Office Action 1: Final Rejection (2023-01-19)
Rejection types: 103 - Obviousness, 112 - Written Description/Enablement

CLAIMS:
What is claimed is:

1\. A flow cytometry method for assaying a target of a sample, comprising:
i) providing to the sample:
1) a molecular probe, comprising a binding moiety conjugated to a first oligonucleotide sequence; and
2) a detectable component, comprising a signal generating moiety conjugated to a second oligonucleotide sequence that is complementary to the first oligonucleotide sequence of the molecular probe;
ii) binding the target in the sample with the binding moiety of the molecular probe;
iii) hybridizing the first oligonucleotide sequence of the molecular probe with the second oligonucleotide sequence of the detectable component; and
iv) detecting a signal generated from the hybridized detectable component using flow cytometry; wherein the method is characterized by one or more of the following:
1) the conjugation between the first oligonucleotide sequence and the binding moiety and conjugation between the second oligonucleotide sequence and the signal generating moiety, comprises one or more covalent bond linkages, comprising a hydrazone, oxime, triazine, or other covalent bond, wherein the formation of the conjugates are at least 90% efficient; and
2) the binding moiety comprises a strong binding affinity for the target.

2\. The method of claim 1, wherein the mode of addition comprises:
i) the molecular probe and the detectable component are combined together and hybridized prior to contacting the sample;
ii) the molecular probe is combined with the sample prior to the addition of the detectable component; or iii) the detectable component is combined with the sample prior to the addition of the molecular probe.

3\. The method of claim 1, wherein the method comprises:
i) the molecular probe binding the target prior to hybridizing with the detectable component; or
ii) the molecular probe hybridizing with the detectable component prior to binding the target.

4\. A flow cytometry method for assaying a target of a sample, comprising:
i) providing to the sample:
1) a molecular probe, comprising a binding moiety conjugated to a first oligonucleotide sequence;
2) a detectable component, comprising a signal generating moiety conjugated to a second oligonucleotide sequence; and
3) a universal adapter, comprising an oligonucleotide sequence having a first sequence segment complementary to the first oligonucleotide sequence of the molecular probe and a second sequence segment complementary to the second oligonucleotide sequence of the detectable component;
ii) binding the target in the sample with the binding moiety of the molecular probe;
iii) hybridizing the first oligonucleotide sequence of the molecular probe to the first oligonucleotide sequence segment of the universal adapter;
iv) hybridizing the second oligonucleotide sequence of the detectable component to the second oligonucleotide sequence segment of the universal adapter; and
v) detecting a signal generated from the hybridized detectable component using flow cytometry; wherein the method is characterized by one or more of the following: 1) the conjugation between the first oligonucleotide sequence and the binding moiety and conjugation between the second complementary oligonucleotide sequence and the signal generating moiety, comprises one or more covalent bond linkages, comprising a hydrazone, oxime, triazine, or other covalent bond, wherein the formation of the conjugates are at least 90% efficient; and
2) the binding moiety comprises a strong binding affinity for the target.

5\. The method of claim 4, wherein the mode of addition comprises:
i) the molecular probe, the universal adapter, and the detectable component are combined together and hybridized prior to contacting the sample;
ii) the molecular probe and the universal adapter are combined together and hybridized prior to contacting the sample;
iii) the detectable component and the universal adapter are combined together and hybridized prior to contacting the sample;
iv) the molecular probe, alone or in combination with the detectable component, is combined with the sample prior to the addition of the universal adapter; or
v) the universal adapter is combined with the sample prior to the addition of the molecular probe and/or the detectable component.

6\. The method of claim 4, wherein the method comprises:
i) the molecular probe hybridizing with the universal adapter prior to said molecular probe binding the target;
ii) the molecular probe hybridizing with the universal adapter after said molecular probe binds the target;
iii) the detectable component hybridizing with the universal adapter prior to the molecular probe binding the target; iv) the detectable component hybridizing with the universal adapter after the molecular probe binds the target;
v) the universal adapter hybridizing with the molecular probe and hybridizing with the detectable component prior to said molecular probe binding the target; or
vi) the universal adapter hybridizing with the molecular probe and hybridizing with the detectable component after said molecular probe binds the target.

7\. The method of claim 1, wherein:
i) the assay comprises a singleplex or multiplex assay; and
ii) the assay further detects, measures, or quantifies the level of binding and/or amount of the target present in the sample with one or more of the following:
immunomagnetic cellular depletion, immunomagnetic cell capture, array, bead array, multiplex bead array, microarray, antibody array, cellular array, chemiluminescence, infrared, microscopy, imaging, high content screening (HCS), mass cytometry, lateral flow immunoassay, immunodetection, immunoturbidity, latex agglutination, gold particle agglutination, visual inspection, a change in light transmittance through said sample, increased light transmittance through said sample, immunohistochemistry (IHC), immunocytochemistry (ICC), in situ hybridization (ISH), enzyme immuno-assay (EIA), enzyme linked immuno-assay (ELISA), ELISpot, a blotting method, a Western blot, a Southern blot, a Southwestern blot, labeling inside an electrophoresis system, labeling on a surface, labeling on an array, PCR amplification, elongation followed by PCR amplification, immunoprecipitation, co- immunoprecipitation, chromatin immunoprecipitation, pretargeting imaging, therapeutic agent, or combinations thereof.

