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 .
Information Disclosure Statement
The information disclosure statement (IDS) filed 04 December 2023 is considered, initialed, and attached hereto.
Claim Status
Claims 1-77 are canceled.
Claims 78-97 are pending and under examination.
Specification
The use of the terms 9°N, New England Biolabs, Ampligase, Epicentre, Vent, KlenTaq, Cascade blue, Texas red, Sigma, EMD Millipore, TrueBlack, Biotium, Amersham, Bodipy, Alexa Fluor, Molecular Probes, Marina Blue, Oregon Green, Clarity, Visium, 10x Genomics, Label-IT, Mirus Bio, Triton X-100, Tween-20, Teflon, Zeonor, Illumina, Megalign, DNASTAR, 454 Life Sciences, SOLiD, Life Technologies, TruSeq, HiSeq, HeliScope, PacBio, Pacific Biosciences, Ion Torrent, Complete Genomics, and Oxford Nanopore Technologies, which are trade names or marks used in commerce, has been noted in this application. The terms should be accompanied by the generic terminology; furthermore the terms should be capitalized wherever it appears or, where appropriate, include a proper symbol indicating use in commerce such as ™, SM , or ® following the term.
Although the use of trade names and marks used in commerce (i.e., trademarks, service marks, certification marks, and collective marks) are permissible in patent applications, the proprietary nature of the marks should be respected and every effort made to prevent their use in any manner which might adversely affect their validity as commercial marks.
Claim Rejections - 35 USC § 112(b) - Indefiniteness
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claims 83 and 92-94 are rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
Claim 83 recites the limitation "the concentration of DdrB protein" in line 1. There is insufficient antecedent basis for this limitation in the claim. For the purpose of examination, claim 83 is interpreted as the method of claim 78 wherein the single strand annealing protein is DdrB protein and the concentration of the DdrB protein used is at least 10nm.
Claim 92 recites the limitation "the substrate" in line 3. There is insufficient antecedent basis for this limitation in the claim. Claims 93 and 94 are also rejected due to their dependency on claim 92.
Claim 93 recites the limitation "the second nucleic acid strand" in line 1. There is insufficient antecedent basis for this limitation in the claim. For the purpose of examination, “the second nucleic acid strand” in claim 93 is interpreted as referring to a nucleic acid strand that comprises the second nucleic acid sequence. Claim 94 is also rejected due to its dependency on claim 93.
Claim 94 recites the limitation “the second nucleic acid strand” in lines 2-3 and 5-6. There is insufficient antecedent basis for this limitation in the claim. For the purpose of examination, “the second nucleic acid strand” in claim 94 is interpreted the same as it is interpreted for claim 93 above: as referring to a nucleic acid strand that comprises the second nucleic acid sequence.
Claim 94 recites the limitations “a fourth nucleic acid strand” and “a fifth nucleic acid strand”. However, within claim 94 and the claims on which it depends (93, 92, and 78), neither a first nucleic acid strand nor a third nucleic acid strand are defined, and the second nucleic acid strange as used in claims 93 and 94 lacks antecedent basis (see rejections of claims 93 and 94 above). Therefore, it is unclear the relationship these strands have to the first nucleic acid sequence and second nucleic acid sequence in the method of claim 78. For the purpose of examination, the fourth nucleic acid strand and the fifth nucleic acid strand are interpreted as two separate nucleic acid strands and that the first, second, and third nucleic acid sequences are not on these two separate nucleic acid strands.
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 78, 80, and 88 are rejected under 35 U.S.C. 102(a)(1) and 102(a)(2) as being anticipated by Akhavan-Tafti (US 2007/0003968, published 4 January 2007).
