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 .
This is in response to the applicant’s reply filed January 5, 2026. In the applicant’s reply; claim 21 was amended, and claims 1-20, 22 and 24 were cancelled. Claims 21, 23, and 25-43 are pending in this application.
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and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114.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 April 26, 2010 has been entered.
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 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.
Terminal Disclaimer
The terminal disclaimer filed on April 14, 2025 disclaiming the terminal portion of any patent granted on this application which would extend beyond the expiration date of US Patent 11,995,828 and US Patent 11,434,532 has been reviewed and is accepted. The terminal disclaimer has been recorded.
Response to Arguments
Applicants' amendments filed on January 5, 2026 have been fully considered. The amendments overcome the following rejections set forth in the office action mailed on July 7, 2025.
Applicant’s filing of a terminal disclaimer on April 14, 2025 overcomes the rejections of Claims 21-39 on the ground of nonstatutory double patenting as being unpatentable over claims 1-10 of U.S. Patent No. US 11,995,828 B2 issued from US Application 18/052880, and the rejection is hereby withdrawn.
Applicant’s filing of a terminal disclaimer on April 14, 2025 overcomes the rejections of Claims 21-39 on the ground of nonstatutory double patenting as being unpatentable over claims 1-31 of US Patent 11,434,532 issued from US Application 17/327,588, and the rejection is hereby withdrawn.
Applicant's arguments have been fully considered but they are not persuasive.
Applicant argues “Drmanac does not describe "detecting said first optical signal and said second optical signal with an imaging system, wherein said imaging system comprises a resolution of up to about 300 nanometers per pixel of said imaging system" as recited, inter alia, in amended claim 21. Instead, Drmanac merely describes that "a density is selected such that at least a majority of single molecules have a nearest neighbor distance of 300 nm or greater ......"See Drmanac, ¶ [0035]. Drmanac discusses density to "ensure that at least seventy percent of single molecules have a nearest neighbor distance of 300 nm or greater ...”
Examiner respectfully disagrees. It is respectfully requested from the applicant, in preparing the responses, to fully consider the references in entirety as potentially teaching all or part of the claimed invention, as well as the context of the passage as taught by the prior art or disclosed by the examiner. It appears that Applicant did not fully consider the teachings of Drmanac in [0035] and rather focused on alternative embodiments than the embodiment that would be most pertinent to the claimed features.
[0035] FIG. 1B illustrates a section (1102) of a surface of a random array of single molecules, such as single stranded polynucleotides. Such molecules under conventional conditions (a conventional DNA buffer, e.g. TE, SSC, SSPE, or the like, at room temperature) form random coils that roughly fill a spherical volume in solution having a diameter of from about 100 to 300 nm…. In one aspect, whenever scanning electron microscopy is employed, for example, with molecule-specific probes having gold nanoparticle labels, e.g. Nie et al (2006), Anal. Chem., 78: 1528-1534, which is incorporated by reference, a density is selected such that at least a majority of single molecules have a nearest neighbor distance of 50 nm or greater; and in another aspect, such density is selected to ensure that at least seventy percent of single molecules have a nearest neighbor distance of 100 nm or greater. In another aspect, whenever optical microscopy is employed, for example with molecule-specific probes having fluorescent labels, a density is selected such that at least a majority of single molecules have a nearest neighbor distance of 200 nm or greater; and in another aspect, such density is selected to ensure that at least seventy percent of single molecules have a nearest neighbor distance of 200 nm or greater….
Examiner has cited particular columns and line numbers or figures in the references as applied to the claims below for the convenience of the applicant. This was cited by Examiner in the Office Action, and Applicant did not address this section of Drmanac’s teachings in their response. Examiner encourages applicant to fully consider the references in entirety as potentially teaching all or part of the claimed invention when drafting a response to the Office Action, as well as the context of the passage as taught by the prior art or disclosed by the examiner.
Applicant argues that Drmanac does not teach “(c) processing a first peak intensity of said first optical signal at a first peak location and a second peak intensity of said second optical signal at a second peak location”
Examiner respectfully disagrees. It is respectfully requested from the applicant, in preparing the responses, to fully consider the references in entirety as potentially teaching all or part of the claimed invention, as well as the context of the passage as taught by the prior art or disclosed by the examiner. Drmananc clearly teaches processing a first peak intensity of said first optical signal at a first peak location and a second peak intensity of said second optical signal at a second peak location in [0221]
(Drmanac: [0221] By overlaying the images obtained from successive hybridization of3 probes, as shown in FIG. 4, it can be seen that most of the arrayed molecules that hybridized with the adaptor probe would only hybridize to either the amplicon 1 probe ( e.g. "A" in FIG. 4) or the amplicon 2 probe ( e.g. "B" in FIG. 4), with very few that would hybridize to both. This specific hybridization pattern demonstrates that each spot on the array contains only one type of sequence, [0234] "Microarray" or "array" refers to a solid phase support having a surface, usually planar or substantially planar, which carries an array of sites containing nucleic acids, such that each member site of the array comprises identical copies of immobilized oligonucleotides or polynucleotides and is spatially defined and not overlapping with other member sites of the array; that is, the sites are spatially discrete. In some cases, sites of a microarray may also be spaced apart as well as discrete; that is, different sites do not share boundaries, but are separated by inter-site regions, usually free of bound nucleic acids. Spatially defined hybridization sites may additionally be "addressable" in that its location and the identity of its immobilized oligonucleotide are known or predetermined, for example, prior to its use. [0016] In one embodiment of this aspect, the step of identifying includes the steps of (a) hybridizing one or more probes from a first set of probes to the random array under conditions that permit the formation of perfectly matched duplexes between the one or more probes and complementary sequences on target concatemers; (b) hybridizing one or more probes from a second set of probes to the random array under conditions that permit the formation of perfectly matched duplexes between the one or more probes and complementary sequences on target concatemers; (c) ligating probes from the first and second sets hybridized to a target concatemer at contiguous sites; (d) identifying the sequences of the ligated first and second probes; and (e) repeating steps (a through (d) until the sequence of the target polynucleotide can be determined from the identities of the sequences of the ligated probes.)
Examiner has cited particular columns and line numbers or figures in the references as applied to the claims below for the convenience of the applicant. Although the specified citations are representative of the teachings in the art and are applied to the specific limitations within the individual claim, other passages and figures may apply as well. It is respectfully requested from the applicant, in preparing the responses, to fully consider the references in entirety as potentially teaching all or part of the claimed invention, as well as the context of the passage as taught by the prior art or disclosed by the examiner.
Applicant argues “Drmanac does not teach or disclose "processing said first optical signal and said second optical signal to distinguish said first analyte from said second analyte”.
Examiner respectfully disagrees. Examiner has cited particular columns and line numbers or figures in the references as applied to the claims below for the convenience of the applicant. Although the specified citations are representative of the teachings in the art and are applied to the specific limitations within the individual claim, other passages and figures may apply as well. It is respectfully requested from the applicant, in preparing the responses, to fully consider the references in entirety as potentially teaching all or part of the claimed invention, as well as the context of the passage as taught by the prior art or disclosed by the examiner. Drmanac clearly teaches in paragraphs [0016]-[0017] an iterative approach to using several probes to distinguish a first and second analyte.
[0016] In one embodiment of this aspect, the step of identifying includes the steps of (a) hybridizing one or more probes from a first set of probes to the random array under conditions that permit the formation of perfectly matched duplexes between the one or more probes and complementary sequences on target concatemers; (b) hybridizing one or more probes from a second set of probes to the random array under conditions that permit the formation of perfectly matched duplexes between the one or more probes and complementary sequences on target concatemers; (c) ligating probes from the first and second sets hybridized to a target concatemer at contiguous sites; (d) identifying the sequences of the ligated first and second probes; and (e) repeating steps (a through (d) until the sequence of the target polynucleotide can be determined from the identities of the sequences of the ligated probes. [0117] In one aspect, a sequencing method for use with the invention for determining sequences in a plurality of DNA or RNA fragments comprises the following steps: (a) generating a plurality of polynucleotide molecules each comprising a concatemer of a DNA or RNA fragment; (b) forming a random array of polynucleotide molecules fixed to a surface at a density such that at least a majority of the target concatemers are optically resolvable; and (c) identifying a sequence of at least a portion of each DNA or RNA fragment in resolvable polynucleotides using at least one chemical reaction of an optically detectable reactant. [0153] Arrays and sequencing methods of the invention used may be used for large-scale identification of polymorphisms using mismatch cleavage techniques. Several approaches to mutation detection employ a heteroduplex in which the mismatch itself is utilized for cleavage recognition.
This was cited by Examiner in the Office Action, and Applicant did not address this section of Drmanac’s teachings in their response. Examiner encourages applicant to fully consider all of the teachings of Drmanac when drafting a response to the Office Action.
Double Patenting
The nonstatutory double patenting rejection is based on a judicially created doctrine grounded in public policy (a policy reflected in the statute) so as to prevent the unjustified or improper timewise extension of the “right to exclude” granted by a patent and to prevent possible harassment by multiple assignees. A nonstatutory double patenting rejection is appropriate where the conflicting claims are not identical, but at least one examined application claim is not patentably distinct from the reference claim(s) because the examined application claim is either anticipated by, or would have been obvious over, the reference claim(s). See, e.g., In re Berg, 140 F.3d 1428, 46 USPQ2d 1226 (Fed. Cir. 1998); In re Goodman, 11 F.3d 1046, 29 USPQ2d 2010 (Fed. Cir. 1993); In re Longi, 759 F.2d 887, 225 USPQ 645 (Fed. Cir. 1985); In re Van Ornum, 686 F.2d 937, 214 USPQ 761 (CCPA 1982); In re Vogel, 422 F.2d 438, 164 USPQ 619 (CCPA 1970); In re Thorington, 418 F.2d 528, 163 USPQ 644 (CCPA 1969).
A timely filed terminal disclaimer in compliance with 37 CFR 1.321(c) or 1.321(d) may be used to overcome an actual or provisional rejection based on nonstatutory double patenting provided the reference application or patent either is shown to be commonly owned with the examined application, or claims an invention made as a result of activities undertaken within the scope of a joint research agreement. See MPEP § 717.02 for applications subject to examination under the first inventor to file provisions of the AIA as explained in MPEP § 2159. See MPEP § 2146 et seq. for applications not subject to examination under the first inventor to file provisions of the AIA . A terminal disclaimer must be signed in compliance with 37 CFR 1.321(b).
The filing of a terminal disclaimer by itself is not a complete reply to a nonstatutory double patenting (NSDP) rejection. A complete reply requires that the terminal disclaimer be accompanied by a reply requesting reconsideration of the prior Office action. Even where the NSDP rejection is provisional the reply must be complete. See MPEP § 804, subsection I.B.1. For a reply to a non-final Office action, see 37 CFR 1.111(a). For a reply to final Office action, see 37 CFR 1.113(c). A request for reconsideration while not provided for in 37 CFR 1.113(c) may be filed after final for consideration. See MPEP §§ 706.07(e) and 714.13.
The USPTO Internet website contains terminal disclaimer forms which may be used. Please visit www.uspto.gov/patent/patents-forms. The actual filing date of the application in which the form is filed determines what form (e.g., PTO/SB/25, PTO/SB/26, PTO/AIA /25, or PTO/AIA /26) should be used. A web-based eTerminal Disclaimer may be filled out completely online using web-screens. An eTerminal Disclaimer that meets all requirements is auto-processed and approved immediately upon submission. For more information about eTerminal Disclaimers, refer to www.uspto.gov/patents/apply/applying-online/eterminal-disclaimer.
Claims 21, 23, and 25-43 are rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1-20 of U.S. Patent No. 12,060,608 issued from co-pending application 18/181,440. Although the claims at issue are not identical, they are not patentably distinct from each other because they are both directed towards methods for processing optical signals and distinguishing a first and second analyte by analyzing a first and second overlapping peak intensity. The patented claims are narrower in scope and anticipate the broader claims of the instant invention.