8\. The method of claim 1, wherein the method further comprises:
i) preparing the molecular probe; and ii) preparing the detectable component;
wherein the prepared molecular probe and prepared detectable component have at least 90% purity.
9\. The method of claim 1, wherein the method further comprises preparing and isolating the molecular probe, comprising: i) providing the binding moiety;
ii) conjugating the binding moiety with at least one first oligonucleotide sequence at greater than 90% efficiency to form binding moiety-oligonucleotide conjugates; and
iii) isolating the binding moiety-oligonucleotide conjugates from the conjugation mixture by binding, retaining, and/or retarding a substantial portion of:
1) the conjugates, removing a substantial portion of the unconjugated first oligonucleotide sequence in a wash step followed by release of the bound, retained, and/or retarded conjugates; or
2) the unconjugated first oligonucleotide sequences, followed by collecting a substantial portion of the non-bound, non-retained, and/or non-retarded conjugates in a wash step.

10\. The method of claim 9, wherein the isolation step utilizes an immobilized binder, chromatography, affinity chromatography, size exclusion chromatography, HPLC, reverse-phase chromatography, electrophoresis, capillary electrophoresis, polyacrylamide gel electrophoresis, agarose gel electrophoresis, free flow electrophoresis, differential centrifugation, thin layer chromatography, immunoprecipitation, hybridization, solvent extraction, dialysis, filtration, diafiltration, tangential flow filtration, ion exchange chromatography, hydrophobic interaction chromatography, or combinations thereof.
11\. The method of claim 1, wherein the method further comprises preparing and isolating the detectable component, comprising:
i) providing a plurality of the signal generating moiety;
ii) conjugating the second oligonucleotide sequence with at least one of the plurality of the signal generating moiety at greater than 90% efficiency to form signal generating moiety-second oligonucleotide conjugates; and
iii) isolating the signal generating moiety-second oligonucleotide conjugates from the conjugation mixture by binding, retaining, and/or retarded a substantial portion of:
1) the conjugates, removing a substantial portion of the unconjugated second oligonucleotide sequences in a wash step followed by release of the bound, retained, and/or retarded conjugates; or
2) the unconjugated second oligonucleotide sequences, followed by collecting a substantial portion of the non-bound, non-retained, and/or
non-retarded conjugates in a wash step.

12\. The method of claim 1, wherein the detectable component comprises a scaffold conjugated to the second oligonucleotide sequence, and wherein said scaffold comprises one or more signal generating moieties.

13\. The method of claim 1, wherein the scaffold comprises a dendrimer, a polysaccharide, a dextran, a protein, a peptide, a further oligonucleotide sequence, a portion of the second oligonucleotide sequence that is not complementary to the first oligonucleotide sequence of the molecular probe, a polymer, a hydrophilic polymer, a bead, a nanoparticle, or
combinations or derivatives thereof.

14\. The method of claim 1, wherein the binding moiety comprises an antibody, a monoclonal antibody, a polyclonal antibody, an enzyme, a protein, a peptide, a carbohydrate, a nuclear receptor, a small molecule, an aptamer, a chelator, or combinations or derivatives thereof.
15\. The method of claim 1, wherein the target is a biological target.

16\. The method of claim 15, wherein the biological target comprises an antigen, a pathogen, a protein, a peptide, an epitope, a carbohydrate-containing molecule, a small molecule, or
combinations or derivatives thereof.

17\. The method of claim 1, wherein the signal generating moiety or the one or more signal generating moieties of the detectable component or the hybridized detectable component, comprises one or more of the following:
a directly detectable signal generating moiety, an indirectly detectable signal generating moiety, a fluorescent dye, a fluorophore, a fluorochrome, a chromophore, a biofluorescent protein, a luminescent species, a chemiluminescent compound, a electrochemiluminescent label, a bioluminescent label, a phosphorescent species, a fluorophore labeled DNA dendrimer, Quantum Dot, a tandem dye, a FRET dye, a heavy atom, a spin label, a radioactive isotope, a nanoparticle, a light scattering nanoparticle or microsphcre, a diffracting particle, a polymer, a polymer particle, a bead, a solid surface, a Raman particle, a metal particle, a stable isotope, a heavy metal chelate, a magnetic particle, an RF 1D tag, a microbarcode particle, an enzyme, an enzyme substrate, a molecule specifically recognized by another substance carrying a label or reacts with a substance carrying a label, an antibody, an antibody fragment, an antigen, a nucleic acid, a nucleic acid analog, oligonucleotide, oligonucleotide analog, complementary oligonucleotide, complementary oligonucleotide analog, a ligand, a protein, a peptide ligand, a protein substrate, a receptor; a substrate, a secondary reporter, a hapten, or combinations or derivatives thereof.

