (h). However, using the same sample would involve adding additional first spike-in sequences to the sample when spike-in sequences have already been added, and where initial spike-ins and samples were already amplified and sequenced. It is thus unclear how using this same sample again would allow for detection of minimal residual disease as claimed. It is recommended the Applicant amend the claim to recite the use of first and second samples for a subject at the first and second time points, respectively.
Claims 2, 4-5, 7, 12, 15-16, 20, 28-36, and 38 are rejected based on their dependence on rejected claim 1.
It is noted that claims 12, 15-16, 20, 34, and 38 also recite “the nucleic acid sample,” and may need to be amended based on Applicant’s response to the rejections of claim 1.
Claim 20 is also indefinite because it depends on claim 8, which has been canceled. The scope of the claim is therefore unclear. The claim will be interpreted as though it depends on claim 1.
Claim Rejections - 35 USC § 112(d)
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.
Claim 20 is 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.
Claim 20 is rejected because as written, it depends on claim 8, which has been canceled. Therefore, claim 20 does not depend on a claim previously set forth, and so is of improper dependent forms. See MPEP 608.01(m) and (n).
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 § 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.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
Claims 1-2, 4-5, 7, 12, 15-16, 20, 28-30, and 33-34 are rejected under 35 U.S.C. 103 as being unpatentable over Christians et al. (US 2017/0275691 A1), in view of Nodine (WO 2018/138334 A1), in view of Mercer (US 2018/0148778 A1), and in view of Zimmerman et al. (US 2019/0316184 A1).
Regarding claims 1, 2, and 7, Christians teaches methods involving the addition of known concentrations of synthetic nucleic acids to a sample and performing sequencing assays (Abstract). These synthetic sequences may be spike-in sequences that have the same lengths and GC content as a target sequence (paras. 92-93). The spike-in sequences can have adapters (para. 143) and these adapters can be primer binding sites (para. 112), thereby indicating that the spike-in sequences can have primer binding sites. Christians also teaches that these adapters may be attached to target nucleic acids (para. 112). These targets may be from a human subject (paras. 6 and 39), where human nucleic acids can specifically be analyzed (paras. 100, 128, 147). Paras. 178-179 and 412 specifically note human genomic sequences can be mapped to, indicated that human genomic DNA can be used. Target nucleic acids may be amplified by PCR (para. 391), and qPCR may be used at any step in the method (para. 156). It would be obvious to the ordinary artisan to create the same primer binding site adapters on both the spike-in sequences and the target sequences so that only one set of PCR primers is needed, simplifying the amplification process. Sequencing methods and bioinformatics analysis of results can occur (para. 259). Christians recites comparing the input and output yield of spike-in sequences to the known output of sample sequences to determine sample input yield (paras. 183 and 199). This yield teaching is taught specifically in the context of quantified sequence reads for target and spike-in sequences (para. 410).
Specifically, Christians teaches that the human subjects analyzed can have or be at risk of having cancer (paras. 117-118), and notes that the target nucleic acid can derive from cancer tissue (para. 127). Sequencing analyses can specifically be associated with detecting cancerous or pre-cancerous variants (paras. 278 and 280). Para. 94 also teaches tracking nucleic acid loss in a sample over time, and para. 168 teaches measuring target nucleic acid abundance over time to identify disease states (para. 168). Para. 282 states that nucleic acid data can be taken at different times over the course of a treatment, in order to monitor and adjust said treatment as needed. Thus, Christians encompasses measuring target nucleic acid abundance in cancer patients over time, and particularly in conjunction with treatment protocols.
However, Christians does not specifically teach that the spike-in sequences have no more than 40 continuous nucleotides in common with the target sequence and that this continuous region is no more than half the length of the spike-in sequence, nor does this reference teach that the spike-in sequences are randomly generated or the result of segmenting and rearranging the first target sequence. Additionally, Christians does not teach methods of creating and evaluating error distributions, nor does the reference teach detecting the amount of minimal residual disease in a sample.