Claim Rejections - 35 USC § 112
The following is a quotation of the second paragraph of 35 U.S.C. 112:
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 21, 23, and 25-43 are rejected under 35 U.S.C. 112, second paragraph, as being indefinite for the following reasons. Independent Claim 21 lines 5-12 are now amended to now recite the following:
(b) detecting said first optical signal and said second optical signal with an imaging system, wherein said imaging system comprises a resolution of up to about 300 nanometers per pixel of said imaging system; and
(c) processing a first peak intensity of said first optical signal at a first peak location, and a second peak intensity of said second optical signal at a second peak location to distinguish said first analyte from said second analyte, wherein said first optical signal and said second optical signal at least partially overlap.
These amendments lead to indefiniteness. First, with respect to overall scope, there is a lack of clarity as to the scope for how “a resolution of up to about 300 nanometers per pixel of said imaging system” is to be interpreted as the actual terminology for “of up to about” are relative terms that are considered to be indefinite. Second, if both the “first optical signal and said second optical signal with an imaging system” and are both within the same resolution, it would appear that inherently that the signals emitted would inherently overlap, and it is unclear whether the scope that the applicant argues in the “Remarks” are actually present in the claimed features or if additional amendments must be made to support the intended arguments. Lastly, it is also unclear if the amendments actually do overcome the prior art or rejection of record, as they currently appear to be the same scope as the claims that were previously presented in the previous office action.
Claim Rejections - 35 USC § 102
The following is a quotation of the appropriate paragraphs of 35 U.S.C. 102 that form the basis for the rejections under this section made in this Office action:
A person shall be entitled to a patent unless –
(a)(1) the claimed invention was patented, described in a printed publication, or in public use, on sale, or otherwise available to the public before the effective filing date of the claimed invention.
(a)(2) the claimed invention was described in a patent issued under section 151, or in an application for patent published or deemed published under section 122(b), in which the patent or application, as the case may be, names another inventor and was effectively filed before the effective filing date of the claimed invention.
Claims 21, 23, and 25-43 are rejected under 35 U.S.C. 102(a)(2) as being anticipated by Drmanac et. al. (US PGPub US 2017 /0152554 A1, hereby referred to as “Drmanac”, filed on February 6, 2017 with provisional priority dating to June 15, 2005).
Consider Claim 21. (New)
Drmanac teaches:
21. (New) A method of distinguishing a first analyte from a second analyte, said method, comprising: (Drmanac: abstract, A high density DNA array comprising a patterned surface, said surface comprising a pattern of small DNA binding regions separated by a non-DNA binding surface, wherein the DNA binding regions comprise DNA capture chemistry and the non-DNA binding surface does not have the DNA capture chemistry wherein more than 50% of the DNA binding regions in the array have single informative DNA species. [0016]-[0018], [0035] FIG. 1B illustrates a section (1102) of a surface of a random array of single molecules, such as single stranded polynucleotides. Figure 1B)
(a) binding a first probe to said first analyte to generate a first optical signal; (Drmanac: [0017], [0035] FIG. 1B illustrates a section (1102) of a surface of a random array of single molecules, such as single stranded polynucleotides…. Some embodiments utilize a support with a grid of regions with DNA capture chemistry separated by surface without DNA capture chemistry, each region being 0.1-10 micrometer with center to center distance of about 0.2 to 20 um. In some embodiments, the source DNA is all sequence variants of given length 8 to 20 base. [0067] In one example each 6mm x 6mm array consists of 36 million 250-500 nm square regions at 1 micrometer pitch. Hydrophobic or other surface or physical barriers may be used to prevent mixing different reactions between unit arrays.)
-; and a second probe to said second analyte to generate a second optical signal (Drmanac: [0016] In one embodiment of this aspect, the step of identifying includes the steps of (a) hybridizing one or more probes from a first set of probes to the random array under conditions that permit the formation of perfectly matched duplexes between the one or more probes and complementary sequences on target concatemers; (b) hybridizing one or more probes from a second set of probes to the random array under conditions that permit the formation of perfectly matched duplexes between the one or more probes and complementary sequences on target concatemers; (c) ligating probes from the first and second sets hybridized to a target concatemer at contiguous sites; (d) identifying the sequences of the ligated first and second probes; and (e) repeating steps (a through (d) until the sequence of the target polynucleotide can be determined from the identities of the sequences of the ligated probes. [0117] In one aspect, a sequencing method for use with the invention for determining sequences in a plurality of DNA or RNA fragments comprises the following steps: (a) generating a plurality of polynucleotide molecules each comprising a concatemer of a DNA or RNA fragment; (b) forming a random array of polynucleotide molecules fixed to a surface at a density such that at least a majority of the target concatemers are optically resolvable; and (c) identifying a sequence of at least a portion of each DNA or RNA fragment in resolvable polynucleotides using at least one chemical reaction of an optically detectable reactant. [0153] Arrays and sequencing methods of the invention used may be used for large-scale identification of polymorphisms using mismatch cleavage techniques. Several approaches to mutation detection employ a heteroduplex in which the mismatch itself is utilized for cleavage recognition.)
(b) detecting said first optical signal and said second optical signal with an imaging system, (Drmanac: [0040] The proposed high density structured random DNA array chip will have capture oligonucleotides concentrated in small, segregated capture cells aligned into a rectangular grid formation (FIG. 5). Most importantly, each capture cell or binding site will be surrounded by an inert surface and will have a sufficient but limited number of capture molecules (100-400). Each capture molecule will bind one copy of the matching adaptor sequence on the RCR produced DNA concatemer. Since each concatemer contains over 1000 copies of the adapter sequence, it will quickly saturate the binding site upon contact and prevent other concatemers from binding, resulting in exclusive attachment of one RCR product per binding site or spot. Tie proper concentration of RCR products and sufficient reaction time will ensure that almost every spot on the array contains one and only one unique DNA target. [0153] Mismatches are usually repaired but the binding action of the enzymes can be used for the selection of fragments through a mobility shift in gel electrophoresis or by protection from exonucleases, [0220])
wherein said imaging system comprises a resolution of up to about 300 nanometers per pixel of said imaging system; and (Drmanac: [0035] In still another aspect, whenever optical microscopy is employed, for example with molecule-specific probes having fluorescent labels, a density is selected such that at least a majority of single molecules have a nearest neighbor distance of 300 nm or greater; and in another aspect, such density is selected to ensure that at least seventy percent of single molecules have a nearest neighbor distance of 300 nm or greater, or 400 nm or greater, or 500 nm or greater, or 600 nm or greater, or 700 nm or greater, or 800 nm or greater. [0042] Having 125-250 nm DNA sites in a regular grid with 250-500 nm center-to-center spacing will provide 20-80 times more DNA samples per surface than arrays with random attached DNA with spots of about 1000 nm in size and 20% usable occupancy. This will result in 20-80 fold lower reagent consumption and 20-80 fold faster readout. Furthermore, attaching RCR products onto this dense grid of capture probe spots ensures that each DNA ball is concentrated on a much smaller surface, increasing the signal and the speed of biochemical assays. Overall, the reduction of DNA attachment spots from 500 nm to 125 nm in size will result in up to 16 fold higher signal intensities. In short, the proposed DNA arrays will provide an order of magnitude lower cost, higher throughput and higher sensitivity than standard random DNA arrays.)
(c) processing a first peak intensity of said first optical signal at a first peak location and a second peak intensity of said second optical signal at a second peak location (Drmanac: [0221] By overlaying the images obtained from successive hybridization of3 probes, as shown in FIG. 4, it can be seen that most of the arrayed molecules that hybridized with the adaptor probe would only hybridize to either the amplicon 1 probe ( e.g. "A" in FIG. 4) or the amplicon 2 probe ( e.g. "B" in FIG. 4), with very few that would hybridize to both. This specific hybridization pattern demonstrates that each spot on the array contains only one type of sequence, [0234] "Microarray" or "array" refers to a solid phase support having a surface, usually planar or substantially planar, which carries an array of sites containing nucleic acids, such that each member site of the array comprises identical copies of immobilized oligonucleotides or polynucleotides and is spatially defined and not overlapping with other member sites of the array; that is, the sites are spatially discrete. In some cases, sites of a microarray may also be spaced apart as well as discrete; that is, different sites do not share boundaries, but are separated by inter-site regions, usually free of bound nucleic acids. Spatially defined hybridization sites may additionally be "addressable" in that its location and the identity of its immobilized oligonucleotide are known or predetermined, for example, prior to its use. [0016] In one embodiment of this aspect, the step of identifying includes the steps of (a) hybridizing one or more probes from a first set of probes to the random array under conditions that permit the formation of perfectly matched duplexes between the one or more probes and complementary sequences on target concatemers; (b) hybridizing one or more probes from a second set of probes to the random array under conditions that permit the formation of perfectly matched duplexes between the one or more probes and complementary sequences on target concatemers; (c) ligating probes from the first and second sets hybridized to a target concatemer at contiguous sites; (d) identifying the sequences of the ligated first and second probes; and (e) repeating steps (a through (d) until the sequence of the target polynucleotide can be determined from the identities of the sequences of the ligated probes.)
to distinguish said first analyte from said second analyte, wherein said first optical signal and said second optical signal at least partially overlap. (Drmanac: [0117] In one aspect, a sequencing method for use with the invention for determining sequences in a plurality of DNA or RNA fragments comprises the following steps: (a) generating a plurality of polynucleotide molecules each comprising a concatemer of a DNA or RNA fragment; (b) forming a random array of polynucleotide molecules fixed to a surface at a density such that at least a majority of the target concatemers are optically resolvable; and (c) identifying a sequence of at least a portion of each DNA or RNA fragment in resolvable polynucleotides using at least one chemical reaction of an optically detectable reactant. [0035] In still another aspect, whenever optical microscopy is employed, for example with molecule-specific probes having fluorescent labels, a density is selected such that at least a majority of single molecules have a nearest neighbor distance of 300 nm or greater; and in another aspect, such density is selected to ensure that at least seventy percent of single molecules have a nearest neighbor distance of 300 nm or greater, or 400 nm or greater, or 500 nm or greater, or 600 nm or greater, or 700 nm or greater, or 800 nm or greater. [0042] Having 125-250 nm DNA sites in a regular grid with 250-500 nm center-to-center spacing will provide 20-80 times more DNA samples per surface than arrays with random attached DNA with spots of about 1000 nm in size and 20% usable occupancy. This will result in 20-80 fold lower reagent consumption and 20-80 fold faster readout. Furthermore, attaching RCR products onto this dense grid of capture probe spots ensures that each DNA ball is concentrated on a much smaller surface, increasing the signal and the speed of biochemical assays. Overall, the reduction of DNA attachment spots from 500 nm to 125 nm in size will result in up to 16 fold higher signal intensities. In short, the proposed DNA arrays will provide an order of magnitude lower cost, higher throughput and higher sensitivity than standard random DNA arrays. [0052] Duplex region (214) of bridging oligonucleotide (210) contains at least a primer binding site for RCR and, in some embodiments, sequences that provide complements to a capture oligonucleotide, which may be the same or different from the primer binding site sequence, or which may overlap the primer binding site sequence. The length of capture oligonucleotides may vary widely, In one aspect, capture oligonucleotides and their complements in a bridging oligonucleotide have lengths in the range of from 10 to 100 nucleotides; and more preferably, in the range of from 10 to 40 nucleotides. In some embodiments, duplex region (214) may contain additional elements, such as an oligonucleotide tag, for example, for identifying the source nucleic acid from which its associated DNA fragment came. [0133] In 2-4 cycles extend to 4-6 base anchor for additional 2-4 bases run 16 different anchors per each array (32-64 physical cycles if 4 colors are used) to determine about 16 possible 8-mers (˜100 bases total) per each fragment (more then enough to map it to the reference (probability that a 100-mer will have a set of 10 8-mers is less than 1 in trillion trillions; (10exp-28). By combining data from different anchors scored in parallel on the same fragment in another array complete sequence of that fragment and by extension to entire genomes may be generated from overlapping 7-10-mers.)
