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 .
2. Applicant’s elected the following species without traverse in the reply filed on February 2, 2026:
Species 3a: dimethyl sulfoxide as the chaotropic compound in the hybridization agent;
Species 3b: the use of a dehybridization agent to remove the labeled oligonucleotide probes;
Species 3c: dimethyl sulfoxide as the chaotropic compound in the dehybridization agent;
Species 3d: a chelating agent in the hybridization agent;
Species 3e: a chelating agent in the dehybridization agent;
Species 3f: fluorescent dyes as the optical labels
After further consideration, the election of species requirements between the chaotropic compounds (species 3a and 3c) are withdrawn.
Claims 1-13, 15, 17, 20-22, 25-28, 30, 32, 34-35, 37, and 39 are currently pending.
Claims 9-10, 21, 26, and 37 are withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to nonelected species, there being no allowable generic or linking claim. Election was made without traverse in the reply filed on February 2, 2026.
Claim Rejections - 35 USC § 112
3. 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.
Claim 39 is 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 39 recites the limitation “the different set of labeled oligonucleotides probes” There is insufficient antecedent basis for this limitation in the claim because although the claim previously refers to “different types of labeled oligonucleotide probes” it does not refer to different sets. Further the claims are indefinite because it is unclear what is required to be present in the second composition. Clarification is requested.
Claim Rejections - 35 USC § 103
4. 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.
5. Claims 1-4, 7, 8, 11-13, 15, 17, 22, 27, 28, 30, 32, and 34-35 are rejected under 35 U.S.C. 103 as being unpatentable over Zhuang (WO 2016/018960 Pub 2/4/2016) in view of Nolan (US Patent 10,370,698 8/6/2019).
Regarding Claim 1, Zhuang teaches the following with respect to Fig 3A (see pages 10-14):
For instance, as is shown in Fig. 3 A, a population of nucleic acids 10 within a cell (represented here by nucleic acids 11, 12, and 13) may be exposed to a population of primary nucleic acid probes 20, including probes 21 and 22. The primary nucleic acid probes may contain, for instance, a target sequence that can recognize a nucleic acid (e.g., a sequence within nucleic acid 11). Probes 21 and 22 may contain the same or different targeting sequences, which may bind to or hybridize with the same or different nucleic acids. As an example, as is shown in Fig. 3 A, probe 21 contains a first targeting sequence 25 which targets the probe to nucleic acid 11, while probe 22 contains a second targeting sequence 26, not identical to the first targeting sequence 25 and which targets the probe to nucleic acid 12. The target sequence may be substantially complementary to at least a portion of a target nucleic acid, and enough of the target sequence may be present such that specific binding of the nucleic acid probe to the target nucleic acid can occur.
Primary nucleic acid probes 20 may also contain one or more "read" sequences. Two such read sequences are used in this example, although in other embodiments, there may be one, three, four, or more read sequences present within a primary nucleic acid probe. The read sequences may all independently be the same or different. In addition, in one set of embodiments, different nucleic acid probes may use one or more common read sequences. For example, more than one read sequence may be combinatorially combined on different nucleic acid probes, thereby producing a relatively large number of different nucleic acid probes that can be separately identified, even though only a relatively small number of read sequences are used. Thus, for example, in Fig. 3 A, probe 21 contains read sequences 27 and 29, while probe 22 contains read sequences 27 and 28, where the two read sequences 27 are identical, and different from read sequences 28 and 29.