Regarding claim 1, Nodine teaches methods related to spike-in oligonucleotides (Abstract). Specifically, Nodine teaches that these spike-in sequences should have a core sequence of at least 8 nucleotides that have mismatches compared to the target sequence (page 5, lines 25-33). This core sequence is surrounded by at least three random nucleotides on each side. Preferably, the random nucleotides comprise 3-7 nucleotides (page 7, para. 2). Therefore, Nodine teaches an embodiment where spike-in sequences share less than 40 nucleotides with a target sequence and where those shared nucleotides are no more than half the length of the spike-in sequence.
Prior to the effective filing date of the claimed invention, it would have been prima facie obvious for one of ordinary skill in the art to use the spike-in sequence length design of Nodine, with a small core sequence corresponding to a target sequence surrounded by sequences that are non-complementary to the target sequence, in the method of Christians. This would be useful for shorter target sequences, as said targets would be more easily distinguished from the spike-in sequences during sequencing and analysis, preventing unclear results when counting sequence reads. There would be a reasonable expectation of success with this modification of the spike-in sequences of Christians because this reference teaches that spike-in sequences can be designed (para. 132), and does teach that these sequences may have portions that are random (para. 143).
However, Nodine does not teach that the spike-in sequences are randomly generated or the result of segmenting and rearranging the first target sequence. Additionally, Nodine does not teach methods of creating and evaluating error distributions, nor does the reference teach the measurement of minimal residual disease.
Regarding claims 1 and 33, Mercer teaches artificial controls for genetic sequencing and quantitation assays (Abstract). In one embodiment, Mercer teaches artificial chromosomes that are a shuffled or rearranged version of a template sequence (para. 123). This allows the artificial sequence to share “little or no sequence identity with any known, naturally occurring sequence, whilst retaining broader characteristics of nucleotide composition that are typical of the original known or natural sequence,” (para. 123). Mercer further teaches that, “Retaining high level nucleotide composition characteristics of a template sequence can be advantageous because sequence-specific features can bias the representation of natural genetic features in next-generation sequencing and analysis,” (para. 124). Mercer additionally teaches that artificial chromosomes may be constructed by reversing a template sequence (para. 126). The reference states, “By substituting nucleotides in a systematic manner, the repeat structure of the sequence can be maintained, the pyrimidine and purine composition can be maintained, and/or the GC content can be maintained, even though the individual nucleotides and the primary sequence may change,” (para. 127).
Prior to the effective filing date of the claimed invention, it would have been prima facie obvious for one of ordinary skill in the art to use the teachings of Mercer to design the spike-in sequences of Christians in view of Nodine. Specifically, this would lead the ordinary artisan to design the spike-in sequences to be rearranged or reversed versions of the target sequence. The ordinary artisan would be motivated to design the spike-in sequences in this manner so that the spike-in sequences and the target sequence could be distinguished from one another while still exhibiting the same overall features (such as pyrimidine and purine composition; Mercer para. 127). Therefore, the spike-in and target sequences are likely to be sequenced with the same efficiency. This would prevent issues associated with sequencing methods biasing towards spike-in or target products. There would be a reasonable expectation of success with this change to the spike-in design in the method of Christians in view of Nodine because Christians teaches that spike-in sequences can be chemically synthesized, and may contain natural or non-natural sequences (para. 146). Thus, these designed spike-in sequences are encompassed in the scope of Christians, and would therefore be compatible with the described methods.
Further regarding claim 1, Mercer teaches error distributions related to sequencing errors. Specifically, Figures 19-21 show analyzing sequencing data to identify sequencing errors, Figure 29B shows the rate of mismatches (i.e. sequencing errors) across sequencing reads (see para. 341), Figure 30A-B shows correct versus erroneous variant calls given particular allele frequencies, Figure 41A-B shows measures of sequencing depth, and Figure 42B-C compares allelic variants to references. Para. 199 of the reference discusses sequencing errors and how to identify them, as well as how this analysis may be useful. In particular, Mercer states, “This analysis also then allows a researcher to normalize or correct systematic sequencing errors within reads from the sample DNA/RNA, providing a far more accurate (both qualitatively and quantitatively) measurement of the target DNA/RNA of interest in the sample. The sequencing error profile of the polynucleotide standards can also be employed to distinguish sequencing errors from genuine nucleotide differences (such as SNPS or nucleotide modifications).” This provides evidence that sequencing error analysis and correction can determine when mismatches are due to SNPs or variants as opposed to true error. Para. 441 discusses false-positive variant calls that are created by sequencing errors and how to further filter these. Specifically, a p-value threshold was developed that predicts a true or false variant call for a particular fraction of a sample at a given sequencing depth (which is shown in Figure 42C). Furthermore, Mercer teaches plotting sequencing depth in genotyping contexts, where a limit of detection is noted and indicates a “confidence with which SNPs are identified,” (Figure 7C and paras. 69 and 377). Figure 26B notes the accuracy of splicing of RNA standard above a user defined threshold (para. 315).