Regarding claim 78, Akhavan-Tafti teaches a method for nucleic acid ligation (“a method” wherein “ligation of the oligonucleotide, polynucleotide or nucleic acid onto another” occurs [0031]), comprising: a) providing a first nucleic acid sequence (“an oligonucleotide, polynucleotide or nucleic acid, having a sequence S2” [0031]) and a second nucleic acid sequence (“another oligonucleotide, polynucleotide or nucleic acid, having a sequence S1” [0031]), wherein the first nucleic acid sequence binds to a first region of a third nucleic acid sequence and the second nucleic acid sequence binds to a second region of the third nucleic acid sequence (“hybridized with a complementary oligonucleotide, polynucleotide or nucleic acid containing adjacent sequences C1 and C2 which are the complements or S1 and S2” [0031], wherein the first nucleic acid sequence, the second nucleic acid sequence, and/or the third nucleic acid sequence are/is bound to a single strand annealing protein (“Single stranded DNA binding proteins can be added to oligonucleotide ligation reactions to improve their efficiency. Their effect is due to their relaxation of any secondary structure that is in the template strand thus allowing the complementary oligonucleotides to bind and ligate” the described single stranded DNA binding proteins are single strand annealing proteins since they allow complementary oligonucleotides to bind/anneal [0054]); and b) ligating the first nucleic acid sequence and the second nucleic acid sequence using the third nucleic acid sequence as a template to generate a ligated oligonucleotide comprising the first nucleic acid sequence and the second nucleic acid sequence (“ligation of the oligonucleotide, polynucleotide or nucleic acid onto another oligonucleotide, polynucleotide or nucleic acid” [0031], also see FIG. 2B).
Regarding claim 80, Akhavan-Tafti teaches the method of claim 78 (as described above), wherein the first nucleic acid sequence and the second nucleic acid sequence are in different molecules (“ligation of the oligonucleotide, polynucleotide or nucleic acid onto another oligonucleotide, polynucleotide or nucleic acid” [0031], also see FIG. 2B).
Regarding claim 88, Akhavan-Tafti teaches the method of claim 78 (as described above), wherein the first nucleic acid sequence and/or the second nucleic acid sequence comprises a ribonucleotide (“Oligomer, oligonucleotide--as used herein will refer to a compound containing a phosphodiester internucleotide linkage and a 5'-terminal monophosphate group. The nucleotides can be the normally occurring ribonucleotides A, C, G, and U” [0025]).
For the reasons above, claim 78, 80, and 88 are anticipated by Akhavan-Tafti.
Claims 78-79, 89, 91, and 95-97 are rejected under 35 U.S.C. 102(a)(1) and 102(a)(2) as being anticipated by Badenhorst et al. (WO 2019/149958, published 8 August 2019), herein Badenhorst.
Regarding claim 78, Badenhorst teaches a method for nucleic acid ligation (“the method involves […] in the next step, the single strands are self-ligated” page 18 lines 16 and 25), comprising: a) providing a first nucleic acid sequence and a second nucleic acid sequence (FIGURE 1, single stranded DNA in the third step, first nucleic acid sequence is the 5’ end and second nucleic acid sequence is the 3’), wherein the first nucleic acid sequence binds to a first region of a third nucleic acid sequence and the second nucleic acid sequence binds to a second region of the third nucleic acid sequence (“a splint is used to enable a double-strand ligase” page 18 line 28), wherein the first nucleic acid sequence, the second nucleic acid sequence, and/or the third nucleic acid sequence are/is bound to a single strand annealing protein (“single-stranded state may be further enhanced by presence of single strand stabilizing gents such as the single-strand binding protein” page 18 lines 22-24); and b) ligating the first nucleic acid sequence and the second nucleic acid sequence using the third nucleic acid sequence as a template to generate a ligated oligonucleotide comprising the first nucleic acid sequence and the second nucleic acid sequence (“the single strands are self-ligated (“circularized”) […] a splint is used to enable a double-strand ligase” page 18 lines 25-28, FIGURE 1, circularized single strand is the ligated oligonucleotide).
Regarding claim 79, Badenhorst teaches the method of claim 78 (as described above), wherein the first nucleic acid sequence and the second nucleic acid sequence are in the same nucleic acid molecule (“the single strands are self-ligated (circularized)”, FIGURE 1).
Regarding claim 89, Badenhorst teaches the method of claim 78 (as described above), wherein the first nucleic acid sequence and/or the second nucleic acid sequence comprises a barcode sequence or a portion thereof (“the invention comprises introduction of barcodes into the target nucleic acids” page 14 lines 8-9; “adaptor molecules are ligated to the target nucleic acid” page 12 lines 3-4 and “adaptors comprise one or more barcodes” page 14 lines 25-26, see FIGURE 1 wherein adaptors are attached to the Insert DNA and are the portions of the first and second nucleic acid sequences that are ligated together to form a circular nucleic acid molecule) and wherein the barcode sequence or a portion thereof is detected in a biological sample; (“a library of circular single-stranded target nucleic acids is sequenced by annealing a sequencing primer to a primer binding site in the adaptor sequence” page 19 lines 2-3; “sequencing individual molecules typically requires molecule barcodes […] the barcode marks the molecule and its progeny” page 14 lines 9-14; “detecting a target nucleic acid in a sample. In some embodiments, the sample is derived from a subject or a patient” page 10 lines 11-12).