After primary nucleic acid probes 20 have been introduced to the sample and allowed to interact with nucleic acids 11, 12, and 13, one or more secondary nucleic acid probes 30 may be applied to the sample to determine the primary nucleic acid probes. The secondary nucleic acid probes may contain a recognition sequence able to recognize one of the read sequences present within the population of primary nucleic acid probes. For instance, the recognition sequence may be substantially complementary to at least a portion of the read sequence, such that the secondary nucleic acid probe is able to bind to or hybridize with corresponding primary nucleic acid probe. For instance, in this example, recognition sequence 35 is able to recognize read sequence 27. In addition, the secondary nucleic acid probes may contain one or more signaling entities 33. For example, a signaling entity may be a fluorescent entity attached to the probe, or a certain sequence of nucleic acids that can be determined in some fashion. More than one secondary sequence may be used, e.g., sequentially. For example, as shown in this figure, the initial secondary probe 30 may be removed (e.g., as discussed below) and a new secondary probe 31 may be added, containing recognition sequence 36 able to recognize read sequence 28 and one or more signaling entities 33. This may also be repeated multiple times, e.g., to determine read sequence 29 or other read sequences that may be present.
The location of the secondary nucleic acid probes 30, 31, etc. may be determined by determining signaling entity 33. For example, if the signaling entity is fluorescent, then fluorescence microscopy can be used to determine the signaling entity. In some
embodiments, imaging of a sample to determine the signaling entity may be used at relatively high resolutions, and in some cases, super-resolution imaging techniques (e.g., resolutions better than the wavelength of visible light or the diffraction limit of light) may be used.
Examples of super-resolution imaging techniques include STORM, or other techniques as discussed herein. In some cases, e.g., with certain super-resolution imaging techniques such as STORM, more than one image of the sample may be acquired.
More than one type of secondary nucleic acid probe may be applied to a cell or other sample. For example, a first secondary nucleic acid probe may be applied that can recognize a first read sequence, then it or its attached signaling entity may be inactivated or removed, and a second secondary nucleic acid probe may be applied that can recognize a second read sequence. This process may be repeated multiple times, each with a different secondary nucleic acid probe, e.g., to determine the read sequences that were present in the various primary nucleic acid probes. Thus, primary nucleic acids within the sample can be determined on the basis of the binding pattern of secondary nucleic acid probes.
For example, a first location within the cell or other sample may exhibit binding of a first secondary probe and a third secondary probe, but not the binding of a second or a fourth secondary probe, while a second location may exhibit a different pattern of binding of various secondary probes. The primary nucleic acid probe that the secondary probes are able to bind to or hybridize with may be determined by considering the pattern of binding of various secondary probes. For instance, referring to Fig. 3A, if a first secondary probe is able to determine read sequence 27, a second secondary probe is able to determine read sequence 28, and a third secondary probe is able to determine read sequence 29, then primary nucleic acid 25 may be determined through the binding of the first and third secondary probes (but not the second secondary probe), while primary nucleic acid 26 may be determined through the binding of the first and second secondary probes (but not the third secondary probe).
Similarly, if it is known that first probe 21 contains target sequence 25 while second probe 22 contains target sequence 26, then nucleic acids 11 and 12 may also be determined within the sample, e.g., spatially, based on the binding pattern of the various secondary nucleic acid probes. In addition, it should be noted that due to the presence of more than one read sequence on the primary nucleic acid probes, even though first probe 21 and second probe 22 contains a common read sequence (read sequence 27), these probes may be distinguished in the sample due to the different binding patterns of the various secondary nucleic acid probes.
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Zhuang teaches that the target nucleic acid may be mRNA (page 10, line 17). Additionally Zhuang discloses the hybridization conditions used to perform Fluorescence In Situ Hybridization (FISH) - primary (encoding) probes. Cells were hydrated in wash buffers (2xSSC, 50% formamide) for 10 minutes, labelled with primary oligos (0.5 nM per sequence) in hybridization buffers (2xSSC, 50% formamide, 1 mg/mL yeast tRNA, and 10% dextran sulfate) overnight at 37 °C, washed with wash buffers at 47 °C for 10 minutes twice, and washed with 2xSSC twice (page 52, lines 22-27). Further Zhang discloses the hybridization conditions for the
secondary probes. Secondary (readout) probes (10 nM) were hybridized in secondary hybridization buffers (2xSSC, 20% formamide, and 10% dextran sulfate) to their primary targets for 30 minutes at 37 °C. Cells remained on the microscope stage during the hybridization. An objective heater was used to maintain the temperature at 37 °C. Cells were washed with secondary wash buffers (2xSSC, 20% formamide) (page 52, lines 28-32).