18\. The method of claim 1, wherein the method comprises an automated system or robotic system.
19\. The method of claim 1, wherein the method further comprises removing the hybridized detectable component or plurality of detectable components from the bound target or plurality of targets, respectively, wherein said removal is by a washing or stripping process.

20\. The method of claim 19, wherein the method further comprises re-probing with a second detectable component or second plurality of detectable components, respectively, wherein said second detectable component comprises at least one second signal generating moieties conjugated to a second oligonucleotide sequence or a complementary second oligonucleotide sequence, or said second plurality of detectable components arc prepared by independently pairing, via conjugation, a second plurality of signal generating moieties and a second plurality of second oligonucleotide sequences or a second plurality of complementary second oligonucleotide sequences. 348 349 350 351 352 353 354 355 CORE/3515781.000201/172760516.1

OFFICE ACTION:
FINAL ACTION
1.	The present application is being examined under the pre-AIA  first to invent provisions.
Preliminary Amendments and Status of the Claims
2.	The preliminary amendment filed 22 February 2022, in which the specification was amended and the sequence listing was provided, has been thoroughly reviewed and entered.
3.	Claims 1-20 are under prosecution.
Information Disclosure Statement
4.	The Information Disclosure Statement filed 28 February 2022 is acknowledged and has been considered.
It is noted that the listing of references in the specification is not a proper information disclosure statement.  37 CFR 1.98(b) requires a list of all patents, publications, or other information submitted for consideration by the Office, and MPEP § 609.04(a) states, the list may not be incorporated into the specification but must be submitted in a separate paper.  Therefore, unless the references have been cited by the examiner on form PTO-892, they have not been considered.
Claim Rejections - 35 USC § 112
5.	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.
6.	Claims 1-20 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 pre-AIA  the applicant regards as the invention.
A.	Claim 1 (upon which claims 2-3 and 7-20 each depend) and claim 4 (upon which claims 5-6 depend) are each indefinite in the term “strong binding affinity,” which is a relative term which renders the claims indefinite.  The term strong binding affinity is not defined by the claims, the specification does not provide a standard for ascertaining the requisite degree, and one of ordinary skill in the art would not be reasonably apprised of the scope of the invention.
B.	It is also noted that none of claims 7, 10, 13-14, 16, or 17 utilize proper Markush language.  A proper Markush claim contains the language selected from the group consisting of A, B, and C.  See MPEP 803.02
C.	The recitations “(HCS),” “(IHC),” “(ICC),” “(ISH),” “(EIA),” and “(ELISA)” in claim 7 is indefinite because it is unclear if the text within the parenthesis is an alternative limitation of the claim or merely a descriptor of other elements of the claims.    In addition, the recitations are acronyms, the meanings of which may change over time, and the specification does not define what is encompassed by the term “CAPS.”
It is suggested the claim be amended to remove all parenthetical limitations and recite only full names or text for each limitation.
D.	Claim 9 (upon which claim 10 depends) is indefinite in each of the following:
I.	The recitation “the conjugation mixture,” which lacks antecedent basis because there is no previous recitation of a “conjugation mixture.”
II.	The recitation “the conjugates,” which lacks antecedent basis in the previous recitation of “binding moiety-oligonucleotide conjugates;” it is also noted that any amendments to the conjugates may affect the similar recitations at the end of iii) a).
III.	The recitation “the unconjugated first oligonucleotide sequence,” which antecedent basis because there is no previous recitation of an “unconjugated first oligonucleotide sequence.”
IV.	It is also suggested that the phrase “at least one first oligonucleotide sequence” in part ii) of claim 9 be amended so as to differentiate the limitation from “the first oligonucleotide sequence” of claim 1.
E.	Claim 11 is indefinite in each of the following:
I.	The recitation “the conjugation mixture,” which lacks antecedent basis because there is no previous recitation of a “conjugation mixture.”
II.	The recitation “the conjugates,” which lacks antecedent basis in the previous recitation of “binding moiety-oligonucleotide conjugates;” it is also noted that any amendments to the conjugates may affect the similar recitations at the end of iii) a).
III.	The recitation “the unconjugated second oligonucleotide sequence,” which lacks antecedent basis because there is no previous recitation of an “unconjugated second oligonucleotide sequence.”