Regarding claim 91, Badenhorst teaches the method of claim 78 (as described above), wherein the first nucleic acid sequence is in a first nucleic acid strand comprising a first barcode sequence (“adaptors comprise one or more barcodes” page 14 lines 25-26), and wherein the first nucleic acid strand is immobilized on a substrate (“adaptors comprise a primer binding site, e.g., an amplification primer binding site” page 12 lines 23-24; “one of the amplification primers may comprise an affinity ligand (e.g., biotin) that will enable the strand to be captured by an affinity capture moiety (e.g., streptavidin) […] the affinity capture utilizes the affinity molecules (e.g., streptavidin) bound to solid support” page 15 lines 21-25).
Regarding claim 95, Badenhorst teaches the method of claim 91 (as described above), wherein the substrate is a bead (“the solid support may be capable of suspension in a solution (e.g., a glass bead, a magnetic bead, a polymer bead or another like particle)” page 15 lines 25-27).
Regarding claim 96, Badenhorst teaches the method of claim 78 (as described above), wherein the method comprises capturing the ligated oligonucleotide on an array (“one of the amplification primers may comprise an affinity ligand (e.g., biotin) that will enable the strand to be captured by an affinity capture moiety (e.g., streptavidin) […] the affinity capture utilizes the affinity molecules (e.g., streptavidin) bound to solid support” page 15 lines 21-25).
Regarding claim 97, Badenhorst teaches the method of claim 96 (as described above), wherein the method comprises sequencing all or a portion of the captured ligated oligonucleotide or a complement thereof (“detecting target nucleic acids in a sample by nucleic acid sequencing” page 17 lines 3-4; “the adaptor or the target specific primer may comprise a sequencing primer binding site which can initiate a sequencing read of each strand” page 17 lines 14-15).
For the reasons given above, claim 78-79, 89, 91, and 95-97 are anticipated by Badenhorst.
Claim Rejections - 35 USC § 103
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
Claims 79, 84-87, and 89-90 are rejected under 35 U.S.C. 103 as being unpatentable over Akhavan-Tafti (US 2007/0003968, published 4 January 2007), as applied to claims 78, 80, and 88 above, in view of Larman et al. (in IDS filed 4 December 2023)(US 2023/0039899, effectively filed date 3 January 2020), herein Larman.
Regarding claim 79, Akhavan-Tafti teaches the method of claim 78. However, Akhavan-Tafti does not teach that the first nucleic acid sequence and the second nucleic acid sequence are in the same nucleic acid molecule. This deficiency is made up for in the teachings of Larman.
Regarding claim 79, Larman teaches a method involving nucleic acid ligation wherein a first nucleic acid sequence and a second nucleic acid sequence are in the same nucleic acid molecule (two probe halves), bind to complementary regions on a third nucleic acid sequence (bridge primer), and are ligated together to form a ligated oligonucleotide (“after adjacent donor and acceptor probe have become ligated, will the two probe halves present the complete 34-nucleotide bridge sequence (17nt from each probe), which is subsequently hybridized by the bridge primer (step 3) and ligated by T4 DNA ligase (step 4)” [0076]; FIG. 2).
Regarding claim 84, Larman teaches a method wherein the first nucleic acid sequence is in a probe, and the method comprises contacting a biological sample with the probe, wherein the third nucleic acid sequence is in a target nucleic acid in the biological sample (“contacting a biological sample comprising the target nucleic acid in a reaction mixture with at least one probe set comprising (i) a first multi-partite probe comprising a 5′ phosphorylated donor probe and a first bridge probe” [0080]), wherein the second nucleic acid sequence is in the same probe or is in a different probe that binds to the target nucleic acid (“and (ii) a second multi-partite probe comprising a 3′ acceptor probe and a second bridge probe” [0080]), wherein the ligating is performed in the biological sample, and wherein the ligated oligonucleotide is a ligated probe comprising the first nucleic acid sequence and the second nucleic acid sequence (“ligating the 5′ phosphorylated donor probe and the 3′ acceptor probe” [0080]; “ligating the first bridge probe and the second bridge probe thereby forming a circularized probe” [0080]; see also FIG. 2 steps 1-3).