Prior to the effective filing date of the claimed invention, it would have been prima facie obvious for one of ordinary skill in the art to use these additional teachings of Mercer in the method of Christians, in view of Nodine, and in view of Mercer described above to arrive at the invention of instant claim 1. In particular, Mercer teaches that by examining sequencing errors, one can determine whether perceived mismatches are due to actual mutations in the target nucleic acids, or due to methodology errors. This in itself would be motivating to the ordinary artisan, as the difference between a true or false variant could have implications for the use of the method in diagnostics. Furthermore, in order to meaningfully interpret the sequencing error data, Mercer recognizes that a threshold or limit for confidence is required, that way the sequencing error determinations have a degree of accuracy. Mercer teaches such thresholds in terms of p-values (associated with a single sequencing depth) or a threshold for a variety of sequencing depths. In considering the method of Christians, in view of Nodine, and in view of Mercer already established above, the ordinary artisan would recognize that finding a threshold for a variety of sequencing depths would have more utility, as the sequencing depth obtained for targets and spike-ins may depend on the particular target sequence used and the particular spike-in created. Thus, combining these teachings of Mercer with the method of Christians, in view of Nodine, and in view of Mercer would provide an analysis that is widely applicable to different targets and allows more accurate conclusions to be drawn from sequencing results. There would be a reasonable expectation of success as Mercer teaches all the aspects of this analysis, showing that they would be possible for the ordinary artisan, and these teachings only add to downstream portions of the method, and would not interfere with the actual manipulation (e.g. any extraction, amplification, and sequencing reactions) of targets and spike-ins.
However, though Mercer recites the use of cancer samples (e.g. paras. 88 and 320), and notes minimal residual disease as a potential application for use with their teachings (paras. 171 and 333), Mercer does not actually teach the calculation of such a value.
Zimmerman teaches the detection of target nucleic acids in cancer samples utilizing methods involving amplification and sequencing (Abstract). The reference also teaches the use of spike calibrators for absolute quantification of nucleic acids (paras. 659 and 812-815). Paras. 665-666 also teach the use of standards related to cancer mutations that can be spiked into polynucleotide samples. In para. 810, Example 3 performs methods to detect minimal residual disease after curative intent treatment, utilizing methods that involve spike-in sequences. Example 8 (starting with para. 837) describes the utility of measuring and detecting variants in nucleic acids while monitoring minimal residual disease following breast cancer treatment (paras. 837-839). This workflow measures disease progression over time by measuring target nucleic acids over time (see Results in paras. 861-875). Paras. 866-867 specifically note the analysis of markers at different time points. Paras. 876-885 describe the utility of detecting MRD in patients with breast cancer in order to monitor disease progression.
Prior to the effective filing date of the claimed invention, it would have been prima facie obvious for one of ordinary skill in the art to use the teachings of Zimmerman to guide the method of Christians, in view of Nodine, and in view of Mercer to use the longitudinal measurements in cancer patients to determine the presence of minimal residual disease in a patient. Christians generally teaches taking longitudinal measurements in association with treatment, and makes clear the use of their methods in cancer patients. As minimal residual disease can affect patient prognosis and treatment plans in the context of cancer, using the teachings of Zimmerman with Christians, in view of Nodine, and in view of Mercer would be motivating to the ordinary artisan, so that potential relapses can be caught early and/or the efficacy of treatment plans can be analyzed and adjusted if need be. This would overall improve patient outcomes on short and long-term scales. There would be a reasonable expectation of success as the methods of Christians, in view of Nodine, and in view of Mercer already teach quantifying target molecules at different time points, and adding minimal residual disease analysis would simply be an additional analysis step that can be successfully performed in this context, as evidenced by Zimmerman.