F.	Claim 13 is indefinite in the limitation “the scaffold,” which lacks antecedent basis because there is no previous recitation of a “scaffold.”
G.	The term small molecule in claims 14 and 16 and the term “heavy atom” on claim 17 are a relative term which renders the claim indefinite.  The terms “small molecule” and “heavy atom” not defined by the claim, the specification does not provide a standard for ascertaining the requisite degree, and one of ordinary skill in the art would not be reasonably apprised of the scope of the invention.
H.	Claims 17 and 19 are each indefinite in the recitation “the hybridized detectable component,” which antecedent basis because there is no previous recitation of a “hybridized detectable component.”
I. 	Claim 17 is indefinite in each of the following”
I.	The recitation “the hybridized detectable component,” which lacks antecedent basis because there is no previous recitation of a “hybridized detectable component.”
II.	The recitation “the one or more signal generating moieties” in lines 1 and 2, which lacks antecedent basis because there is no previous recitation of “one or more signal generating moieties.”
J.	Claim 18 is rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being incomplete for omitting essential steps, such omission amounting to a gap between the steps.  See MPEP § 2172.01.  The omitted steps are:  how the automated system or robotic system is actually utilized in the method.
K.	It is suggested that the phrase “a second oligonucleotide sequence” in claim 20 be amended so as to differentiate the limitation from “the second oligonucleotide sequence” of claim 1.
L.	Applicant is cautioned against introducing new matter in response to the rejections and suggestions discussed above.
Claim Rejections - 35 USC § 103
7.	The following is a quotation of pre-AIA  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, 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.
8.	This application currently names joint inventors. In considering patentability of the claims under pre-AIA  35 U.S.C. 103(a), the examiner presumes that the subject matter of the various claims was commonly owned at the time any inventions covered therein were made absent any evidence to the contrary.  Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and invention dates of each claim that was not commonly owned at the time a later invention was made in order for the examiner to consider the applicability of pre-AIA  35 U.S.C. 103(c) and potential pre-AIA  35 U.S.C. 102(e), (f) or (g) prior art under pre-AIA  35 U.S.C. 103(a).
9.	Clams 1-8 and 12-19 are rejected under 35 U.S.C. 103(a) as being unpatentable over US 7,183,052 B2 Sorge (hereinafter referred to as “Sorge”) in view of US 2010/0159446 A1 Haff et al, (hereinafter referred to as “Haff”).
Regarding claim 1, Sorge teaches a method for assaying a target in a sample, comprising providing to the sample a probe comprising two nucleic acid subunits, wherein at least one subunit comprises a region which is complementary to a target nucleic acid sequence (col. 4, lines 28-31) and the other subunit comprises a signal generating moiety (col. 4, lines 40-43), binding or associating the subunits of the probe with one another in the absence of the target (col. 4, lines 35-37), and binding the target in the sample with the region(s) within the nucleic acid subunits which is complementary to the target (col. 5, lines 7-10).  Sorge goes on to teach that the subunits of the probe are associated with one another by hydrogen or covalent bond formation (col. 6, lines 14-16) and that 80-99% of the probe subunits are associated with one another under conditions used to bind target nucleic acid, which is sufficiently strong to prevent dissociation between the probe subunits (col. 4, lines 56-60; col. 16, lines 51-58), which reads on a 90% efficiency of conjugation of the probe subunits. (The examiner would like to note that the terms “nucleic acid” and “oligonucleotide” are used interchangeably throughout the disclosure of Sorge [col. 31, lines 50-52]).  Sorge also goes on to disclose that complementary nucleic acid sequences (which, in this case, would include the target nucleic acid sequence and at least one nucleic acid subunit comprising a region which is complementary to said target nucleic acid sequence) can anneal by the formation of hydrogen bonds between the complementary bases (col. 5, lines 53-56), which reads on the probe subunit having a strong binding affinity for the target.
Sorge does not disclose that flow cytometry is used to detect the signal generated from the binding of the target to the signal generating moiety of one of the probe subunits.