Thus Zhang teaches a method of contacting a biological sample (a population of nucleic acids in a cell) with a first composition comprising multiple different types of unlabeled oligonucleotide probes (primary nucleic acid probes, 20) that hybridize to RNA species in the sample; (b) contacting the biological sample with a hybridization agent comprising a chaotropic compound (formamide); (c) contacting the biological sample with a second composition comprising labeled oligonucleotide probes (secondary nucleic acid probes 30), wherein the labeled oligonucleotide probes selectively hybridizes to the unlabeled oligonucleotide probes; (d) obtaining at least one image (fluorescence microscopy/STORM) of the biological sample with the labeled oligonucleotide probes (secondary nucleic acid probes 30) bound to the sample; and (e) identifying spatial locations of the RNA species in the sample based on components of the at least one image, wherein the biological sample is contacted with the second composition under isothermal conditions (the second hybridization step was performed at 37ºC and a heater was used to maintain this temperature).
Regarding Claim 2 Zhuang teaches a method wherein after imaging, the secondary nucleic acid probes are removed, and a different secondary nucleic acid probe is added to the sample (page 10, lines 12-13). Thus Zhuang teaches a method further comprising removing the labeled oligonucleotide probes from the sample.
Regarding Claim 3 Zhuang teaches after imaging, the secondary nucleic acid probes are inactivated or removed, and a different secondary nucleic acid probe is added to the sample. This may be repeated multiple times with multiple different secondary nucleic acid probes. The pattern of binding of the various secondary nucleic acid probes may be used to determine the primary nucleic acid probes at locations within the cell or other sample, which can be used to determine mRNA or other nucleic acids that are present (page 10, lines 12-17). Thus Zhuang teaches a method further comprising repeating steps (c)-(e) with a different set of labeled oligonucleotide probes (secondary nucleic acid probes, 31) to identify spatial locations of at least one additional RNA species in the sample.
Regarding Claim 4 Zhuang teaches after imaging, the secondary nucleic acid probes are inactivated or removed, and a different secondary nucleic acid probe is added to the sample (page 10, lines 12-17). Zhuang discloses the hybridization conditions for the secondary probes. Secondary (readout) probes (10 nM) were hybridized in secondary hybridization buffers (2xSSC, 20% formamide, and 10% dextran sulfate) to their primary targets. Thus Zhuang teaches repeating step (b) using a hybridization agent comprising a chaotropic agent (formamide) (page 52, lines 28-32).
Regarding Claim 7 Zhuang teaches a method wherein the chaotropic compound comprises formamide (page 52, lines 22-32).
Regarding Claim 8 Zhuang does not specifically teaches that the weight percentage of the chaotropic compound in the hybridization agent is between 5% and 20%.
However, to have determined the optimum weight percentage of the chaotropic compound in the hybridization solution would have been obvious to one of ordinary skill in the art and well within the skill of the art. As discussed in MPEP 2144.05(b), “(w)here the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation. In re Aller, 220 F.2d 454, 105 USPQ 233, 235 (CCPA 1955).
MPEP 2144.05(b):
“Generally, differences in concentration or temperature will not support the patentability of subject matter encompassed by the prior art unless there is evidence indicating such concentration or temperature is critical. "[W]here the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation." In re Aller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA 1955)”
“A particular parameter must first be recognized as a result-effective variable, i.e., a variable which achieves a recognized result, before the determination of the optimum or workable ranges of said variable might be characterized as routine experimentation. In re Antonie, 559 F.2d 618, 195 USPQ 6 (CCPA 1977).”
Regarding Claim 15 Zhuang does not specifically teaches that the weight percentage of the chaotropic compound in the dehybridization agent is 50% or more.
However, to have determined the optimum weight percentage of the chaotropic compound in the dehybridization solution would have been obvious to one of ordinary skill in the art and well within the skill of the art. As discussed in MPEP 2144.05(b), “(w)here the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation. In re Aller, 220 F.2d 454, 105 USPQ 233, 235 (CCPA 1955).