Therefore, claims 1-2, 7, and 33 are prima facie obvious over Christians, in view of Nodine, in view of Mercer, and in view of Zimmerman.
Regarding claims 4-5, Christians, in view of Nodine, in view of Mercer, and in view of Zimmerman teaches the methods of claims 1-2, as described above. Mercer also teaches that their rearranging/shuffling methods can involve windows. This reference teaches that the window for shuffling a template sequence can correspond to a fixed nucleotide length, and gives examples from 10-1000 or more nucleotides (para. 125). Christians teaches that both the target nucleic acid and the spike-in sequences may be at least 20 to at least 500 nucleotides in the case of the spike-in sequence, with even longer lengths permitted for the target sequences (paras. 129 and 137). These lengths would mean that the rearranging of the target sequence could occur with at least three segments, given the length requirements recited in Mercer para. 125. This would generate rearranged nucleic acid sequences where the nucleic acids within each window were rearranged, but would not involve rearrangement of the windows themselves, as taught in claims 4-5.
Mercer also teaches that the shuffling and reversing techniques can be applied “in any combination or permutation” (para. 128). The reference also teaches that in order to ensure that there is no sequence similarity between an artificial and template sequence, additional editing can be done to sequences (para. 130). Mercer also teaches translocation to rearrange chromosomes (paras. 71 and 150), which would remove or relocate segments of a sequence.
Thus, it would be prima facie obvious for one of ordinary skill in the art to not only rearrange nucleotides within windows as taught in Mercer, but to rearrange the windows themselves in the method of Christians, in view of Nodine, in view of Mercer, and in view of Zimmerman. If a particular window contains a string of a single nucleotide or a repeat sequence, then even if rearranged, that portion of the sequence will likely still have similarity to the target sequence. Thus, the ordinary artisan would recognize that additional editing could be done to specifically move the windows to different locations within the artificial sequence, in order to ensure there is no similarity to the target sequence. As Mercer already teaches translocations (e.g. the movement of segments of a sequence) between two sequences, the ordinary artisan could similarly use ordinary creativity to move segments within a single sequence. By reordering the windows as much as possible from one another, this would also ensure diversity between the spike-in and target sequences.
Therefore, claims 4-5 are prima facie obvious over Christians, in view of Nodine, in view of Mercer, and in view of Zimmerman.
Regarding claim 12, though Christians does teach graphing sequence reads (e.g. Figure 9), this reference does not teach graphing the reads using a standard curve.
Nodine does teach using standard curves to map relative and absolute amounts of spike-in molecules (Figure 5, page 9, Figure 5 description, Figure 1b, and pages 36-37). Figure 5 particularly shows standard curves for multiple replicates (page 9, Figure 5 description). Nodine also teaches using these curves to predict the number of target wild type molecules present in a sample using linear modeling (page 37, para. 1).
Prior to the effective filing date of the claimed invention, it would have been prima facie obvious for one of ordinary skill in the art to use this standard curve methodology in the method of Christians, in view of Nodine, in view of Mercer, and in view of Zimmerman described above. Said method already involves quantifying the amount of target sequence present in a sample using the known input and output yields of the spike-in sequences, and this standard curve method provides a specific, graphical way of doing this. By additionally using linear modeling, any error can additionally be quantified, allowing the ordinary artisan to determine how reasonable their results and conclusions are. It is noted that this would be done for each of the time points of Christians, in view of Nodine, in view of Mercer, and in view of Zimmerman, as quantification of targets in this method is done on a time point basis.
Therefore, claim 12 is prima facie obvious over Christians, in view of Nodine, in view of Mercer, and in view of Zimmerman.