Consider Claim 22. CANCELLED
Consider Claim 23.
Drmanac teaches: 23. (New) The method of claim 21, wherein said first probe is bound to said first analyte in a first cycle, and wherein said second probe is bound to said second analyte in a second cycle. (Drmanac: [0016] In one embodiment of this aspect, the step of identifying includes the steps of (a) hybridizing one or more probes from a first set of probes to the random array under conditions that permit the formation of perfectly matched duplexes between the one or more probes and complementary sequences on target concatemers; (b) hybridizing one or more probes from a second set of probes to the random array under conditions that permit the formation of perfectly matched duplexes between the one or more probes and complementary sequences on target concatemers; (c) ligating probes from the first and second sets hybridized to a target concatemer at contiguous sites; (d) identifying the sequences of the ligated first and second probes; and (e) repeating steps (a through (d) until the sequence of the target polynucleotide can be determined from the identities of the sequences of the ligated probes. [0117], [0153], [0051] In another aspect, primer extension from a genomic DNA template is used to generate a linear amplification of selected sequences greater than 10 kilobases surrounding genomic regions of interest. For example, to create a population of defined-sized targets, 20 cycles of linear amplification is performed with a forward primer followed by 20 cycles with a reverse primer. Before applying the second primer, the first primer is removed with a standard column for long DNA purification or degraded if a few uracil bases are incorporated.)
Consider Claim 24. CANCELLED
Consider Claim 25.
Drmanac teaches: 25. (New) The method of claim 21, wherein said first optical signal and said second optical signal are overlapping. (Drmanac: [0117] In one aspect, a sequencing method for use with the invention for determining sequences in a plurality of DNA or RNA fragments comprises the following steps: (a) generating a plurality of polynucleotide molecules each comprising a concatemer of a DNA or RNA fragment; (b) forming a random array of polynucleotide molecules fixed to a surface at a density such that at least a majority of the target concatemers are optically resolvable; and (c) identifying a sequence of at least a portion of each DNA or RNA fragment in resolvable polynucleotides using at least one chemical reaction of an optically detectable reactant. [0035] In still another aspect, whenever optical microscopy is employed, for example with molecule-specific probes having fluorescent labels, a density is selected such that at least a majority of single molecules have a nearest neighbor distance of 300 nm or greater; and in another aspect, such density is selected to ensure that at least seventy percent of single molecules have a nearest neighbor distance of 300 nm or greater, or 400 nm or greater, or 500 nm or greater, or 600 nm or greater, or 700 nm or greater, or 800 nm or greater. [0042] Having 125-250 nm DNA sites in a regular grid with 250-500 nm center-to-center spacing will provide 20-80 times more DNA samples per surface than arrays with random attached DNA with spots of about 1000 nm in size and 20% usable occupancy. This will result in 20-80 fold lower reagent consumption and 20-80 fold faster readout. Furthermore, attaching RCR products onto this dense grid of capture probe spots ensures that each DNA ball is concentrated on a much smaller surface, increasing the signal and the speed of biochemical assays. Overall, the reduction of DNA attachment spots from 500 nm to 125 nm in size will result in up to 16 fold higher signal intensities. In short, the proposed DNA arrays will provide an order of magnitude lower cost, higher throughput and higher sensitivity than standard random DNA arrays.)
Consider Claim 26.
Drmanac teaches: 26. (New) The method of claim 21, wherein said first analyte and said second analyte are different analytes.(Drmanac: [0009] Compositions of the invention in one form include random arrays of a plurality of different single molecules disposed on a surface, where the single molecules each comprise a macromolecular structure and at least one analyte, such that each macromolecular structure comprises a plurality of attachment functionalities that are capable of forming bonds with one or more functionalities on the surface. In one aspect, the analyte is a component of the macromolecular structure, and in another aspect, the analyte is attached to the macromolecular structure by a linkage between a unique functionality on such structure and a reactive group or attachment moiety on the analyte.)
Consider Claim 27.
Drmanac teaches: 26. (New) The method of claim 21, wherein said first analyte and said second analyte are the same analytes. (Drmanac: [0009] In another aspect, compositions of the invention include random arrays of single molecules disposed on a surface, where the single molecules each comprise a concatemer of at least one target polynucleotide and each is attached to the surface by linkages formed between one or more functionalities on the surface and complementary functionalities on the concatemer. In another form, compositions of the invention include random arrays of single molecules disposed on a surface, where the single molecules each comprise a concatemer of at least one target polynucleotide and at least one adaptor oligonucleotide and each is attached to such surface by the formation of duplexes between capture oligonucleotides on the surface and the attachment oligonucleotides in the concatemer)
Consider Claim 28.
Drmanac teaches: 28. (New) The method of claim 21, wherein said first optical signal or said second optical signal comprises a fluorescent optical signal. (Drmanac: [0035] In still another aspect, whenever optical microscopy is employed, for example with molecule-specific probes having fluorescent labels, a density is selected such that at least a majority of single molecules have a nearest neighbor distance of 300 nm or greater; and in another aspect, such density is selected to ensure that at least seventy percent of single molecules have a nearest neighbor distance of 300 nm or greater, or 400 nm or greater, or 500 nm or greater, or 600 nm or greater, or 700 nm or greater, or 800 nm or greater.)
Consider Claim 29.
Drmanac teaches: 29. (New) The method of claim 21, wherein said first analyte or said second analyte comprises a nucleic acid molecule. (Drmanac: [0234] "Microarray" or "array" refers to a solid phase support having a surface, usually planar or substantially planar, which carries an array of sites containing nucleic acids, such that each member site of the array comprises identical copies of immobilized oligonucleotides or polynucleotides and is spatially defined and not overlapping with other member sites of the array; that is, the sites are spatially discrete. In some cases, sites of a microarray may also be spaced apart as well as discrete; that is, different sites do not share boundaries, but are separated by inter-site regions, usually free of bound nucleic acids. Spatially defined hybridization sites may additionally be "addressable" in that its location and the identity of its immobilized oligonucleotide are known or predetermined, for example, prior to its use.)
Consider Claim 30.
Drmanac teaches: 30. (New) The method of claim 29, wherein said nucleic acid molecule comprises at least 1 kilo- base (kb). (Drmanac: [0234] "Microarray" or "array" refers to a solid phase support having a surface, usually planar or substantially planar, which carries an array of sites containing nucleic acids, such that each member site of the array comprises identical copies of immobilized oligonucleotides or polynucleotides and is spatially defined and not overlapping with other member sites of the array; that is, the sites are spatially discrete. In some cases, sites of a microarray may also be spaced apart as well as discrete; that is, different sites do not share boundaries, but are separated by inter-site regions, usually free of bound nucleic acids. Spatially defined hybridization sites may additionally be "addressable" in that its location and the identity of its immobilized oligonucleotide are known or predetermined, for example, prior to its use.)
Consider Claim 31.
Drmanac teaches: 31. (New) The method of claim 21, wherein said first analyte or said second analyte comprises a protein or a polypeptide. (Drmanac: [0117] In another embodiment, such optically detectable reactant is a nucleoside triphosphate, e.g. a fluorescently labeled nucleoside triphosphate that may be used to extend an oligonucleotide hybridized to a concatemer. In another embodiment, such optically detectable reagent is an oligonucleotide formed by ligating a first and second oligonucleotides that form adjacent duplexes on a concatemer. In another embodiment, such chemical reaction is synthesis of DNA or RNA, e.g. by extending a primer hybridized to a concatemer. In yet another embodiment, the above optically detectable reactant is a nucleic acid binding oligopeptide or polypeptide or protein.)
Consider Claim 32.
Drmanac teaches: 32. (New) The method of claim 21, wherein said first probe or said second probe comprises a labelled nucleotide, an aptamer, an antibody, a polypeptide, an oligonucleotide, or any combination thereof. (Drmanac: [0117] In another embodiment, such optically detectable reactant is a nucleoside triphosphate, e.g. a fluorescently labeled nucleoside triphosphate that may be used to extend an oligonucleotide hybridized to a concatemer. In another embodiment, such optically detectable reagent is an oligonucleotide formed by ligating a first and second oligonucleotides that form adjacent duplexes on a concatemer. In another embodiment, such chemical reaction is synthesis of DNA or RNA, e.g. by extending a primer hybridized to a concatemer. In yet another embodiment, the above optically detectable reactant is a nucleic acid binding oligopeptide or polypeptide or protein.)
Consider Claim 33.
Drmanac teaches: 33. (New) The method of claim 21, wherein said first analyte or said second analyte is immobilized to said substrate. (Drmanac: [0234] “Microarray” or “array” refers to a solid phase support having a surface, usually planar or substantially planar, which carries an array of sites containing nucleic acids, such that each member site of the array comprises identical copies of immobilized oligonucleotides or polynucleotides and is spatially defined and not overlapping with other member sites of the array; that is, the sites are spatially discrete. In some cases, sites of a microarray may also be spaced apart as well as discrete; that is, different sites do not share boundaries, but are separated by inter-site regions, usually free of bound nucleic acids. Spatially defined hybridization sites may additionally be “addressable” in that its location and the identity of its immobilized oligonucleotide are known or predetermined, for example, prior to its use. In some aspects, the oligonucleotides or polynucleotides are single stranded and are covalently attached to the solid phase support, usually by a 5′-end or a 3′-end. In other aspects, oligonucleotides or polynucleotides are attached to the solid phase support non-covalently, e.g. by a biotin-streptavidin linkage, hybridization to a capture oligonucleotide that is covalently bound, and the like)
Consider Claim 34.
Drmanac teaches: 34. (New) The method of claim 21, wherein said first analyte or said second analyte comprises a plurality of analytes. (Drmanac: [0035] In still another aspect, whenever optical microscopy is employed, for example with molecule-specific probes having fluorescent labels, a density is selected such that at least a majority of single molecules have a nearest neighbor distance of 300 nm or greater; and in another aspect, such density is selected to ensure that at least seventy percent of single molecules have a nearest neighbor distance of 300 nm or greater, or 400 nm or greater, or 500 nm or greater, or 600 nm or greater, or 700 nm or greater, or 800 nm or greater. [0042] Having 125-250 nm DNA sites in a regular grid with 250-500 nm center-to-center spacing will provide 20-80 times more DNA samples per surface than arrays with random attached DNA with spots of about 1000 nm in size and 20% usable occupancy. This will result in 20-80 fold lower reagent consumption and 20-80 fold faster readout. Furthermore, attaching RCR products onto this dense grid of capture probe spots ensures that each DNA ball is concentrated on a much smaller surface, increasing the signal and the speed of biochemical assays. Overall, the reduction of DNA attachment spots from 500 nm to 125 nm in size will result in up to 16 fold higher signal intensities. In short, the proposed DNA arrays will provide an order of magnitude lower cost, higher throughput and higher sensitivity than standard random DNA arrays.)
Consider Claim 35.