Regarding claim 85, Larman teaches a method comprising detecting the ligated probe or a product thereof at a location in the biological sample (“amplifying the circularized probe by rolling circle amplification; and (h) detecting the target nucleic acid” [0080]; “The RCA product is in essence a ‘nanoball’ of single stranded DNA containing many copies of the detector sequences. Due to the extensive crosslinking of the surrounding tissue, the nanoball remains trapped in a position that approximates the position of the templating RNA molecule” [0076]; “fluorescently labeled oligos (detector probes) are annealed to the complementary detector sequences” [0076]).
Regarding claim 86, Larman teaches a method wherein the ligated oligonucleotide is a circular probe (“ligating the first bridge probe and the second bridge probe thereby forming a circularized probe” [0080]; see also FIG. 2 step 4).
Regarding claim 87, Larman teaches a method wherein the third nucleic acid sequence is RNA and the first nucleic acid sequence and/or the second nucleic acid sequence comprises DNA (third nucleic acid sequences is RNA: “the LnR acceptor and donor probes anneal to the target RNA sequence, followed by ligation with Rnl2” [0076]; first and/or second nucleic acid sequences comprises DNA: “T4 RNA Ligase 2 (Rnl2), an enzyme that performs RNA-templated ligations of DNA probes with very high efficiency, when the two 3′ bases of the acceptor probe are composed of ribonucleotides” [0075]; further evidence that the acceptor and donor probes comprise DNA is that the ligations of their other ends to form a circular probe with the bridge primer as a template uses T4 DNA ligase “ligated by T4 DNA ligase” [0076]).
Regarding claim 89, Larman teaches a method wherein the first nucleic acid sequence and/or the second nucleic acid sequence comprises a barcode sequence or a portion thereof and wherein the barcode sequence or a portion thereof is detected in a biological sample (“the probe set comprises a barcode unique to the target RNA and wherein sequencing of the barcode detects the target RNA” [0095]).
Regarding claim 90, Larman teaches a method wherein the target nucleic acid is at a location in a biological sample and the ligated probe is generated and amplified at the location in the biological sample, and wherein the ligated probe and/or the product thereof is detected at the location in the biological sample, wherein the product is a rolling circle amplification (RCA) product (“amplifying the circularized probe by rolling circle amplification” [0080]; “the RCA product is in essence a ‘nanoball’” [0080]; “the nanoball remains trapped in a position that approximates the position of the templating RNA molecule” [0080]).
It would have been obvious to one of ordinary skill in the art prior to the effective filing date of the claimed invention to combine the method of oligonucleotide ligation taught by Akhavan-Tafti with the method of Larman that uses oligonucleotide ligation. Akhavan-Tafti’s teaching that single strand annealing proteins can be added to oligonucleotide ligation reactions to improve their efficiency would motivate one of ordinary skill in the art to combine it with Larman’s method of creating a circularized probe at the location of a target RNA using ligation of two multipartite probes in order to obtain the benefit of increase ligation efficiency. One of ordinary skill in the art would have a reasonable expectation of success in this combination because both methods perform similar reactions where a first and second oligonucleotide are ligated together using a third splint oligonucleotide to bring them into proximity based on its ability to hybridize to the first and second oligonucleotides. Therefore, the invention as a whole of claims 79, 84-87, and 89-90 would have been prima facie obvious to one of ordinary skill in the art prior to the effective filing date of the claimed invention.
Claim 81 is rejected under 35 U.S.C. 103 as being unpatentable over Akhavan-Tafti (US 2007/0003968, published 4 January 2007), as applied to claims 78, 80, and 88 above, in view of Van Dyck et al. ("Binding of double-strand breaks in DNA by human Rad52 protein", Nature 398 pages 728-31 (1999)), herein Van Dyck.
Regarding claim 81, Akhavan-Tafti teaches the method of claim 78. However, Akhavan-Tafti does not teach that the ligation efficiency is increased by at least 10% with the bound single strand annealing protein compared to ligation in the absence of the single strand annealing protein. This deficiency is made up for in the teachings of Van Dyck.