However, Haff teaches [0007] a method of assaying a target in a sample, comprising combining the sample with two probes, wherein the each probe comprises a binding moiety for the target that can be covalently associated with a oligonucleotide sequence and a product precursor that can be covalently associated with each oligonucleotide sequence, wherein the two oligonucleotide sequences are capable of hybridizing to one another and bind the target in the sample with the binding moieties of each probe, wherein the hybridization of the two probes causes a reaction between the first and second product precursors of the probes resulting in a detectable reaction product.  (In this case, the examiner interprets the reaction product precursors to be signal generating moieties which are indirectly detected by exposure of the reaction product to a detection system.)   Haff goes on to teach that the detection system comprises a detection component capable of interacting with said reaction product and an amplification component capable of producing a plurality of detectable moieties [0008], wherein the detectable moieties are detected using flow cytometry ([0084]; Figs. 29A and 29B).
It would have been obvious to a person having ordinary skill in the art to combine the teachings of Sorge and Haff in order to produce a method in which the binding moieties have a strong affinity for the target and in which the probes are at least 90% efficiently associated with the binding moieties and signal generating moieties and a person having ordinary skill in the art would have been motivated to do so in order to optimize the detection method to prevent any dissociation of the target-probe system during the detection step of assaying the sample.  Additionally, it would have been obvious to a person having ordinary skill in the art to combine the teachings of Sorge and Haff to use a flow cytometry system for the detection of a signal generating moiety produced by the preceding optimized method, and a person having ordinary skill in the art would have been motivated to do so since this technique is routinely used in the art to detect health disorders.
Regarding claims 2-3, the method of claim 1 is discussed above.  Sorge teaches that the first probe subunit comprising a region which is complementary to a target nucleic acid sequence and a second probe subunit comprising a signal generating moiety are combined together and bound prior to contacting the target nucleic acid contained in a sample (col. 4, lines 35-40).
Regarding claim 4, while Sorge and Haff teach as described above pertaining to instant claim 1, Sorge goes on to teach that the probe has at least two subunits wherein at least one subunit comprises a region or regions that are complementary to a target nucleic acid sequence (col. 4, lines 28-32) which would read on a probe having three nucleic acid subunits bound or associated with one another, wherein at least one subunit (which would act as an adapter) would bind the target nucleic acid sequence.
Regarding claims 5-6, the method of claim 4 is discussed above.  Sorge teaches that the subunits of the probe, which in this case would include the adapter subunit as described regarding claim 4 above, are associated or bound together prior to contacting the target nucleic acid contained in a sample (col. 4, lines 35-40).
Regarding claim 7, the method of claim 1 is discussed above. Sorge teaches a singleplex assay for the detection of β-actin wherein PCR amplification is utilized to measure a target present in the sample (col. 62, lines 25-35; col. 27, lines 10-13).
Regarding claim 8, the method of claim 1 is discussed above.  Haff teaches ([0163]; Figure 13) the preparation of oligonucleotide conjugates comprising complementary nucleic acid sequences and hemipeptides, (wherein the N-terminal hemi-peptide comprises the detectable component fluorescein [0158]), and wherein the oligonucleotide conjugates are first analyzed using HPLC, then purified using an analytic C-18 column, and subsequently analyzed using LC-MS.  While Haff does not specifically disclose that the resultant oligonucleotide conjugates possess 90% purity, the examiner believes that the purification and analysis of the oligonucleotide conjugates as taught in Haff would result in 90% purity of the oligonucleotide conjugates. It is noted that In re Best (195 USPQ 430) and In re Fitzgerald (205 USPQ 594) discuss the support of rejections wherein the prior art discloses subject matter which there is reason to believe naturally includes functions that are newly cited or is identical to a product instantly claimed.  In such a situation the burden is shifted to the applicants to prove that subject matter shown to be in the prior art does not possess characteristic relied on (205 USPQ 594, second column, first full paragraph).
Regarding claims 12 and 13, the method of claim 1 is discussed above. Sorge teaches that the labeled probe which comprises the second oligonucleotide sequence has an incorporated a specific binding partner, wherein said specific binding partner comprises a protein (col. 45, lines 26-64).
Regarding claim 14, the method of claim 1 is discussed above.  Haff teaches that the binding moiety is a protein [0043].
Regarding claims 15 and 16, the method of claim 1 is discussed above.  Sorge teaches that the target is serum (col. 19, lines 7-21), a biological target comprising a protein.
Regarding claim 17, the method of claim 1 is discussed above.  Sorge teaches that the signal generating moiety or the one or more signal generating moieties of one of the probe subunits comprises a fluorophore (col. 46, lines 8-18).