Drmanac teaches: 35. (New) The method of claim 34, wherein said plurality of analytes are provided on said substrate at a density of at least 2 molecules/pm2. (Drmanac: [0035] In still another aspect, whenever optical microscopy is employed, for example with molecule-specific probes having fluorescent labels, a density is selected such that at least a majority of single molecules have a nearest neighbor distance of 300 nm or greater; and in another aspect, such density is selected to ensure that at least seventy percent of single molecules have a nearest neighbor distance of 300 nm or greater, or 400 nm or greater, or 500 nm or greater, or 600 nm or greater, or 700 nm or greater, or 800 nm or greater. [0042] Having 125-250 nm DNA sites in a regular grid with 250-500 nm center-to-center spacing will provide 20-80 times more DNA samples per surface than arrays with random attached DNA with spots of about 1000 nm in size and 20% usable occupancy. This will result in 20-80 fold lower reagent consumption and 20-80 fold faster readout. Furthermore, attaching RCR products onto this dense grid of capture probe spots ensures that each DNA ball is concentrated on a much smaller surface, increasing the signal and the speed of biochemical assays. Overall, the reduction of DNA attachment spots from 500 nm to 125 nm in size will result in up to 16 fold higher signal intensities. In short, the proposed DNA arrays will provide an order of magnitude lower cost, higher throughput and higher sensitivity than standard random DNA arrays.)
Consider Claim 36.
Drmanac teaches: 36. (New) The method of claim 34, wherein said plurality of analytes are provided on said substrate at a density of at least 4 molecules/pm2. (Drmanac: [0035] In still another aspect, whenever optical microscopy is employed, for example with molecule-specific probes having fluorescent labels, a density is selected such that at least a majority of single molecules have a nearest neighbor distance of 300 nm or greater; and in another aspect, such density is selected to ensure that at least seventy percent of single molecules have a nearest neighbor distance of 300 nm or greater, or 400 nm or greater, or 500 nm or greater, or 600 nm or greater, or 700 nm or greater, or 800 nm or greater. [0042] Having 125-250 nm DNA sites in a regular grid with 250-500 nm center-to-center spacing will provide 20-80 times more DNA samples per surface than arrays with random attached DNA with spots of about 1000 nm in size and 20% usable occupancy. This will result in 20-80 fold lower reagent consumption and 20-80 fold faster readout. Furthermore, attaching RCR products onto this dense grid of capture probe spots ensures that each DNA ball is concentrated on a much smaller surface, increasing the signal and the speed of biochemical assays. Overall, the reduction of DNA attachment spots from 500 nm to 125 nm in size will result in up to 16 fold higher signal intensities. In short, the proposed DNA arrays will provide an order of magnitude lower cost, higher throughput and higher sensitivity than standard random DNA arrays.)
Consider Claim 37.
Drmanac teaches: 37. (New) The method of claim 34, wherein said plurality of analytes are provided on said substrate at a density of at least 8 molecules/pm2. (Drmanac: [0035] In still another aspect, whenever optical microscopy is employed, for example with molecule-specific probes having fluorescent labels, a density is selected such that at least a majority of single molecules have a nearest neighbor distance of 300 nm or greater; and in another aspect, such density is selected to ensure that at least seventy percent of single molecules have a nearest neighbor distance of 300 nm or greater, or 400 nm or greater, or 500 nm or greater, or 600 nm or greater, or 700 nm or greater, or 800 nm or greater. [0042] Having 125-250 nm DNA sites in a regular grid with 250-500 nm center-to-center spacing will provide 20-80 times more DNA samples per surface than arrays with random attached DNA with spots of about 1000 nm in size and 20% usable occupancy. This will result in 20-80 fold lower reagent consumption and 20-80 fold faster readout. Furthermore, attaching RCR products onto this dense grid of capture probe spots ensures that each DNA ball is concentrated on a much smaller surface, increasing the signal and the speed of biochemical assays. Overall, the reduction of DNA attachment spots from 500 nm to 125 nm in size will result in up to 16 fold higher signal intensities. In short, the proposed DNA arrays will provide an order of magnitude lower cost, higher throughput and higher sensitivity than standard random DNA arrays.)
Consider Claim 38.
Drmanac teaches: 38. (New) The method of claim 21, wherein said first optical signal or said second optical signal comprises a sequence of optical signals. (Drmanac: [0036] Other factors in selecting sequences for capture oligonucleotides are similar to those considered in selecting primers, hybridization probes, oligonucleotide tags, and the like, for which there is ample guidance, as evidenced by the references cited below in the Definitions section. In some embodiments, a discrete spaced apart region may contain more than one kind of capture oligonucleotide, and each different capture oligonucleotide may have a different length and sequence. In one aspect of embodiments employing regular arrays of discrete spaced apart regions, sequences of capture oligonucleotides are selected so that sequences of capture oligonucleotide at nearest neighbor regions have different sequences. In a rectilinear array, such configurations are achieved by rows of alternating sequence types. In other embodiments, a surface may have a plurality of subarrays of discrete spaced apart regions wherein each different subarray has capture oligonucleotides with distinct nucleotide sequences different from those of the other subarrays. A plurality of subarrays may include 2 subarrays, or 4 or fewer subarrays, or 8 or fewer subarrays, or 16 or fewer subarrays, or 32 or fewer subarrays, or 64 of fewer subarrays. In still other embodiments, a surface may include 5000 or fewer subarrays. In one aspect, capture oligonucleotides are attached to the surface of an array by a spacer molecule, e.g. polyethylene glycol, or like inert chain, as is done with microarrays, in order to minimize undesired affects of surface groups or interactions with the capture oligonucleotides or other reagents.)
Consider Claim 39.
Drmanac teaches: 39. (New) The method of claim 21, wherein said sequence of optical signals are determined or obtained over a plurality of cycles. (Drmanac: [0051] In another aspect, primer extension from a genomic DNA template is used to generate a linear amplification of selected sequences greater than 10 kilobases surrounding genomic regions of interest. For example, to create a population of defined-sized targets, 20 cycles of linear amplification is performed with a forward primer followed by 20 cycles with a reverse primer. Before applying the second primer, the first primer is removed with a standard column for long DNA purification or degraded if a few uracil bases are incorporated. [0128] In a similar way the 6 bases from the right side of the 12mer can be decoded by using a fixed oligonucleotide and 5-prime labeled probes. In the above described system 6 cycles are required to define 6 bases of one side of the 12mer. With redundant cycle analysis of bases distant to the ligation site this may increase to 7 or 8 cycles. In total then, complete sequencing of the 12mer could be accomplished with 12-16 cycles of ligation. Partial or complete sequencing of arrayed DNA by combining two distinct types of libraries of detector probes. In this approach one set has probes of the general type N3-8B4-6 (anchors) that are ligated with the first 2 or 3 or 4 probes/probe pools from the set BN6-8, NBN5-7, N2BN4-6, and N3BN3-5.The main requirement is to test in a few cycles a probe from the first set with 2-4 or even more probes from the second set to read longer continuous sequence such as 5-6+3-4=8-10 in just 3-4 cycles.)
Consider Claim 40.
Drmanac teaches: 40. (New) The method of claim 21, wherein said first probe comprises a first probe set, and wherein said second probe comprises a second probe set. (Drmanac: [0016] In one embodiment of this aspect, the step of identifying includes the steps of (a) hybridizing one or more probes from a first set of probes to the random array under conditions that permit the formation of perfectly matched duplexes between the one or more probes and complementary sequences on target concatemers; (b) hybridizing one or more probes from a second set of probes to the random array under conditions that permit the formation of perfectly matched duplexes between the one or more probes and complementary sequences on target concatemers; (c) ligating probes from the first and second sets hybridized to a target concatemer at contiguous sites; (d) identifying the sequences of the ligated first and second probes; and (e) repeating steps (a through (d) until the sequence of the target polynucleotide can be determined from the identities of the sequences of the ligated probes. [0117] In one aspect, a sequencing method for use with the invention for determining sequences in a plurality of DNA or RNA fragments comprises the following steps: (a) generating a plurality of polynucleotide molecules each comprising a concatemer of a DNA or RNA fragment; (b) forming a random array of polynucleotide molecules fixed to a surface at a density such that at least a majority of the target concatemers are optically resolvable; and (c) identifying a sequence of at least a portion of each DNA or RNA fragment in resolvable polynucleotides using at least one chemical reaction of an optically detectable reactant. [0153] Arrays and sequencing methods of the invention used may be used for large-scale identification of polymorphisms using mismatch cleavage techniques. Several approaches to mutation detection employ a heteroduplex in which the mismatch itself is utilized for cleavage recognition.)
Consider Claim 41.
Drmanac teaches: 41. (New) The method of claim 21, wherein said processing comprises distinguishing a first peak intensity of said first optical signal of said first analyte from a second peak intensity of said second optical signal of said second analyte. (Drmanac: [0113] One approach for efficient low cost assay reaction is to apply reaction mixes in a thin layer such as droplets or layers of about one to a few microns, but preferably less than 10 microns, in size/thickness. In a lxlx 1 micron volume designated for a lxlmicron spot area, in 1 pmol/lul (luM concentration) there would be about 1000 molecules of probe in close proximity to 1-1000 copies of DNA. Using up to 100-300 molecules of probes would not significantly reduce the probe concentration and it would provide enough reacted probes to get significant signal. This approach may be used in an open reaction chamber that may stay open or closed for removal and washing of the probes and enzyme. [0165] In one aspect, methods of the invention permit large-scale measurement of splice variants with the following steps: (a) Prepare full length first strand cDNA for targeted or all mRNAs. (b) Circularize the generated full length ( or all) first strand cDNA molecules by incorporating an adapter sequence. ( c) By using primer complementary to the adapter sequence perform rolling circle replication (RCR) of cDNA circles to form concatemers with over 100 copies of initial cDNA. (d) Prepare random arrays by attaching RCR produced "cDNA balls" to glass surface coated with capture oligonucleotide complementary to a portion of the adapter sequence; with an advanced submicron patterned surface one mm2 can have between 1-10 million cDNA spots; note that the attachment is a molecular process and does not require robotic spotting of individual "cDNA balls" or concatemers. (e) Starting from pre-made universal libraries of 4096 6-mers and 1024 labeled 5-mers, use a sophisticated computer program and a simple robotic pipettor to create 40-80 pools of about 200 6-mers and 20 5-mers for testing all 10,000 or more exons in targeted 1000 or more up to all known genes in the sample organism/tissue. (f) In a 4-8 hour process, hybridize/ligate all probe pools in 40-80 cycles on the same random array using an automated microscope-like instrument with a sensitive 10-mega pixel)
Consider Claim 42.
Drmanac teaches: 42. (New) The method of claim 21, further comprising generating an oversampled image of said first optical signal and said second optical signal. (Drmanac: [0113] One approach for efficient low cost assay reaction is to apply reaction mixes in a thin layer such as droplets or layers of about one to a few microns, but preferably less than 10 microns, in size/thickness. In a lxlx 1 micron volume designated for a lxlmicron spot area, in 1 pmol/lul (luM concentration) there would be about 1000 molecules of probe in close proximity to 1-1000 copies of DNA. Using up to 100-300 molecules of probes would not significantly reduce the probe concentration and it would provide enough reacted probes to get significant signal. This approach may be used in an open reaction chamber that may stay open or closed for removal and washing of the probes and enzyme. [0165] In one aspect, methods of the invention permit large-scale measurement of splice variants with the following steps: (a) Prepare full length first strand cDNA for targeted or all mRNAs. (b) Circularize the generated full length ( or all) first strand cDNA molecules by incorporating an adapter sequence. ( c) By using primer complementary to the adapter sequence perform rolling circle replication (RCR) of cDNA circles to form concatemers with over 100 copies of initial cDNA. (d) Prepare random arrays by attaching RCR produced "cDNA balls" to glass surface coated with capture oligonucleotide complementary to a portion of the adapter sequence; with an advanced submicron patterned surface one mm2 can have between 1-10 million cDNA spots; note that the attachment is a molecular process and does not require robotic spotting of individual "cDNA balls" or concatemers. (e) Starting from pre-made universal libraries of 4096 6-mers and 1024 labeled 5-mers, use a sophisticated computer program and a simple robotic pipettor to create 40-80 pools of about 200 6-mers and 20 5-mers for testing all 10,000 or more exons in targeted 1000 or more up to all known genes in the sample organism/tissue. (f) In a 4-8 hour process, hybridize/ligate all probe pools in 40-80 cycles on the same random array using an automated microscope-like instrument with a sensitive 10-mega pixel)
Consider Claim 43.