Regarding claim 81, Van Dyck teaches that human Rad52 is a single strand annealing protein, since it binds to single stranded DNA (“hRad52 exhibits preferential binding to single-stranded DNA” page 3 paragraph 3) and is involved in annealing (“Rad52 anneals complementary single strands” page 4 paragraph 6), and that ligation efficiency is increased by at least 10% with the bound single strand annealing protein compared to ligation in the absence of the single strand annealing protein (“most hRad52-facilitated events were intermolecular and approximately 65% of the DNA was converted into multimeric linear forms (Fig. 2a, lane 4) […] In the absence of hRad52, ligation resulted primarily in the formation of circular (covalently closed and nicked circular) species (Fig. 2a, lane 3). The stimulatory effect of hRad52 on DNA ligation was particularly striking with blunt-ended DNA (Fig. 2b: compare lanes 3 and 4)” page 3 paragraph 4 and Figure 2, in the case of 2a using linear DNA molecules with cohesive ends the ligation efficiency of the ligation reaction to obtain the linear multimer products with Rad52 was ~65% while they are nearly undetectable in the absence of Rad52, a clearly >10% difference).
It would have been obvious to one of ordinary skill in the art prior to the effective filing date of the claimed invention to perform the simple substitution of the Rad52 protein taught by Van Dyck for the single strand annealing protein in the method of Akhavan-Tafti, thereby obtaining the ligation efficiency of claim 81. One of ordinary skill in the art would be able to substitute the Rad52 protein of Van Dyck for the single strand annealing protein of Akhavan-Tafti and would expect that the results of the substitution would be predictable because Rad52 is taught by Van Dyck to be a single strand annealing protein with a known function of annealing complementary single strands (see claim 82 rejections above), so Rad52 would be used in the method of Akhavan-Tafti merely to perform its known function. Therefore, the invention as a whole of claims 81 would have been prima facie obvious to one of ordinary skill in the art prior to the effective filing date of the claimed invention.
Claims 82-83 are rejected under 35 U.S.C. 103 as being unpatentable over Akhavan-Tafti (US 2007/0003968, published 4 January 2007), as applied to claims 78, 80, and 88 above, in view of Xu et al. ("DdrB stimulates single-stranded DNA annealing and facilitates RecA-independent DNA repair in Deinococcus radiodurans”, DNA Repair (Amst). 9(7) pages 805-12 (2010)), herein Xu.
Regarding claim 82, Akhavan-Tafti teaches the method of claim 78. However, Akhavan-Tafti does not teach that the single strand annealing protein is DdrB or RecT.
Regarding claim 82, Xu teaches that DdrB is a single strand annealing protein that promotes annealing (“incubation of the complementary Фx174 DNA strands with DdrB resulted in the time-dependent appearance of a DNA product that migrated more slowly than the input DNA strands, with a portion accumulating in the wells. The new DNA species were proposed to be the product of DNA strand annealing because they were completely resistant to treatment with SDS and proteinase K” page 4 paragraph 3; Fig. 2).
Regarding claim 83, Xu teaches that the concentration of DdrB protein used is at least 10nM (“Denatured Φx174 complementary strands (37.5 μM) were incubated with 4.0 μM DdrB (in lanes 4–8)” Fig. 2a caption; “the 48-mer oligonucleotide (oligo-1; 200 nM) and a complementary oligonucleotide (oligo-2; 200 nM) were incubated in 2 ml of reaction buffer containing 0.2 μM DAPI with 200 nM DdrB protein (closed squares)” Fig. 2b caption).
It would have been obvious to one of ordinary skill in the art prior to the effective filing date of the claimed invention to perform the simple substitution of the DdrB protein taught by Xu for the single strand annealing protein in the method of Akhavan-Tafti. One of ordinary skill in the art would be able to substitute the DdrB protein of Xu for the single strand annealing protein of Akhavan-Tafti and would expect that the results of the substitution would be predictable because DdrB is shown by Xu to be a single strand annealing protein that binds to ssDNA (Xu Figure 1) that stimulates single-strand annealing (Xu Figure 2), so DdrB would be used in the method of Akhavan-Tafti merely to perform its known function. Therefore, the invention as a whole of claims 82-83 would have been prima facie obvious to one of ordinary skill in the art prior to the effective filing date of the claimed invention.
Claims 92-94 are rejected under 35 U.S.C. 103 as being unpatentable over Akhavan-Tafti (US 2007/0003968, published 4 January 2007), as applied to claims 78, 80, and 88 above, in view of Ramachandran Iyer et al. (in IDS filed 4 December 2023)(US 2022/0010367, published 13 January 2022), herein Ramachandran Iyer.
Regarding claim 92, Akhavan-Tafti teaches the method of claim 78. However, Akhavan-Tafti does not teach that the second nucleic acid sequence comprises a barcode sequence or portion thereof, wherein the barcode sequence is a spatial barcode associated with a location on the substrate. This deficiency is made up for in the teaching of Ramachandran Iyer.