MPEP 2144.05(b):
“Generally, differences in concentration or temperature will not support the patentability of subject matter encompassed by the prior art unless there is evidence indicating such concentration or temperature is critical. "[W]here the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation." In re Aller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA 1955)”
“A particular parameter must first be recognized as a result-effective variable, i.e., a variable which achieves a recognized result, before the determination of the optimum or workable ranges of said variable might be characterized as routine experimentation. In re Antonie, 559 F.2d 618, 195 USPQ 6 (CCPA 1977).”
Regarding Claim 17 Zhuang discloses a hybridization solution containing 2xSSC, 50% formamide, and 10% dextran sulfate (page 52, lines 22-32). Thus Zhuang teaches a method wherein a hybridization agent comprises a buffer (2xSSC).
Regarding Claim 27 Zhuang teaches that in some cases, more than one type of (primary) nucleic acid probe may be applied to a sample, e.g., simultaneously. For example, there may be at least 2, at least 5, at least 10, at least 25, at least 50, at least 75, at least 100, at least 300, at least 1,000, at least 3,000, at least 10,000, or at least 30,000 distinguishable nucleic acid probes that are applied to a sample, e.g., simultaneously or sequentially (page 18, lines 23-28). Thus Zhuang teaches a method wherein the first composition comprises 20 or more different types of unlabeled oligonucleotide probes.
Regarding Claim 30 Zhuang teaches a method wherein each primary probe comprises a target regions that is at least 5 bp and a read sequence that is at least 5 bp (pages 18-20). Thus Zhuang teaches a method wherein each type of unlabeled oligonucleotide probe comprises at least 5 bases.
Regarding Claim 32 Zhuang teaches a method wherein each secondary probe contains a recognition sequence able to bind to or hybridize with a read sequence of a primary probe. Zhuang teaches each recognition sequences is at least 5 bp (page 22). Thus Zhuang teaches a method wherein each type of labeled oligonucleotide probe comprises at least 5 bases.
Regarding Claims 34-35 Zhuang teaches that the secondary nucleic acid probes include one or more signaling entities. If more than one nucleic acid probe is used, the signaling entities may each be the same or different. In certain embodiments, a signaling entity is any entity able to emit light. For instance, in one embodiment, the signaling entity is fluorescent. Non-limiting examples of signaling entities include fluorescent entities (fluorophores) or phosphorescent entities, for example, cyanine dyes (e.g., Cy2, Cy3, Cy3B, Cy5, Cy5.5, Cy7, etc.), Alexa Fluor dyes, Atto dyes, photo swtichable dyes, photoactivatable dyes, fluorescent dyes, metal nanoparticles, semiconductor nanoparticles or "quantum dots", fluorescent proteins such as GFP (Green Fluorescent Protein), or photoactivabale fluorescent proteins, such as PAGFP, PSCFP, PSCFP2, Dendra, Dendra2, EosFP, tdEos, mEos2, mEos3, PAmCherry, PAtagRFP, mMaple, mMaple2, and mMaple3 (pages 30-31). Thus Zhuang teaches a method wherein each type of labeled oligonucleotide probe comprise a different optical label and wherein the different optical labels comprise different fluorescent dyes, different chromogenic moieties, and different quantum dot-based species.
While Zhuang teaches contacting the sample with a second composition of labeled oligonucleotide probes (secondary nucleic acid probes), Zhuang does not teach that the second composition comprises multiple different types of labeled oligonucleotide probes (clm 1). Zhuang does not teach a method further comprising removing the labeled oligonucleotide probes from the sample by exposing the sample to a dehybridization agent to dehybridize the labeled oligonucleotide probes from the unlabeled oligonucleotide probes (claim 11). Zhuang does not teach a method wherein the dehybridization agent comprises a chaotropic compound (clm 12). Zhuang does not teach a method wherein the chaotropic compound of the dehybridization agent comprises dimethyl sulfoxide or formamide (clm 13). Zhuang does not teach a method wherein the dehybridization agent comprises at least one member selected from the group consisting of a buffer agent, a salt, and a surfactant (clm 22). Zhuang does not teach a method wherein the second composition comprises 4 or more different types of labeled oligonucleotide probes (clm 28).