Drmanac teaches: 43. (New) The method of claim 21, wherein said processing comprises applying nearest neighbor variable regression to said first optical signal and said second optical signal to distinguish said first analyte from said second analyte. (Drmanac: [0113] One approach for efficient low cost assay reaction is to apply reaction mixes in a thin layer such as droplets or layers of about one to a few microns, but preferably less than 10 microns, in size/thickness. In a lxlx 1 micron volume designated for a lxlmicron spot area, in 1 pmol/lul (luM concentration) there would be about 1000 molecules of probe in close proximity to 1-1000 copies of DNA. Using up to 100-300 molecules of probes would not significantly reduce the probe concentration and it would provide enough reacted probes to get significant signal. This approach may be used in an open reaction chamber that may stay open or closed for removal and washing of the probes and enzyme. [0165] In one aspect, methods of the invention permit large-scale measurement of splice variants with the following steps: (a) Prepare full length first strand cDNA for targeted or all mRNAs. (b) Circularize the generated full length ( or all) first strand cDNA molecules by incorporating an adapter sequence. ( c) By using primer complementary to the adapter sequence perform rolling circle replication (RCR) of cDNA circles to form concatemers with over 100 copies of initial cDNA. (d) Prepare random arrays by attaching RCR produced "cDNA balls" to glass surface coated with capture oligonucleotide complementary to a portion of the adapter sequence; with an advanced submicron patterned surface one mm2 can have between 1-10 million cDNA spots; note that the attachment is a molecular process and does not require robotic spotting of individual "cDNA balls" or concatemers. (e) Starting from pre-made universal libraries of 4096 6-mers and 1024 labeled 5-mers, use a sophisticated computer program and a simple robotic pipettor to create 40-80 pools of about 200 6-mers and 20 5-mers for testing all 10,000 or more exons in targeted 1000 or more up to all known genes in the sample organism/tissue. (f) In a 4-8 hour process, hybridize/ligate all probe pools in 40-80 cycles on the same random array using an automated microscope-like instrument with a sensitive 10-mega pixel)
Claim Rejections - 35 USC § 103
The following is a quotation of 35 U.S.C. 103(a) which forms the basis for all obviousness rejections set forth in this Office action:
(a) A patent may not be obtained though the invention is not identically disclosed or described as set forth in section 102 of this title, if the differences between the subject matter sought to be patented and the prior art are such that the subject matter as a whole would have been obvious at the time the invention was made to a person having ordinary skill in the art to which said subject matter pertains. Patentability shall not be negatived by the manner in which the invention was made.
Claims 21, 23, and 25-43 are rejected under 35 U.S.C. 103(a) as being unpatentable over Drmanac et. al. (US PGPub US 2017 /0152554 A1, hereby referred to as “Drmanac”, filed on February 6, 2017 with provisional priority dating to June 15, 2005), in view of Ryu et al. (US PGPub US2005/0239113 A1), hereby referred to as “Ryu”.
Consider Claim 21. (New)
Drmanac teaches:
21. (New) A method of distinguishing a first analyte from a second analyte, said method, comprising: (Drmanac: abstract, A high density DNA array comprising a patterned surface, said surface comprising a pattern of small DNA binding regions separated by a non-DNA binding surface, wherein the DNA binding regions comprise DNA capture chemistry and the non-DNA binding surface does not have the DNA capture chemistry wherein more than 50% of the DNA binding regions in the array have single informative DNA species. [0016]-[0018], [0035] FIG. 1B illustrates a section (1102) of a surface of a random array of single molecules, such as single stranded polynucleotides. Figure 1B)
(a) binding a first probe to said first analyte to generate a first optical signal; (Drmanac: [0017], [0035] FIG. 1B illustrates a section (1102) of a surface of a random array of single molecules, such as single stranded polynucleotides…. Some embodiments utilize a support with a grid of regions with DNA capture chemistry separated by surface without DNA capture chemistry, each region being 0.1-10 micrometer with center to center distance of about 0.2 to 20 um. In some embodiments, the source DNA is all sequence variants of given length 8 to 20 base. [0067] In one example each 6mm x 6mm array consists of 36 million 250-500 nm square regions at 1 micrometer pitch. Hydrophobic or other surface or physical barriers may be used to prevent mixing different reactions between unit arrays.)
-; and a second probe to said second analyte to generate a second optical signal (Drmanac: [0016] In one embodiment of this aspect, the step of identifying includes the steps of (a) hybridizing one or more probes from a first set of probes to the random array under conditions that permit the formation of perfectly matched duplexes between the one or more probes and complementary sequences on target concatemers; (b) hybridizing one or more probes from a second set of probes to the random array under conditions that permit the formation of perfectly matched duplexes between the one or more probes and complementary sequences on target concatemers; (c) ligating probes from the first and second sets hybridized to a target concatemer at contiguous sites; (d) identifying the sequences of the ligated first and second probes; and (e) repeating steps (a through (d) until the sequence of the target polynucleotide can be determined from the identities of the sequences of the ligated probes. [0117] In one aspect, a sequencing method for use with the invention for determining sequences in a plurality of DNA or RNA fragments comprises the following steps: (a) generating a plurality of polynucleotide molecules each comprising a concatemer of a DNA or RNA fragment; (b) forming a random array of polynucleotide molecules fixed to a surface at a density such that at least a majority of the target concatemers are optically resolvable; and (c) identifying a sequence of at least a portion of each DNA or RNA fragment in resolvable polynucleotides using at least one chemical reaction of an optically detectable reactant. [0153] Arrays and sequencing methods of the invention used may be used for large-scale identification of polymorphisms using mismatch cleavage techniques. Several approaches to mutation detection employ a heteroduplex in which the mismatch itself is utilized for cleavage recognition.)
(b) detecting said first optical signal and said second optical signal with an imaging system, (Drmanac: [0040] The proposed high density structured random DNA array chip will have capture oligonucleotides concentrated in small, segregated capture cells aligned into a rectangular grid formation (FIG. 5). Most importantly, each capture cell or binding site will be surrounded by an inert surface and will have a sufficient but limited number of capture molecules (100-400). Each capture molecule will bind one copy of the matching adaptor sequence on the RCR produced DNA concatemer. Since each concatemer contains over 1000 copies of the adapter sequence, it will quickly saturate the binding site upon contact and prevent other concatemers from binding, resulting in exclusive attachment of one RCR product per binding site or spot. Tie proper concentration of RCR products and sufficient reaction time will ensure that almost every spot on the array contains one and only one unique DNA target. [0153] Mismatches are usually repaired but the binding action of the enzymes can be used for the selection of fragments through a mobility shift in gel electrophoresis or by protection from exonucleases, [0220])
wherein said imaging system comprises a resolution of up to about 300 nanometers per pixel of said imaging system; and (Drmanac: [0035] In still another aspect, whenever optical microscopy is employed, for example with molecule-specific probes having fluorescent labels, a density is selected such that at least a majority of single molecules have a nearest neighbor distance of 300 nm or greater; and in another aspect, such density is selected to ensure that at least seventy percent of single molecules have a nearest neighbor distance of 300 nm or greater, or 400 nm or greater, or 500 nm or greater, or 600 nm or greater, or 700 nm or greater, or 800 nm or greater. [0042] Having 125-250 nm DNA sites in a regular grid with 250-500 nm center-to-center spacing will provide 20-80 times more DNA samples per surface than arrays with random attached DNA with spots of about 1000 nm in size and 20% usable occupancy. This will result in 20-80 fold lower reagent consumption and 20-80 fold faster readout. Furthermore, attaching RCR products onto this dense grid of capture probe spots ensures that each DNA ball is concentrated on a much smaller surface, increasing the signal and the speed of biochemical assays. Overall, the reduction of DNA attachment spots from 500 nm to 125 nm in size will result in up to 16 fold higher signal intensities. In short, the proposed DNA arrays will provide an order of magnitude lower cost, higher throughput and higher sensitivity than standard random DNA arrays.)
(c) processing a first peak intensity of said first optical signal at a first peak location and a second peak intensity of said second optical signal at a second peak location (Drmanac: [0221] By overlaying the images obtained from successive hybridization of3 probes, as shown in FIG. 4, it can be seen that most of the arrayed molecules that hybridized with the adaptor probe would only hybridize to either the amplicon 1 probe ( e.g. "A" in FIG. 4) or the amplicon 2 probe ( e.g. "B" in FIG. 4), with very few that would hybridize to both. This specific hybridization pattern demonstrates that each spot on the array contains only one type of sequence, [0234] "Microarray" or "array" refers to a solid phase support having a surface, usually planar or substantially planar, which carries an array of sites containing nucleic acids, such that each member site of the array comprises identical copies of immobilized oligonucleotides or polynucleotides and is spatially defined and not overlapping with other member sites of the array; that is, the sites are spatially discrete. In some cases, sites of a microarray may also be spaced apart as well as discrete; that is, different sites do not share boundaries, but are separated by inter-site regions, usually free of bound nucleic acids. Spatially defined hybridization sites may additionally be "addressable" in that its location and the identity of its immobilized oligonucleotide are known or predetermined, for example, prior to its use. [0016] In one embodiment of this aspect, the step of identifying includes the steps of (a) hybridizing one or more probes from a first set of probes to the random array under conditions that permit the formation of perfectly matched duplexes between the one or more probes and complementary sequences on target concatemers; (b) hybridizing one or more probes from a second set of probes to the random array under conditions that permit the formation of perfectly matched duplexes between the one or more probes and complementary sequences on target concatemers; (c) ligating probes from the first and second sets hybridized to a target concatemer at contiguous sites; (d) identifying the sequences of the ligated first and second probes; and (e) repeating steps (a through (d) until the sequence of the target polynucleotide can be determined from the identities of the sequences of the ligated probes.)
to distinguish said first analyte from said second analyte, wherein said first optical signal and said second optical signal at least partially overlap. (Drmanac: [0117] In one aspect, a sequencing method for use with the invention for determining sequences in a plurality of DNA or RNA fragments comprises the following steps: (a) generating a plurality of polynucleotide molecules each comprising a concatemer of a DNA or RNA fragment; (b) forming a random array of polynucleotide molecules fixed to a surface at a density such that at least a majority of the target concatemers are optically resolvable; and (c) identifying a sequence of at least a portion of each DNA or RNA fragment in resolvable polynucleotides using at least one chemical reaction of an optically detectable reactant. [0035] In still another aspect, whenever optical microscopy is employed, for example with molecule-specific probes having fluorescent labels, a density is selected such that at least a majority of single molecules have a nearest neighbor distance of 300 nm or greater; and in another aspect, such density is selected to ensure that at least seventy percent of single molecules have a nearest neighbor distance of 300 nm or greater, or 400 nm or greater, or 500 nm or greater, or 600 nm or greater, or 700 nm or greater, or 800 nm or greater. [0042] Having 125-250 nm DNA sites in a regular grid with 250-500 nm center-to-center spacing will provide 20-80 times more DNA samples per surface than arrays with random attached DNA with spots of about 1000 nm in size and 20% usable occupancy. This will result in 20-80 fold lower reagent consumption and 20-80 fold faster readout. Furthermore, attaching RCR products onto this dense grid of capture probe spots ensures that each DNA ball is concentrated on a much smaller surface, increasing the signal and the speed of biochemical assays. Overall, the reduction of DNA attachment spots from 500 nm to 125 nm in size will result in up to 16 fold higher signal intensities. In short, the proposed DNA arrays will provide an order of magnitude lower cost, higher throughput and higher sensitivity than standard random DNA arrays. [0052] Duplex region (214) of bridging oligonucleotide (210) contains at least a primer binding site for RCR and, in some embodiments, sequences that provide complements to a capture oligonucleotide, which may be the same or different from the primer binding site sequence, or which may overlap the primer binding site sequence. The length of capture oligonucleotides may vary widely, In one aspect, capture oligonucleotides and their complements in a bridging oligonucleotide have lengths in the range of from 10 to 100 nucleotides; and more preferably, in the range of from 10 to 40 nucleotides. In some embodiments, duplex region (214) may contain additional elements, such as an oligonucleotide tag, for example, for identifying the source nucleic acid from which its associated DNA fragment came. [0133] In 2-4 cycles extend to 4-6 base anchor for additional 2-4 bases run 16 different anchors per each array (32-64 physical cycles if 4 colors are used) to determine about 16 possible 8-mers (˜100 bases total) per each fragment (more then enough to map it to the reference (probability that a 100-mer will have a set of 10 8-mers is less than 1 in trillion trillions; (10exp-28). By combining data from different anchors scored in parallel on the same fragment in another array complete sequence of that fragment and by extension to entire genomes may be generated from overlapping 7-10-mers.)