Regarding claim 92, Ramachandran Iyer teaches a method of generating a ligated oligonucleotide by ligating two oligonucleotides together using a third oligonucleotide as a splint (“to generate probes [ligated oligonucleotide] using oligonucleotides, a primer complementary to a portion of an oligonucleotide [first nucleic acid strand having the first nucleic acid sequence]” […] “can hybridize to the oligonucleotide and extend the oligonucleotide (using the oligonucleotide as a template) to form a duplex and to create a 3′ overhang [the newly made strand is the third nucleic acid sequence]” […] “a capture probe [ligated oligonucleotide] can be generated by adding additional oligonucleotides [second nucleic acid strand having the second nucleic acid sequence] to the end of the 3′ overhang (e.g., via splint oligonucleotide mediated ligation)” [0553]). Further, Ramachandran Iyer teaches this method wherein the second nucleic acid sequence comprises a barcode sequence, wherein the barcode sequence is a spatial barcode associated with a location on a substrate (“the array has a plurality of capture probes comprising spatial barcodes. These spatial barcodes and their relationship to the locations on the array can be determined” [0555]; “a plurality of second oligonucleotides comprising two or more different barcode sequences can be ligated onto a plurality of first oligonucleotides that comprise the same barcode sequence, thereby generating two or more different species of barcodes” [0548]).
Regarding claim 93, Ramachandran Iyer teaches the method described above for claim 92 wherein the second nucleic acid strand further comprises a splint sequence (“The additional oligonucleotide (e.g., a sequence or a portion of sequence of a capture domain) [the second nucleic acid strand] can include a sequence compatible for hybridizing [a splint sequence] or ligating with an analyte of interest in a biological sample. An analyte of interest can also be used as a splint oligonucleotide to ligate additional oligonucleotides onto a probe” [0554]).
Regarding claim 94, Ramachandran Iyer teaches the method described above for claim 93 further comprising providing a fourth nucleic acid strand and a fifth nucleic acid strand, wherein the splint sequence of the second nucleic acid strand hybridizes to a third hybridization region in the fifth nucleic acid strand (“The additional oligonucleotide (e.g., a sequence or a portion of sequence of a capture domain) [the second nucleic acid strand] can include a sequence compatible for hybridizing [a splint sequence] or ligating with an analyte of interest [the fifth nucleic acid strand] in a biological sample. An analyte of interest can also be used as a splint oligonucleotide” [0554]), and the fourth nucleic acid strand comprises a fourth nucleic acid sequence that hybridizes to a fourth hybridization domain in the fifth nucleic acid strand (“An analyte of interest can also be used as a splint oligonucleotide [the fifth nucleic acid strand] to ligate additional oligonucleotides [the fourth nucleic acid strand] onto a probe. When using a splint oligonucleotide to assist in the ligation of additional oligonucleotides, an additional oligonucleotide can include a sequence [fourth nucleic acid sequence] that is complementary to the sequence [fourth hybridization domain] of the splint oligonucleotide” [0554]). Regarding claim 94, as discussed in the 35 U.S.C. 102 rejection in view of Akhavan-Tafti above, Akhavan-Tafti teaches the use of single strand annealing proteins in ligation reactions such as the ones taught by Ramachandran Iyer using the second, fourth, and fifth nucleic acid strands.
It would have been obvious to one of ordinary skill in the art prior to the effective filing date of the claimed invention to combine the method of oligonucleotide ligation taught by Akhavan-Tafti with the method of Ramachandran Iyer that uses oligonucleotide ligation. Akhavan-Tafti’s teaching that single strand annealing proteins can be added to oligonucleotide ligation reactions to improve their efficiency would motivate one of ordinary skill in the art to combine it with Ramachandran Iyer’s method of generating and modifying capture probes using oligonucleotide ligation in order to obtain the benefit of increase ligation efficiency. One of ordinary skill in the art would have a reasonable expectation of success in this combination because both methods perform similar reactions where a first and second oligonucleotide are ligated together using a third splint oligonucleotide to bring them into proximity based on its ability to hybridize to the first and second oligonucleotides. Therefore, the invention as a whole of claims 92-94 would have been prima facie obvious to one of ordinary skill in the art prior to the effective filing date of the claimed invention.
Conclusion
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/JEFFREY BELLAH/Examiner, Art Unit 1683
/ANNE M. GUSSOW/Supervisory Patent Examiner, Art Unit 1683