However Nolan teaches a method for analyzing a sample, comprising the following steps, performed in order: (a) obtaining: i. a plurality of capture agents that are each linked to a different oligonucleotide; and ii. a corresponding plurality of labeled nucleic acid probes, wherein each of the labeled nucleic acid probes specifically hybridizes with only one of the oligonucleotides of (a)(i); (b) labeling a sample with the plurality of capture agents of (a)(i); (c) specifically hybridizing a first sub-set of the labeled nucleic acid probes of (a)(ii) with the sample, wherein the probes in the first sub-set are distinguishably labeled, to produce labeled probe/oligonucleotide duplexes; (d) reading the sample to obtain an image showing the binding pattern for each of the probes hybridized in step (c); (e) inactivating or removing the labels that are associated with the sample in step (c), leaving the plurality of capture agents of (b) and their associated oligonucleotides still bound to the sample; and (f) repeating steps (c) and (d) multiple times with a different sub-set of the labeled nucleic acid probes of (a)(ii), each repeat followed by step (e) except for the final repeat, to produce a plurality of images of the sample, each image corresponding to a sub-set of labeled nucleic acid probes used in (c) (col 20, lines 35-62). Nolan further teaches that the probes are removed in step (e) using formamide (col 21, lines 40-47). Nolan further teaches that the hybridized dye-labeled oligonucleotides are removed using an 80% formamide solution with 2 mM Tris pH=7.5, 2 mM MgCl.sub.2, 25 mM NaCl and 0.02% (v/v) TritonX (col 27, lines 33-40). Further Nolan teaches that after the sample has been bound to the capture agents, the method may involve specifically hybridizing a first sub-set of the labeled nucleic acid probes with the sample, wherein the probes in the first sub-set are distinguishably labeled, to produce labeled probe/oligonucleotide duplexes. By “sub-set” is meant at least two, e.g., two, three or four and the term “distinguishably labeled” means that the labels can be separately detected, even if they are at the same location (col 8, lines 24-30).
Accordingly, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of Zhuang by contacting the biological sample with a second composition comprising multiple different types of labeled oligonucleotide probes (i.e., at least 4) as suggested by Nolan. One of skill in the art would have been motivated to use multiple different types of labeled oligonucleotide probes particularly in instances where each of the labeled nucleic acid probes specifically hybridizes with only one of the oligonucleotides for the benefit of reducing the number of times that the steps need to be repeated in order to decode the encoding probes. Further it would have been obvious to modify the method of Zhuang by using a dehybridization solution comprising a chaotropic compound, a salt, and a surfactant to remove the secondary oligonucleotides as suggested by Nolan. In the instant case Nolan discloses a dehybridization solution that allows for the probes hybridized in step c to be removed from the sample by denaturation (i.e., by un-annealing the labeled probes from the oligonucleotides and washing them away), leaving the capture agents of (b) and their associated oligonucleotides still bound to the sample. Based on the teachings of Wang, the skilled artisan would have recognized that the use of the dehybridization solution would have been an effective way to remove the secondary probes of Zhuang so that the primary probes could then bind to additional secondary probes until all the primary probes were read.
6. Claim 5 is rejected under 35 U.S.C. 103 as being unpatentable over Zhuang (WO 2016/018960 Pub 2/4/2016) in view of Nolan (US Patent 10,370,698 8/6/2019) as applied to claims 1-3 above and in further view of Cai (US 2016/0369329 Pub 12/22/2016).
The teachings of Zhuang and Nolan are presented above.
The combined references do not teach that prior to repeating steps (c)-(e): contacting the biological sample with a third composition comprising multiple different types of unlabeled oligonucleotide probes that hybridize to RNA species in the sample, wherein the multiple different types of unlabeled oligonucleotide probes of the third composition are also present in the first composition.