Regarding claim 18, the method of claim 1 is discussed above.  Sorge teaches that the method comprises the use of a PCR assay to detect a target nucleic acid sequence, wherein the reactions are assayed in an AB17700 spectrofluorometric thermocycler, which reads on an automated system.  Moreover, the examiner would like to note that In re Venner (120 USPQ 193,194) discusses the support of rejections wherein the prior art discloses a manual activity which accomplishes the same result as an instantly claimed automated or mechanical means.
Regarding claim 19, the method of claim 1 is discussed above.  Sorge teaches that hybridized nucleic acid subunits are removed from bound target nucleic acid, wherein said removal is by a washing process (col. 18, line 65-col. 19, line 1).
10.	Claims 9-11 and 20 are rejected under 35 U.S.C. 103(a) as being unpatentable over Sorge in view of Haff, as applied to claims 1 and 19 above, and in further view of Tyagi and Kramer, Nature Biotechnology, 14, pp. 303-308, March 1996,  (hereinafter referred to as “Tyagi”).
Regarding claims 9-11 and 20, the methods of claims 1 and 19 are discussed above in Section 9.
Neither Sorge nor Haff specifically teach that the preparation and isolation of the molecular probe and the detectable components or the re-probing of said sample with a second detectable component or plurality of second detectable components.
However, Tyagi teaches (“Synthesis of molecular beacons”, pg. 307, right col., last para.-pg. 308, left col., 1st para.) a method of preparing a molecular probe with a detectable component, comprising providing the oligonucleotide sequence capable of binding a target nucleic acid and the fluorophore 5-(2'-aminoethyl)aminonapthalene-1-sulfonic acid (EDANS).  Upon incubation of the mixture of the oligonucleotide sequence with the fluorophore EDANS, the reaction mixture was precipitated with sodium acetate and ethanol, thereby removing unreacted EDANS.  The resulting oligonucleotides which were then covalently bound to EDANS were then dissolved in triethylammonium acetate and fractionated using a C-18 reverse-phase column, wherein fractions absorbed at 260, 336, and 490 nm were isolated and assessed for characteristic fluorescence of EDANS.  Moreover, Tyagi goes on to point out (pg. 307, right col., 1st full para.) that their method of preparing a molecular probe with a detectable component allows for the detection of probe-target hybrids without having to separate them from unhybridized probes.  However, Tyagi does not specifically teach a 90% efficiency of the formation of the oligonucleotide conjugates (i.e., claims 9-11).
Tyagi also teaches that a single sample can be probed for several different targets using a method which comprises probing with a plurality of molecular probes, which are specific for different target sequences in the sample (pg. 307, right col., 4th full para.), wherein the probes can comprise a plurality of detectable components which can emit or can be excited by light at different wavelengths (i.e., claim 20; pg. 307, right col., 3rd full para.)
It would have been obvious to one skilled in the art that the precipitation of an oligonucleotide conjugate followed by the reverse-phase chromatography isolation of said conjugate, and a subsequent assessment of the isolated conjugate for characteristic EDANS fluorescence would ensure the isolation of a highly-efficient oligonucleotide conjugate.  It would have also been obvious to one having ordinary skill in the art that said isolation steps could be repeated until a desired efficiency of 90% was reached.  Moreover, it would have been obvious to one skilled in the art to combine the teachings of Sorge, Hall, and Tyagi to produce an oligonucleotide probe with the capability of binding a target nucleic acid and/or with a detectable component, and a person having ordinary skill in the art would have been motivated to do so in order to produce highly-efficient probes which are extraordinarily target-specific, wherein Tyagi discloses (pg. 307, right col., 1st full para.) that the probes produced form their disclosure ignore nucleic acid target sequences that differ by as little as a single nucleotide.
Conclusion
11.	No claim is allowed.
12.	This is a continuation of Applicant's earlier Application No. 13/302,877.  All claims are drawn to the same invention claimed in the earlier application and could have been finally rejected on the grounds and art of record in the next Office action if they had been entered in the earlier application.  Accordingly, THIS ACTION IS MADE FINAL even though it is a first action in this case.  See MPEP § 706.07(b).  Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
13.	A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action.  In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any extension fee pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action.  In no, however, event will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
14.