Even if Drmanac does not specifically teach: wherein said first optical signal and said second optical signal at least partially overlap
Ryu teaches:
21. (New) A method of distinguishing a first analyte from a second analyte, said method, comprising: (Ryu: abstract, The present invention provides methods and devices for high sensitivity and high speed microarray optical imaging. The methods include using patterned excitation to obtain a series of images and analyzing the images to resolve probe intensities which reflect the hybridization or binding between target and probes. Probe feature information and patterned excitation (structured illumination) information are incorporated into the analysis. [0004]-[0010] [0004] In one aspect of the invention, a method for microarray detection is provided. In one aspect of the invention, methods and devices are provided for microarray detection using a series of structured, textured, or patterned excitation (referred herein as patterned excitation) images to achieve subpixel resolution in detecting probe intensities. The microarray can be a nucleic acid probe array such as a spotted array (e.g., with cDNA or short oligonucleotide probes), high density in situ synthesized arrays (such as the GeneChip® high density probe arrays manufactured by Affymetrix, Inc., Santa Clara, Calif.). The microarrays can also be protein or peptide arrays. Typically, the density of the microarrays is higher than 500, 5000, 50000, or 500,000 different probes per cm2. The feature size of the probes (synthesis area or immobilization area) is typically smaller than 500, 150, 25, 9, 5, 3 or 1 μm2.)
(a) binding a first probe to said first analyte to generate a first optical signal and a second probe to said second analyte to generate a second optical signal; (Ryu: [0016] FIG. 3. Excitation using a single laser beam. The figure shows a single laser beam used as an excitation source. The angle θ defined in the figure is typically 75 degrees in the experiment. [0017] FIG. 4. CCD image of an Affymetrix standard microarray with 18 μm probe spacing excited by a single laser beam. A single laser beam (FIG. 3) with 6 mW optical power and 0.7 mm beam diameter was used as an excitation source and the CCD imaging setup in FIG. 2 was used to record an image of a standard Affymetrix array. The angle between the beam and a line perpendicular to the horizontal surface (the angle θ in FIG. 3) was 75 degrees. This results in an estimated optical power of 1.6 mW/mm2 on the top surface of the fused silica scan window. In this image acquisition, the gain of the CCD camera was turned on to enhance the detection of the low intensity probes, while saturating the high and mid intensity probes. The image shown is the average of 30 repeated acquisitions, each with 520 msec exposure time.)
(b) detecting said first optical signal and said second optical signal with an imaging system, (Ryu: [0018] FIG. 5. Direct imaging of the interference pattern. To directly verify the generation of the high resolution optical pattern formed by the interference of two laser beams, a high power microscope objective with 100× magnification (model NT38-344, Edmund Industrial Optics, Barrington, N.J.) was used. The objective was positioned such that the focal plane of the objective lies in the region where the two beams overlap (bright spot in the photograph). [0019] FIG. 6. Magnified image of the produced interference pattern projected onto the wall of the laboratory. Interference pattern made by a pair of laser beams is a sinusoidal brightness grating, also known as fringe pattern. The distance between adjacent two peak (or valley) intensities (the distance D defined in the figure) corresponds to the pitch or feature size of the interference pattern. Combined with the direction of the fringe pattern, this feature size of the interference pattern defines the spatial frequency of the pattern, that is, the vector k in the figure, in units of μm−1. As will be explained in the following figure, the vector k is determined by the directions and the wavelength of the beams.)
wherein said imaging system comprises a resolution of up to about 300 nanometers per pixel of said imaging system; and (Ryu: [0023] FIG. 10. The brightness of the pixel that contains the calibration sphere encodes the sub-pixel position of the interference pattern. The box represents an area corresponding to a single pixel of the CCD camera that has the size of 1.6 μm in the image plane and the circle in the figure left represents a 100 nm diameter fluorescent sphere located inside the pixel. The figure on the left also shows a particular interference pattern overlaid on top of the sphere. The figure on the right shows the gray value of the same pixel on the left, indicating the brightness of the sphere illuminated by this particular excitation pattern on the left. If the interference pattern is translated relative to the fixed sphere, this will result in the systematic change in the brightness of the sphere, which is demonstrated experimentally in the next figure.)
(c) processing a first peak intensity of said first optical signal at a first peak location and a second peak intensity of said second optical signal at a second peak location (Ryu: [0008], [0019] FIG. 6. Magnified image of the produced interference pattern projected onto the wall of the laboratory. Interference pattern made by a pair of laser beams is a sinusoidal brightness grating, also known as fringe pattern. The distance between adjacent two peak (or valley) intensities (the distance D defined in the figure) corresponds to the pitch or feature size of the interference pattern. Combined with the direction of the fringe pattern, this feature size of the interference pattern defines the spatial frequency of the pattern, that is, the vector k in the figure, in units of μm−1. As will be explained in the following figure, the vector k is determined by the directions and the wavelength of the beams.)
to distinguish said first analyte from said second analyte, wherein said first optical signal and said second optical signal at least partially overlap. (Examiner Note: the spatial resolving of sub-pixel intensities in the CCD image demonstrates overlapping optical signals to assess and distinguish different adjacent peak intensities; Ryu: [0008] Information about different excitation patterns may include spatial frequency information such as orientation and spacing between adjacent peak intensities. In some embodiments, the analyzing steps include extracting cosine parameters to obtain IDC, (DC component of intensity values), IAC (AC component of intensity values), and φ (timing information, where the peak intensity appears) of pixel intensities. In a preferred embodiment, the analyzing step includes constructing a system of linear equations that relate the pixel intensities, subpixel weighting functions, and unknown subpixel intensities. For example, the linear equations may be as follows:
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where Ii (m,n) is the unknown subpixel intensities; Wi(m,n, k) is the weighting function within i-th pixel for k-th frame at a subpixel location (m,n); and bi(k) is the sequence of gray intensity values of i-th pixel. The equations may be solved to obtain subpixel intensities. [0009] The weighting function Wi(m,n, k) can be calculated, for example, using pattern calibration parameters as: EDC+EAC·cos(kx·x+ky·y+φ), where EDC and EAC are DC and AC components of the pattern intensities, respectively; kx and ky are x and y components of the pattern spatial frequency, respectfully; and the φ represents subpixel position of the pattern. Alternatively, the weighting function Wi(m,n, k) is calculated by solving the equation
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using data obtained with reference samples with known subpixel intensities. [0010] In another aspect of the invention, the intensity values are estimated using optimization methods. In some embodiments, the subpixel intensities are estimated with probe feature information as constraints. For example, the regularity of the probe features is used as constraints. The dynamic range the probe intensities can also be used. One particularly preferred method is to minimize
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Liner programmingg is a preferred method for estimating the intensity values. [0026]-[0030], [0105]-[0114], Figures 14-17, [0030] FIG. 17 demonstrates spatially resolving subpixel probe intensities. The figure on the left is a conventional CCD image of a small region of a microarray with 5 μm probe spacing imaged using 6.4 μm image pixel size under conventional CCD imaging conditions; the image at the middle is a computer reconstructed image of the same region of the array acquired using the same lens and CCD with pattern excitation. Comparing the two images demonstrates the improvement in resolution, resolving subpixel intensities with the patterned excitation. The image on the right was acquired using 1.6 μm pixel size under conventional CCD imaging condition, showing a good correspondence with the patterned excitation image acquired at 6.4 μm pixel.)
It would have been obvious before the effective filing date of the claimed invention was made to one of ordinary skill in the art to modify Drmanac with Ryu as they are both directed towards methods and devices for high sensitivity optical imaging and analysis. The determination of obviousness is predicated upon the following findings: One skilled in the art would have been motivated to modify Drmanac in in order to leverage known techniques for in situ imaging of target-probe hybridization that are disclosed by Ryu. Furthermore, the prior art collectively includes each element claimed (though not all in the same reference), and one of ordinary skill in the art could have combined the elements in the manner explained above using known engineering design, interface and programming techniques, without changing a “fundamental” operating principle of Drmanac for large-scale molecular imaging analysis, while the teaching of Ryu continues to perform the same function as originally taught prior to being combined, in order to produce the repeatable and predictable result of ensuring improved optimization of intensity values to more easily distinguish target-probe combinations in the overall field of in situ hybridization. It is for at least the aforementioned reasons that the examiner has reached a conclusion of obviousness with respect to the claim in question.
Consider Claim 23.
The combination of Drmanac and Ryu teaches: 23. (New) The method of claim 21, wherein said first probe is bound to said first analyte in a first cycle, and wherein said second probe is bound to said second analyte in a second cycle. (Drmanac: [0016] In one embodiment of this aspect, the step of identifying includes the steps of (a) hybridizing one or more probes from a first set of probes to the random array under conditions that permit the formation of perfectly matched duplexes between the one or more probes and complementary sequences on target concatemers; (b) hybridizing one or more probes from a second set of probes to the random array under conditions that permit the formation of perfectly matched duplexes between the one or more probes and complementary sequences on target concatemers; (c) ligating probes from the first and second sets hybridized to a target concatemer at contiguous sites; (d) identifying the sequences of the ligated first and second probes; and (e) repeating steps (a through (d) until the sequence of the target polynucleotide can be determined from the identities of the sequences of the ligated probes. [0117], [0153], [0051] In another aspect, primer extension from a genomic DNA template is used to generate a linear amplification of selected sequences greater than 10 kilobases surrounding genomic regions of interest. For example, to create a population of defined-sized targets, 20 cycles of linear amplification is performed with a forward primer followed by 20 cycles with a reverse primer. Before applying the second primer, the first primer is removed with a standard column for long DNA purification or degraded if a few uracil bases are incorporated.)
Consider Claim 25.
The combination of Drmanac and Ryu teaches: 25. (New) The method of claim 21, wherein said first optical signal and said second optical signal are overlapping. (Drmanac: [0117] In one aspect, a sequencing method for use with the invention for determining sequences in a plurality of DNA or RNA fragments comprises the following steps: (a) generating a plurality of polynucleotide molecules each comprising a concatemer of a DNA or RNA fragment; (b) forming a random array of polynucleotide molecules fixed to a surface at a density such that at least a majority of the target concatemers are optically resolvable; and (c) identifying a sequence of at least a portion of each DNA or RNA fragment in resolvable polynucleotides using at least one chemical reaction of an optically detectable reactant. [0035] In still another aspect, whenever optical microscopy is employed, for example with molecule-specific probes having fluorescent labels, a density is selected such that at least a majority of single molecules have a nearest neighbor distance of 300 nm or greater; and in another aspect, such density is selected to ensure that at least seventy percent of single molecules have a nearest neighbor distance of 300 nm or greater, or 400 nm or greater, or 500 nm or greater, or 600 nm or greater, or 700 nm or greater, or 800 nm or greater. [0042] Having 125-250 nm DNA sites in a regular grid with 250-500 nm center-to-center spacing will provide 20-80 times more DNA samples per surface than arrays with random attached DNA with spots of about 1000 nm in size and 20% usable occupancy. This will result in 20-80 fold lower reagent consumption and 20-80 fold faster readout. Furthermore, attaching RCR products onto this dense grid of capture probe spots ensures that each DNA ball is concentrated on a much smaller surface, increasing the signal and the speed of biochemical assays. Overall, the reduction of DNA attachment spots from 500 nm to 125 nm in size will result in up to 16 fold higher signal intensities. In short, the proposed DNA arrays will provide an order of magnitude lower cost, higher throughput and higher sensitivity than standard random DNA arrays.)