However Cai teaches a method comprising steps of: (a) performing a first contacting step that involves contacting a cell comprising a plurality of nucleic acids with a first plurality of detectably labeled oligonucleotides, each of which targets a nucleic acid and is labeled with a detectable moiety, so that the composition comprises at least: (i) a first oligonucleotide targeting a first nucleic acid and labeled with a first detectable moiety; and (ii) a second oligonucleotide targeting a second nucleic acid and labeled with a second detectable moiety; (b) imaging the cell after the first contacting step so that interaction by oligonucleotides of the first plurality with their targets is detected; (c) performing a second contacting step that involves contacting the cell with a second plurality of detectably labeled oligonucleotides, which second plurality includes oligonucleotides targeting overlapping nucleic acids that are targeted by the first plurality, so that the second plurality comprises at least: (i) a third oligonucleotide, optionally identical in sequence to the first oligonucleotide, targeting the first nucleic acid; and (ii) a fourth oligonucleotide, optionally identical in sequence to the second oligonucleotide (paras 0007-0011).
Accordingly, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of Zhuang and Nolan by contacting the biological sample with a third composition comprising multiple different types of unlabeled oligonucleotide probes that hybridize to RNA species in the sample, wherein the multiple different types of unlabeled oligonucleotide probes of the third composition are also present in the first composition. In the instance case Cai teaches performing first and second hybridization steps wherein the oligonucleotide probes of the second hybridization step are also present in the first hybridization. One of ordinary skill in the art would have been motivated to rehybridize the biological sample with a third composition of probes comprising probes that were present in the first composition for the benefit of being able detect these RNA species since the probes in the first composition may not have all hybridized to their respective targets during the first hybridization and/or some of the probes from the first composition may have become dehybridized during the dehybridization/removal steps.
7. Claims 6 and 20 are rejected under 35 U.S.C. 103 as being unpatentable over Zhuang (WO 2016/018960 Pub 2/4/2016) in view of Nolan (US Patent 10,370,698 8/6/2019) as applied to claim 1 above and in further view of Bodary (US 2006/0199181 Pub 9/7/2006).
The teachings of Zhuang and Nolan are presented above.
The combined references do not teach a method wherein the chaotropic compound in the hybridization solution comprises dimethylsulfoxide (DMSO) (clm 6). The combined references do not teach a method wherein the hybridization solution comprises at least one chelating agent (clm 20).
However Bodary teaches they compared hybridization signals obtained using a first hybridization buffer (Buffer 1) comprising 50% (v/v) formamide, 5xSSC buffer and a second hybridization buffer (Buffer 2) comprising 2.4 M TEACl, 50 mM Tris, 2 mM EDTA, pH 8.0, with 20% formamide/5% (v/v) DMSO. Bodary teaches that the hybridization signal found with 2.4 M TEACl, 50 mM Tris, 2 mM EDTA, pH 8.0, with 20% formamide/5% (v/v) DMSO was increased 3-5 fold over the signal obtained in hybridization buffer lacking TEACl and DMSO (para 0172). It is noted that EDTA is a chelating agent.
Accordingly, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of Zhuang and Nolan by using a hybridization buffer that comprises dimethylsulfoxide (DMSO) and EDTA as suggested by Bodary. One of skill in the art would have been motivated to perform hybridization with a solution comprising DMSO and EDTA particularly since Boday teaches that the hybridization signal found with 2.4 M TEACl, 50 mM Tris, 2 mM EDTA, pH 8.0, with 20% formamide/5% (v/v) DMSO was increased 3-5 fold over the signal obtained in hybridization buffer lacking TEACl and DMSO (para 0172).
8. Claim 25 is rejected under 35 U.S.C. 103 as being unpatentable over Zhuang (WO 2016/018960 Pub 2/4/2016) in view of Nolan (US Patent 10,370,698 8/6/2019) as applied to claims 1, 2, 11, and 12 above and in further view of Krieg (US 2020/0190505 Filed 3/12/2019).