APPLICANT REBUTTAL: REMARKS
Claims 1-3, 7-11, 13-14, and 16-19 have been amended. Claims 4-6, 12, and 20 have been canceled. New claims 21-31 have been added. Claims 1-3, 7-11, 13-19, and 21-31 will be pending upon entry of this amendment.
Support for amendments to the amended claims and the new claims can be found throughout the specification (e.g., in paragraphs [0006], [0035], [0135], [0150], [0155], [0179], [0189], [0219], [0244], [0246], [0281], [0801], and FIG. 19) and the claims as originally filed.
No new matter is being added with these amendments.
Rej
ections Under 35 U.S.C
Reconsideration is respectfully requested of the rejection of claims 1-20 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 of the invention.
The Office argues that claims 1 and 4 are each indefinite in the term "strong binding affinity," which is a relative term which renders the claims indefinite.
The Office argues that none of claims 7, 10, 13-14, 16, or 17 utilize proper Markush language.
The Office argues that the recitations "(HCS)," "(IHC)," "(ICC)," "(ISH)," "(EIA)," and "(ELISA)" in claim 7 are indefinite because it is unclear if the text within the parentheses is an alternative limitation of the claim or merely a descriptor of other elements of the claims. The Office further argues that the recitations are acronyms, the meanings of which may change over time.
Regarding claim 9, the Office argues that the recitations "the conjugation mixture," "the conjugates," and "the unconjugated first oligonucleotide sequence" all lack antecedent basis. The Office further argues that the phrase "at least one first oligonucleotide sequence" in part ii) of claim 9 be amended so as to differentiate the limitation from "the first oligonucleotide sequence" of claim 1.
The Office argues that claim 11 is indefinite for the recitation "the conjugation mixture," "the conjugate," and "the unconjugated second oligonucleotide sequence" lacking antecedent basis.
The Office argues that claim 13 is indefinite because the limitation "the scaffold" lacks antecedent basis.
The Office argues that the terms "small molecule" in claims 14 and 16 and "heavy atom" in claim 17 are relative terms which render the claims indefinite.
The Office argues that claims 17 and 19 are each indefinite for the recitation of "the hybridized detectable component" lacking antecedent basis.
The Office argues that claim 17 is further indefinite for the recitation "the one or more signal generating moieties" lacking antecedent basis.
Regarding claims 1 and 4, the term "strong binding affinity" has been removed from claim 1, and claim 4 has been canceled.
Regarding Markush language for claims 7, 10, 13-14, 16, and 17, claims 7, 10, 13, 16, and 17 have been amended to recite proper Markush language. Claim 14 as amended recites two options for the antibody in the alternative.
Regarding the parenthetical recitations in claim 7, "(HCS)," "(IHC)," "(ICC)," "(ISH)," "(EIA)," and "(ELISA)" have been removed.
Regarding the antecedent basis of claim 9, claim 9 has been amended to add
antecedent basis by reciting "a conjugation mixture," "a binding moiety-oligonucleotide conjugate," and "unconjugated first oligonucleotide." Part ii) of claim 9 has also been amended to recite "the first oligonucleotide" to match the limitation in claim 1.
Regarding the antecedent basis of claim 11, claim 11 now recites "a conjugation mixture comprising a signal generator-second oligonucleotide conjugate" and "unconjugated second oligonucleotide."
Regarding the antecedent basis of claim 13, claim 1 has been amended to recite "a scaffold molecule," and claim 13 has been amended to recite "the scaffold molecule."
Regarding the indefinite phrases of claims 14 and 16-17, "small molecule" and
"heavy atom" have been deleted from claims 14 and 17. "Small molecule" as used in claim 16 is defined in the specification in at least paragraph [0130].
Regarding the antecedent basis of claims 17 and 19, claims 17 and 19 have been amended to recite "a hybridized detectable component." Claim 17 has been further amended to recite "the plurality of signal generators," which has antecedent basis in claim 11.
Accordingly, Applicants respectfully request withdrawal of these rejections.
Reconsideration is respectfully requested of the rejection of claim 18 under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA), second paragraph as being incomplete for omitting essential steps.
The Office argues that the omitted steps are: how the automated system or robotic system is actually utilized in the method.
The Office also suggests that the phrase "a second oligonucleotide sequence" in
claim 20 be amended so as to differentiate the limitation from "the second oligonucleotide sequence" of claim 1.
Claim 18 has been amended to recite the method of claim 1, wherein the method comprises an automated system or robotic system that performs one or more steps of the claimed method.
Claim 18 has been amended to recite how the automated system or robotic system is utilized in the method.
Claim 20 has been canceled.
Accordingly, Applicants respectfully request withdrawal of these rejections.
Rej
ections Under 35 U.
S.C X103
1. So
rge
and
Haff
Reconsideration is respectfully requested of the rejection of claims 1-8 and 12-19 under 35 U.S.C. 103(a) as being unpatentable over US 7,183,052 B2 Sorge in view of US 2010/0159446 Al Haff et al.
The Office argues that Sorge teaches a method for assaying a target in a sample,
comprising providing to the sample a probe comprising two nucleic acid subunits, wherein at least one subunit comprises a region which is complementary to a target nucleic acid sequence and the other subunit comprises a signal generating moiety, binding or associating the subunits of the probe with one another in the absence of the target, and binding the target in the sample with the region(s) within the nucleic acid subunits which is complementary to the target. The Office argues that Sorge goes on to teach that the subunits of the probe are associated with one another by hydrogen or covalent bond
formation and that 80-99% of the probe subunits are associated with one another under conditions used to bind target nucleic acid, which is sufficiently strong to prevent dissociation between the probe subunits, which reads on a 90% efficiency of conjugation of the probe subunits. The Office argues that the terms "nucleic acid" and "oligonucleotide" are used interchangeably throughout the disclosure of Sorge. The Office argues that Sorge also goes on to disclose that complementary nucleic acid sequences can anneal by the formation of hydrogen bonds between the complementary bases, which reads on the probe subunit having a strong binding affinity for the target.