Consider Claim 26.
The combination of Drmanac and Ryu teaches: 26. (New) The method of claim 21, wherein said first analyte and said second analyte are different analytes.(Drmanac: [0009] Compositions of the invention in one form include random arrays of a plurality of different single molecules disposed on a surface, where the single molecules each comprise a macromolecular structure and at least one analyte, such that each macromolecular structure comprises a plurality of attachment functionalities that are capable of forming bonds with one or more functionalities on the surface. In one aspect, the analyte is a component of the macromolecular structure, and in another aspect, the analyte is attached to the macromolecular structure by a linkage between a unique functionality on such structure and a reactive group or attachment moiety on the analyte.)
Consider Claim 27.
The combination of Drmanac and Ryu teaches: 26. (New) The method of claim 21, wherein said first analyte and said second analyte are the same analytes. (Drmanac: [0009] In another aspect, compositions of the invention include random arrays of single molecules disposed on a surface, where the single molecules each comprise a concatemer of at least one target polynucleotide and each is attached to the surface by linkages formed between one or more functionalities on the surface and complementary functionalities on the concatemer. In another form, compositions of the invention include random arrays of single molecules disposed on a surface, where the single molecules each comprise a concatemer of at least one target polynucleotide and at least one adaptor oligonucleotide and each is attached to such surface by the formation of duplexes between capture oligonucleotides on the surface and the attachment oligonucleotides in the concatemer)
Consider Claim 28.
The combination of Drmanac and Ryu teaches: 28. (New) The method of claim 21, wherein said first optical signal or said second optical signal comprises a fluorescent optical signal. (Drmanac: [0035] In still another aspect, whenever optical microscopy is employed, for example with molecule-specific probes having fluorescent labels, a density is selected such that at least a majority of single molecules have a nearest neighbor distance of 300 nm or greater; and in another aspect, such density is selected to ensure that at least seventy percent of single molecules have a nearest neighbor distance of 300 nm or greater, or 400 nm or greater, or 500 nm or greater, or 600 nm or greater, or 700 nm or greater, or 800 nm or greater.)
Consider Claim 29.
The combination of Drmanac and Ryu teaches: 29. (New) The method of claim 21, wherein said first analyte or said second analyte comprises a nucleic acid molecule. (Drmanac: [0234] "Microarray" or "array" refers to a solid phase support having a surface, usually planar or substantially planar, which carries an array of sites containing nucleic acids, such that each member site of the array comprises identical copies of immobilized oligonucleotides or polynucleotides and is spatially defined and not overlapping with other member sites of the array; that is, the sites are spatially discrete. In some cases, sites of a microarray may also be spaced apart as well as discrete; that is, different sites do not share boundaries, but are separated by inter-site regions, usually free of bound nucleic acids. Spatially defined hybridization sites may additionally be "addressable" in that its location and the identity of its immobilized oligonucleotide are known or predetermined, for example, prior to its use.)
Consider Claim 30.
The combination of Drmanac and Ryu teaches: 30. (New) The method of claim 29, wherein said nucleic acid molecule comprises at least 1 kilo- base (kb). (Drmanac: [0234] "Microarray" or "array" refers to a solid phase support having a surface, usually planar or substantially planar, which carries an array of sites containing nucleic acids, such that each member site of the array comprises identical copies of immobilized oligonucleotides or polynucleotides and is spatially defined and not overlapping with other member sites of the array; that is, the sites are spatially discrete. In some cases, sites of a microarray may also be spaced apart as well as discrete; that is, different sites do not share boundaries, but are separated by inter-site regions, usually free of bound nucleic acids. Spatially defined hybridization sites may additionally be "addressable" in that its location and the identity of its immobilized oligonucleotide are known or predetermined, for example, prior to its use.)
Consider Claim 31.
The combination of Drmanac and Ryu teaches: 31. (New) The method of claim 21, wherein said first analyte or said second analyte comprises a protein or a polypeptide. (Drmanac: [0117] In another embodiment, such optically detectable reactant is a nucleoside triphosphate, e.g. a fluorescently labeled nucleoside triphosphate that may be used to extend an oligonucleotide hybridized to a concatemer. In another embodiment, such optically detectable reagent is an oligonucleotide formed by ligating a first and second oligonucleotides that form adjacent duplexes on a concatemer. In another embodiment, such chemical reaction is synthesis of DNA or RNA, e.g. by extending a primer hybridized to a concatemer. In yet another embodiment, the above optically detectable reactant is a nucleic acid binding oligopeptide or polypeptide or protein.)
Consider Claim 32.
The combination of Drmanac and Ryu teaches: 32. (New) The method of claim 21, wherein said first probe or said second probe comprises a labelled nucleotide, an aptamer, an antibody, a polypeptide, an oligonucleotide, or any combination thereof. (Drmanac: [0117] In another embodiment, such optically detectable reactant is a nucleoside triphosphate, e.g. a fluorescently labeled nucleoside triphosphate that may be used to extend an oligonucleotide hybridized to a concatemer. In another embodiment, such optically detectable reagent is an oligonucleotide formed by ligating a first and second oligonucleotides that form adjacent duplexes on a concatemer. In another embodiment, such chemical reaction is synthesis of DNA or RNA, e.g. by extending a primer hybridized to a concatemer. In yet another embodiment, the above optically detectable reactant is a nucleic acid binding oligopeptide or polypeptide or protein.)
Consider Claim 33.
The combination of Drmanac and Ryu teaches: 33. (New) The method of claim 21, wherein said first analyte or said second analyte is immobilized to said substrate. (Drmanac: [0234] “Microarray” or “array” refers to a solid phase support having a surface, usually planar or substantially planar, which carries an array of sites containing nucleic acids, such that each member site of the array comprises identical copies of immobilized oligonucleotides or polynucleotides and is spatially defined and not overlapping with other member sites of the array; that is, the sites are spatially discrete. In some cases, sites of a microarray may also be spaced apart as well as discrete; that is, different sites do not share boundaries, but are separated by inter-site regions, usually free of bound nucleic acids. Spatially defined hybridization sites may additionally be “addressable” in that its location and the identity of its immobilized oligonucleotide are known or predetermined, for example, prior to its use. In some aspects, the oligonucleotides or polynucleotides are single stranded and are covalently attached to the solid phase support, usually by a 5′-end or a 3′-end. In other aspects, oligonucleotides or polynucleotides are attached to the solid phase support non-covalently, e.g. by a biotin-streptavidin linkage, hybridization to a capture oligonucleotide that is covalently bound, and the like)
Consider Claim 34.
The combination of Drmanac and Ryu teaches: 34. (New) The method of claim 21, wherein said first analyte or said second analyte comprises a plurality of analytes. (Drmanac: [0035] In still another aspect, whenever optical microscopy is employed, for example with molecule-specific probes having fluorescent labels, a density is selected such that at least a majority of single molecules have a nearest neighbor distance of 300 nm or greater; and in another aspect, such density is selected to ensure that at least seventy percent of single molecules have a nearest neighbor distance of 300 nm or greater, or 400 nm or greater, or 500 nm or greater, or 600 nm or greater, or 700 nm or greater, or 800 nm or greater. [0042] Having 125-250 nm DNA sites in a regular grid with 250-500 nm center-to-center spacing will provide 20-80 times more DNA samples per surface than arrays with random attached DNA with spots of about 1000 nm in size and 20% usable occupancy. This will result in 20-80 fold lower reagent consumption and 20-80 fold faster readout. Furthermore, attaching RCR products onto this dense grid of capture probe spots ensures that each DNA ball is concentrated on a much smaller surface, increasing the signal and the speed of biochemical assays. Overall, the reduction of DNA attachment spots from 500 nm to 125 nm in size will result in up to 16 fold higher signal intensities. In short, the proposed DNA arrays will provide an order of magnitude lower cost, higher throughput and higher sensitivity than standard random DNA arrays.)
Consider Claim 35.
The combination of Drmanac and Ryu teaches: 35. (New) The method of claim 34, wherein said plurality of analytes are provided on said substrate at a density of at least 2 molecules/pm2. (Drmanac: [0035] In still another aspect, whenever optical microscopy is employed, for example with molecule-specific probes having fluorescent labels, a density is selected such that at least a majority of single molecules have a nearest neighbor distance of 300 nm or greater; and in another aspect, such density is selected to ensure that at least seventy percent of single molecules have a nearest neighbor distance of 300 nm or greater, or 400 nm or greater, or 500 nm or greater, or 600 nm or greater, or 700 nm or greater, or 800 nm or greater. [0042] Having 125-250 nm DNA sites in a regular grid with 250-500 nm center-to-center spacing will provide 20-80 times more DNA samples per surface than arrays with random attached DNA with spots of about 1000 nm in size and 20% usable occupancy. This will result in 20-80 fold lower reagent consumption and 20-80 fold faster readout. Furthermore, attaching RCR products onto this dense grid of capture probe spots ensures that each DNA ball is concentrated on a much smaller surface, increasing the signal and the speed of biochemical assays. Overall, the reduction of DNA attachment spots from 500 nm to 125 nm in size will result in up to 16 fold higher signal intensities. In short, the proposed DNA arrays will provide an order of magnitude lower cost, higher throughput and higher sensitivity than standard random DNA arrays.)
Consider Claim 36.
The combination of Drmanac and Ryu teaches: 36. (New) The method of claim 34, wherein said plurality of analytes are provided on said substrate at a density of at least 4 molecules/pm2. (Drmanac: [0035] In still another aspect, whenever optical microscopy is employed, for example with molecule-specific probes having fluorescent labels, a density is selected such that at least a majority of single molecules have a nearest neighbor distance of 300 nm or greater; and in another aspect, such density is selected to ensure that at least seventy percent of single molecules have a nearest neighbor distance of 300 nm or greater, or 400 nm or greater, or 500 nm or greater, or 600 nm or greater, or 700 nm or greater, or 800 nm or greater. [0042] Having 125-250 nm DNA sites in a regular grid with 250-500 nm center-to-center spacing will provide 20-80 times more DNA samples per surface than arrays with random attached DNA with spots of about 1000 nm in size and 20% usable occupancy. This will result in 20-80 fold lower reagent consumption and 20-80 fold faster readout. Furthermore, attaching RCR products onto this dense grid of capture probe spots ensures that each DNA ball is concentrated on a much smaller surface, increasing the signal and the speed of biochemical assays. Overall, the reduction of DNA attachment spots from 500 nm to 125 nm in size will result in up to 16 fold higher signal intensities. In short, the proposed DNA arrays will provide an order of magnitude lower cost, higher throughput and higher sensitivity than standard random DNA arrays.)
Consider Claim 37.