The teachings of Zhuang and Nolan are presented above.
The combined references do not teach a method wherein the dehybridization agent comprises at least one chelating agent.
However Krieg discloses a dehybridization solution that comprises a formamide solution, for example, a formamide dehybridization buffer (e.g., FDB, 95% (v/v) formamide, 5 mM EDTA, pH 8.1) (para 0054). It is noted that EDTA is a chelating agent.
Accordingly, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of Zhuang and Nolan by using a dehybridization agent that comprises at least one chelating agent as suggested by Trayes. One of skill in the art would have been motivated to perform dehybridization with a solution comprising EDTA for the benefit of facilitating the separation of nucleic acid strands.
9. Claim 39 is rejected under 35 U.S.C. 103 as being unpatentable over Zhuang (WO 2016/018960 Pub 2/4/2016) in view of Nolan (US Patent 10,370,698 8/6/2019) as applied to claims 1-3 above and in further view of Gizzi (Molecular Cell 4/4/2019 74 212-222).
The teachings of Zhuang and Nolan are presented above.
Additionally Zhuang teaches that images of the same sample region in different rounds of hybridization were registered by rotating and translating the image to align the two fiducial beads within the same image that were most similar in location after a coarse initial alignment via image correlation. All images were aligned to a coordinate system established by the images collected in the first round of hybridization. The quality of this alignment was determined from the residual distance between five additional fiducial beads, and alignment error was typically -20 nm (page 81, lines 8-13). Thus Zhuang teaches registering at least one image of a biological sample with multiple different types of labeled oligonucleotide probes bound to the sample and at least one image of the biological sample with a different set of labeled oligonucleotide probes bound to the sample based on the measurement signals corresponding to the fiducial.
The combined references do not teach a method wherein the second composition comprises a first type of labeled oligonucleotide probe among the multiple different types of labeled oligonucleotide probes; the different set of labeled oligonucleotide probes comprises the first type of labeled oligonucleotide probe; the at least one image of the biological sample with the multiple different types of labeled oligonucleotide probes bound to the sample comprises a first image comprising a measurement signal corresponding to the first type of labeled oligonucleotide probe; the at least one image of the biological sample with the different set of labeled oligonucleotide probes bound to the sample comprises a second image comprising a measurement signal corresponding to the first type of labeled oligonucleotide probe; and the method further comprises registering the at least one image of the biological sample with the multiple different types of labeled oligonucleotide probes bound to the sample and the at least one image of the biological sample with the different set of labeled oligonucleotide probes bound to the sample based on the measurement signals corresponding to the first type of labeled oligonucleotide probe in the first and second images.
However Gizzi teaches a method of sequential labeling and imaging of multiple DNA loci. Gizzi teaches that a primary library containing thousands of oligonucleotides targeting multiple genomic loci was designed and produced by high-throughput DNA synthesis (STAR Methods). The subset of oligonucleotides targeting each genomic locus (hereafter called bar code) contained unique tails with specific sequences that could be independently read by complementary, fluorescently labeled oligonucleotide probes (hereafter called readout probes; Figures 1A and S1A). An additional bar code (hereafter called fiducial bar code), visible in all rounds of hybridization, was used for image registration and drift correction (STAR Methods) (page 214 and Fig 1).
Accordingly, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of Zhuang and Nolan by using a labeled oligonucleotide probe that is visible in the first image and the second image and acts as a fiducial to aid in image registration as suggested by Gizzi. In the instant case Zhuang teaches using beads a fiducials for image registration whereas Gizzi teaches using a barcode as a fiducial. The claims would have been obvious because the substitution of one known element for another would have yielded predictable results to one of ordinary skill in the art at the time of the invention.
10. Any inquiry concerning this communication or earlier communications from the examiner should be directed to AMANDA HANEY whose telephone number is (571)272-8668. The examiner can normally be reached Monday-Friday, 8:15am-4:45pm EST.
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/AMANDA HANEY/Primary Examiner, Art Unit 1682
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