Regarding claim 15, as noted above, Christians teaches that multiple spike-in sequences can be used, and specifically teaches up to 10 such sequences (para. 152). Christians also teaches that spike-in sequences can be added at varying concentrations (para. 151). Prior to the effective filing date of the claimed invention, it would have been prima facie obvious for one of ordinary skill in the art to use multiple spike-in sequences at different concentrations with the same primer binding sequences, as specified in claim 15. One set of primer binding sequences would mean that only one set of PCR primers is required, which would equate to less materials needed to perform the method and a potentially more efficient protocol, as only one set of thermocycling conditions would be required.
Therefore, claim 15 is prima facie obvious over Christians, in view of Nodine, in view of Mercer, and in view of Zimmerman.
Regarding claim 16, as stated above in the rejection of claim 12, though Christians does not teach graphing the reads using a standard curve, Nodine does teach this using standard curves to map relative and absolute amounts of spike-in molecules (Figure 5, page 9, Figure 5 description, Figure 1b, and pages 36-37). This reference also teaches using these curves to predict the number of target wild type molecules present in a sample using linear modeling (page 37, para. 1). In particular, the example of pages 36-37 deals with eight distinct spike-in sequences.
Prior to the effective filing date of the claimed invention, it would have been prima facie obvious for one of ordinary skill in the art to use this standard curve methodology in the method of Christians, in view of Nodine, in view of Mercer, and in view of Zimmerman described above. Said method already involves quantifying the amount of target sequence present in a sample using the known input and output yields of the spike-in sequences, and this standard curve method provides a specific, graphical way of doing this. By using linear modeling, any error can be quantified, allowing the ordinary artisan to determine how reasonable their results and conclusions are. Additionally, Nodine shows that this method is compatible with multiple spike-in sequences, giving the ordinary artisan a reasonable expectation of success in performing this analysis.
Therefore, claim 16 is prima facie obvious over Christians, in view of Nodine, in view of Mercer, and in view of Zimmerman.
Regarding claim 20, Christians specifically teaches that gene expression may be quantified through detection or sequencing of one or more target nucleic acids of interest (para. 285).
Regarding claim 28, Christians teaches that the spike-in sequence can contain random sequences (para. 143).
Regarding claims 29-30, Christians teaches that spike-in sequences can have GC contents and/or lengths that match those of the target sequences (para. 93).
Regarding claim 34, Christians teaches that at any point in the method, qPCR can be used to quantify sample loss (para. 156). It would therefore be obvious to one of ordinary skill in the art that qPCR could also be used to quantify the amount of nucleic acid originally present in the sample, in order to compare later quantitative measurements to this original value and determine if sample loss is occurring. This would be useful because if sample loss is occurring during the method, it may indicate issues with equipment or reagents, and would produce faulty results.
Therefore, the method of claim 34 is prima facie obvious over Christians, in view of Nodine, in view of Mercer, and in view of Zimmerman.
Claims 31-32, 35-36, and 38 are rejected under 35 U.S.C. 103 as being unpatentable over Christians et al. (US 2017/0275691 A1), in view of Nodine (WO 2018/138334 A1), in view of Mercer (US 2018/0148778 A1), in view of Zimmerman et al. (US 2019/0316184 A1), and further in view of Wu et al. (US 10,465,232 B1).
Regarding claims 31-32, as stated above, Christians teaches that the spike-in sequences can contain random sequences (para. 143). Christians also teaches that spike-in sequences may be different to the target sequence (i.e. would not align to the target sequence; para. 150). However, Christians does not teach about the secondary structure of the spike-in sequences relative to the target sequence, or about amplification efficiency.
Nodine teaches that spike-in sequences can have varying sequencing efficiencies due to their different secondary structures, but does not teach about amplification efficiency (page 36, para. 2).
Zimmerman does not provide any teachings regarding nucleic acid secondary structure, and though the reference does provide information about bias in amplification efficiencies (see paras. 281, 343, 345, and 354, for example), this is generally in the context of correcting for bias in such efficiencies, and is not related to designing sequences while keeping amplification efficiency in mind.
Wu teaches methods for quantifying the efficiency of nucleic acid extraction from a sample via the use of spike-in sequences (Abstract). The spike-in sequences can be selected based on multiple attributes – namely secondary structure and ability to amplify by PCR (column 12, para. 3).