The combination of Drmanac and Ryu teaches: 37. (New) The method of claim 34, wherein said plurality of analytes are provided on said substrate at a density of at least 8 molecules/pm2. (Drmanac: [0035] In still another aspect, whenever optical microscopy is employed, for example with molecule-specific probes having fluorescent labels, a density is selected such that at least a majority of single molecules have a nearest neighbor distance of 300 nm or greater; and in another aspect, such density is selected to ensure that at least seventy percent of single molecules have a nearest neighbor distance of 300 nm or greater, or 400 nm or greater, or 500 nm or greater, or 600 nm or greater, or 700 nm or greater, or 800 nm or greater. [0042] Having 125-250 nm DNA sites in a regular grid with 250-500 nm center-to-center spacing will provide 20-80 times more DNA samples per surface than arrays with random attached DNA with spots of about 1000 nm in size and 20% usable occupancy. This will result in 20-80 fold lower reagent consumption and 20-80 fold faster readout. Furthermore, attaching RCR products onto this dense grid of capture probe spots ensures that each DNA ball is concentrated on a much smaller surface, increasing the signal and the speed of biochemical assays. Overall, the reduction of DNA attachment spots from 500 nm to 125 nm in size will result in up to 16 fold higher signal intensities. In short, the proposed DNA arrays will provide an order of magnitude lower cost, higher throughput and higher sensitivity than standard random DNA arrays.)
Consider Claim 38.
The combination of Drmanac and Ryu teaches: 38. (New) The method of claim 21, wherein said first optical signal or said second optical signal comprises a sequence of optical signals. (Drmanac: [0036] Other factors in selecting sequences for capture oligonucleotides are similar to those considered in selecting primers, hybridization probes, oligonucleotide tags, and the like, for which there is ample guidance, as evidenced by the references cited below in the Definitions section. In some embodiments, a discrete spaced apart region may contain more than one kind of capture oligonucleotide, and each different capture oligonucleotide may have a different length and sequence. In one aspect of embodiments employing regular arrays of discrete spaced apart regions, sequences of capture oligonucleotides are selected so that sequences of capture oligonucleotide at nearest neighbor regions have different sequences. In a rectilinear array, such configurations are achieved by rows of alternating sequence types. In other embodiments, a surface may have a plurality of subarrays of discrete spaced apart regions wherein each different subarray has capture oligonucleotides with distinct nucleotide sequences different from those of the other subarrays. A plurality of subarrays may include 2 subarrays, or 4 or fewer subarrays, or 8 or fewer subarrays, or 16 or fewer subarrays, or 32 or fewer subarrays, or 64 of fewer subarrays. In still other embodiments, a surface may include 5000 or fewer subarrays. In one aspect, capture oligonucleotides are attached to the surface of an array by a spacer molecule, e.g. polyethylene glycol, or like inert chain, as is done with microarrays, in order to minimize undesired affects of surface groups or interactions with the capture oligonucleotides or other reagents.)
Consider Claim 39.
The combination of Drmanac and Ryu teaches: 39. (New) The method of claim 21, wherein said sequence of optical signals are determined or obtained over a plurality of cycles. (Drmanac: [0051] In another aspect, primer extension from a genomic DNA template is used to generate a linear amplification of selected sequences greater than 10 kilobases surrounding genomic regions of interest. For example, to create a population of defined-sized targets, 20 cycles of linear amplification is performed with a forward primer followed by 20 cycles with a reverse primer. Before applying the second primer, the first primer is removed with a standard column for long DNA purification or degraded if a few uracil bases are incorporated. [0128] In a similar way the 6 bases from the right side of the 12mer can be decoded by using a fixed oligonucleotide and 5-prime labeled probes. In the above described system 6 cycles are required to define 6 bases of one side of the 12mer. With redundant cycle analysis of bases distant to the ligation site this may increase to 7 or 8 cycles. In total then, complete sequencing of the 12mer could be accomplished with 12-16 cycles of ligation. Partial or complete sequencing of arrayed DNA by combining two distinct types of libraries of detector probes. In this approach one set has probes of the general type N3-8B4-6 (anchors) that are ligated with the first 2 or 3 or 4 probes/probe pools from the set BN6-8, NBN5-7, N2BN4-6, and N3BN3-5.The main requirement is to test in a few cycles a probe from the first set with 2-4 or even more probes from the second set to read longer continuous sequence such as 5-6+3-4=8-10 in just 3-4 cycles.)
Consider Claim 40.
The combination of Drmanac and Ryu teaches: 40. (New) The method of claim 21, wherein said first probe comprises a first probe set, and wherein said second probe comprises a second probe set. (Drmanac: [0016] In one embodiment of this aspect, the step of identifying includes the steps of (a) hybridizing one or more probes from a first set of probes to the random array under conditions that permit the formation of perfectly matched duplexes between the one or more probes and complementary sequences on target concatemers; (b) hybridizing one or more probes from a second set of probes to the random array under conditions that permit the formation of perfectly matched duplexes between the one or more probes and complementary sequences on target concatemers; (c) ligating probes from the first and second sets hybridized to a target concatemer at contiguous sites; (d) identifying the sequences of the ligated first and second probes; and (e) repeating steps (a through (d) until the sequence of the target polynucleotide can be determined from the identities of the sequences of the ligated probes. [0117] In one aspect, a sequencing method for use with the invention for determining sequences in a plurality of DNA or RNA fragments comprises the following steps: (a) generating a plurality of polynucleotide molecules each comprising a concatemer of a DNA or RNA fragment; (b) forming a random array of polynucleotide molecules fixed to a surface at a density such that at least a majority of the target concatemers are optically resolvable; and (c) identifying a sequence of at least a portion of each DNA or RNA fragment in resolvable polynucleotides using at least one chemical reaction of an optically detectable reactant. [0153] Arrays and sequencing methods of the invention used may be used for large-scale identification of polymorphisms using mismatch cleavage techniques. Several approaches to mutation detection employ a heteroduplex in which the mismatch itself is utilized for cleavage recognition.)
Consider Claim 41.
The combination of Drmanac and Ryu teaches: 41. (New) The method of claim 21, wherein said processing comprises distinguishing a first peak intensity of said first optical signal of said first analyte from a second peak intensity of said second optical signal of said second analyte. (Drmanac: [0113] One approach for efficient low cost assay reaction is to apply reaction mixes in a thin layer such as droplets or layers of about one to a few microns, but preferably less than 10 microns, in size/thickness. In a lxlx 1 micron volume designated for a lxlmicron spot area, in 1 pmol/lul (luM concentration) there would be about 1000 molecules of probe in close proximity to 1-1000 copies of DNA. Using up to 100-300 molecules of probes would not significantly reduce the probe concentration and it would provide enough reacted probes to get significant signal. This approach may be used in an open reaction chamber that may stay open or closed for removal and washing of the probes and enzyme. [0165] In one aspect, methods of the invention permit large-scale measurement of splice variants with the following steps: (a) Prepare full length first strand cDNA for targeted or all mRNAs. (b) Circularize the generated full length ( or all) first strand cDNA molecules by incorporating an adapter sequence. ( c) By using primer complementary to the adapter sequence perform rolling circle replication (RCR) of cDNA circles to form concatemers with over 100 copies of initial cDNA. (d) Prepare random arrays by attaching RCR produced "cDNA balls" to glass surface coated with capture oligonucleotide complementary to a portion of the adapter sequence; with an advanced submicron patterned surface one mm2 can have between 1-10 million cDNA spots; note that the attachment is a molecular process and does not require robotic spotting of individual "cDNA balls" or concatemers. (e) Starting from pre-made universal libraries of 4096 6-mers and 1024 labeled 5-mers, use a sophisticated computer program and a simple robotic pipettor to create 40-80 pools of about 200 6-mers and 20 5-mers for testing all 10,000 or more exons in targeted 1000 or more up to all known genes in the sample organism/tissue. (f) In a 4-8 hour process, hybridize/ligate all probe pools in 40-80 cycles on the same random array using an automated microscope-like instrument with a sensitive 10-mega pixel)
Consider Claim 42.
The combination of Drmanac and Ryu teaches: 42. (New) The method of claim 21, further comprising generating an oversampled image of said first optical signal and said second optical signal. (Drmanac: [0113] One approach for efficient low cost assay reaction is to apply reaction mixes in a thin layer such as droplets or layers of about one to a few microns, but preferably less than 10 microns, in size/thickness. In a lxlx 1 micron volume designated for a lxlmicron spot area, in 1 pmol/lul (luM concentration) there would be about 1000 molecules of probe in close proximity to 1-1000 copies of DNA. Using up to 100-300 molecules of probes would not significantly reduce the probe concentration and it would provide enough reacted probes to get significant signal. This approach may be used in an open reaction chamber that may stay open or closed for removal and washing of the probes and enzyme. [0165] In one aspect, methods of the invention permit large-scale measurement of splice variants with the following steps: (a) Prepare full length first strand cDNA for targeted or all mRNAs. (b) Circularize the generated full length ( or all) first strand cDNA molecules by incorporating an adapter sequence. ( c) By using primer complementary to the adapter sequence perform rolling circle replication (RCR) of cDNA circles to form concatemers with over 100 copies of initial cDNA. (d) Prepare random arrays by attaching RCR produced "cDNA balls" to glass surface coated with capture oligonucleotide complementary to a portion of the adapter sequence; with an advanced submicron patterned surface one mm2 can have between 1-10 million cDNA spots; note that the attachment is a molecular process and does not require robotic spotting of individual "cDNA balls" or concatemers. (e) Starting from pre-made universal libraries of 4096 6-mers and 1024 labeled 5-mers, use a sophisticated computer program and a simple robotic pipettor to create 40-80 pools of about 200 6-mers and 20 5-mers for testing all 10,000 or more exons in targeted 1000 or more up to all known genes in the sample organism/tissue. (f) In a 4-8 hour process, hybridize/ligate all probe pools in 40-80 cycles on the same random array using an automated microscope-like instrument with a sensitive 10-mega pixel)
Consider Claim 43.
The combination of Drmanac and Ryu teaches: 43. (New) The method of claim 21, wherein said processing comprises applying nearest neighbor variable regression to said first optical signal and said second optical signal to distinguish said first analyte from said second analyte. (Drmanac: [0113] One approach for efficient low cost assay reaction is to apply reaction mixes in a thin layer such as droplets or layers of about one to a few microns, but preferably less than 10 microns, in size/thickness. In a lxlx 1 micron volume designated for a lxlmicron spot area, in 1 pmol/lul (luM concentration) there would be about 1000 molecules of probe in close proximity to 1-1000 copies of DNA. Using up to 100-300 molecules of probes would not significantly reduce the probe concentration and it would provide enough reacted probes to get significant signal. This approach may be used in an open reaction chamber that may stay open or closed for removal and washing of the probes and enzyme. [0165] In one aspect, methods of the invention permit large-scale measurement of splice variants with the following steps: (a) Prepare full length first strand cDNA for targeted or all mRNAs. (b) Circularize the generated full length ( or all) first strand cDNA molecules by incorporating an adapter sequence. ( c) By using primer complementary to the adapter sequence perform rolling circle replication (RCR) of cDNA circles to form concatemers with over 100 copies of initial cDNA. (d) Prepare random arrays by attaching RCR produced "cDNA balls" to glass surface coated with capture oligonucleotide complementary to a portion of the adapter sequence; with an advanced submicron patterned surface one mm2 can have between 1-10 million cDNA spots; note that the attachment is a molecular process and does not require robotic spotting of individual "cDNA balls" or concatemers. (e) Starting from pre-made universal libraries of 4096 6-mers and 1024 labeled 5-mers, use a sophisticated computer program and a simple robotic pipettor to create 40-80 pools of about 200 6-mers and 20 5-mers for testing all 10,000 or more exons in targeted 1000 or more up to all known genes in the sample organism/tissue. (f) In a 4-8 hour process, hybridize/ligate all probe pools in 40-80 cycles on the same random array using an automated microscope-like instrument with a sensitive 10-mega pixel)
Conclusion
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TAHMINA N. ANSARI
Examiner
Art Unit 2674
January 22, 2026
/TAHMINA N ANSARI/Primary Examiner, Art Unit 2674