Prior to the effective filing date of the claimed invention, it would have been prima facie obvious for one of ordinary skill in the art to use the teachings of Wu in the invention of Christians, in view of Nodine, in view of Mercer, and in view of Zimmerman to arrive at the invention of claims 31-32. Specifically, the teachings of Wu regarding choosing spike-in sequences for their secondary structure would lead the ordinary artisan to look for this quality when choosing spike-in sequences that are random and different to the target region (i.e. a human genomic sequence, as taught by Christians). Wu teaches that part of the design process for spike-in sequences can involve testing the functionality of desired attributes (page 38, para. 3), and so this would motivate the ordinary artisan to test out multiple designs and choose those which work best for their purposes. This also relates to Wu’s teachings regarding the ability of spike-in sequences to amplify via PCR. The ordinary artisan would be motivated to choose spike-in sequences that have similar amplification efficiency to the target sequence so that there would be an expectation of similar yields for both the spike-in and target sequences after PCR is complete. If the spike-in sequence did not amplify well under conditions that the target sequence did amplify well, then it would be difficult to obtain spike-in sequence reads and use the associated data to quantify the amount of target sequence. There would be a reasonable expectation of success when designing spike-in sequences and testing them for desired attributes because this is a well-known practice in the art, as evidenced by Wu.
Therefore, the methods of claims 31-32 are prima facie obvious over Christians, in view of Nodine, in view of Mercer, and in view of Zimmerman, and further in view of Wu.
Regarding claims 35-36, the instant specification does not provide a specific definition for “amplification efficiency.” However, page 30, para. 5 states, “The spike is [designed] to have high similarity between the GC content, length and features of it and the genomic region of interest giving rise to a highly similar, if not identical, amplification efficiency in the PCR reaction.”
Christians teaches that spike-in sequences can have GC contents and/or lengths that match those of the target sequences (para. 93). Additionally, the spike-in sequences of Christians, in view of Nodine, in view of Mercer, and in view of Zimmerman have the same distribution of nucleotides as in the target sequences, as noted above. Wu additionally provides reason to examine amplification efficiency, as noted in the rejection of claims 31-32 above.
Therefore, if the spike-in and target sequences of Christians, in view of Nodine, in view of Mercer, and in view of Zimmerman have the same nucleotides, GC content, and length, they should have highly similar or identical amplification efficiencies. Wu further teaches examining amplification efficiencies, and so it would be possible for the ordinary artisan to design the spike-in sequences to ensure that they have identical amplification efficiencies to target sequences. The ordinary artisan would be motivated to choose spike-in sequences that have identical amplification efficiencies to the target sequences so that there would be an expectation of the same yields for both the spike-in and target sequences after PCR is complete. If the spike-in sequence did not amplify well under conditions that the target sequence did amplify well under, then it would be difficult to obtain spike-in sequence reads and use the associated data to quantify the amount of target sequence. There would be a reasonable expectation of success when designing spike-in sequences and testing them for desired attributes because this is a well-known practice in the art, as evidenced by Wu.
Therefore, claims 35-36 are prima facie obvious over Christians, in view of Nodine, in view of Mercer, in view of Zimmerman, and further in view of Wu.
Regarding claim 38, as noted above, para. 138 of Christians teaches that spike-in sequences can be used to measure yield for target sequences. Specifically, this paragraph teaches, “comparing a known input with a measured output of the spike-ins can enable inferences of unknown input (e.g., in the sample) with its measured output.” As noted above in para. 36 and the rejection of claims 35-36, it would be prima facie obvious to design the spike-in and target sequences to have the same amplification and sequencing efficiencies. Using these teachings together, this means that the ratio of the known amount of spike-in added to the number of spike-in reads would equal the ratio of the amount of target sequence added to the number of target reads. In solving for the amount of target added, the equation of instant claim 38 would be generated.
Therefore, claim 38 is prima facie obvious over Christians, in view of Nodine, in view of Mercer, and in view of Zimmerman, and further in view of Wu.
Conclusion
No claims are currently allowable.
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/FRANCESCA FILIPPA GIAMMONA/Examiner, Art Unit 1681
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