The Office admits that Sorge does not disclose that flow cytometry is used to detect the signal generated from the binding of the target to the signal generating moiety of one of the probe subunits. The Office adds Haff and argues that Haff teaches a method of assaying a target in a sample, comprising combining the sample with two probes, wherein each probe comprises a binding moiety for the target that can be covalently associated with a oligonucleotide sequence and a product precursor that can be covalently associated with each oligonucleotide sequence, wherein the two oligonucleotide sequences are capable of hybridizing to one another and bind the target in the sample with the binding moieties of each probe, wherein the hybridization of the two probes causes a reaction between the first and second product precursors of the probes resulting in a detectable reaction product. The Office argues that Haff goes on to teach that the detection system comprises a detection component capable of interacting with said reaction product and an amplification component capable of producing a plurality of detectable moieties, wherein the detectable moieties are detected using flow cytometry.
The Office argues that it would have been obvious to a person having ordinary skill in the art to combine the teachings of Sorge and Haff in order to produce a method in which the binding moieties have a strong affinity for the target and in which the probes are at least 90% efficiently associated with the binding moieties and signal generating moieties and a person having ordinary skill in the art would have been motivated to do so in order to optimize the detection method to prevent any dissociation of the target-probe system during the detection step of assaying the sample. The Office argues that it would have been obvious to a person having ordinary skill in the art to combine the teachings of Sorge and Haff to use a flow cytometry system for the detection of a signal generating moiety
produced by the preceding optimized method, and a person having ordinary skill in the art would have been motivated to do so since this technique is routinely used in the art to detect health disorders.
Claim 1 as amended recites a flow cytometry method for detecting a target of a sample, comprising:
i) mixing the sample with a molecular probe and a detectable component, wherein: 1) the molecular probe comprises a binding moiety conjugated to a first oligonucleotide sequence, wherein a. the binding moiety comprises an antibody, an antibody-fragment, an enzyme, a protein, a peptide, a carbohydrate, a nuclear receptor, a chelator, or combinations or derivatives thereof; and b. the conjugation between the binding moiety and the first oligonucleotides comprises one or more covalent bond linkages selected from the group consisting of a hydrazine, an oxime, and a triazine; and 2) the detectable component is not immobilized on a solid support and comprises a signal generator conjugated to a second oligonucleotide having a sequence complementary to the sequence of the first oligonucleotide, wherein a. the signal generator comprises a scaffold molecule further conjugated to more than one signal generating moieties, the scaffold molecule comprising a dendrimer, a polysaccharide, or combinations or derivatives thereof; and b. the second oligonucleotide is directly conjugated to the scaffold molecule of the signal generator via one or more covalent bond linkages selected from the group consisting of a hydrazine, an oxime, and a triazine;
ii) binding the target in the sample with the binding moiety of the molecular probe;
iii) hybridizing the first oligonucleotide with the second oligonucleotide;
iv) reducing the potential for cross-talk by adding to the molecular probe and the detectable component an oligonucleotide comprising at least one of: 1) an unconjugated oligonucleotide having a sequence complementary to the sequence of the first oligonucleotide; and 2) an unconjugated oligonucleotide having a sequence complementary to the sequence of the second oligonucleotide; then
v) detecting a signal generated from the detectable component hybridized to the target- bound molecular probe using flow cytometry, wherein the hybridized detectable component is not immobilized on a solid support.
Sorge's binding component is a nucleic acid sequence. The target binding
component in Sorge's method is a nucleic acid sequence, called the "target nucleic acid binding sequence" (a subunit of Sorge's probe), which "...binds specifically to target nucleic acid" (see Sorge col. 7, lines 16-34). In contrast, the binding moiety of the pending claims comprises an antibody, an antibody-fragment, an enzyme, a protein, a peptide, a carbohydrate, a nuclear receptor, a chelator, or combinations or derivatives thereof.
Applicants contend that Haff fails to cure the deficiencies of Sorge. Haff does not teach or suggest providing a detectable component (comprising a signal generator) to the sample, as specified in the pending claims. Moreover, Haffs additional steps can be a major source of detection inefficiency. For example, Haffs method must first generate a reaction product (a required step that can diminish detection efficiency) and then detect the reaction product. Haffs detection step is limited to the efficiency by which the reaction product was first generated, thereby making it less reflective of the amount of target present in the sample. Furthermore, both processes of either 1) recognizing the reaction product and then generating a signal, or 2) amplification followed by detection, have their own inefficiencies, further compounding Haffs detection inefficiency.
Even assuming that Haff is combinable with Sorge, Applicants submit that Haff also fails to teach the hybridization specified in the pending claims. Specifically, the methods of the pending claims "connect" the detectable component (containing the signal generator) to the target bound molecular probe via hybridization. In contrast, Haffs method does not link the target bound probe to the "detectable" reaction product generated via hybridization. Rather, to connect the entity capable of generating a signal for detection to Haffs target bound probe, Haff requires a reaction to form at least one conjugate bond (or require chemical transformation of a masked precursor for subsequence recognition) (see Haff paragraphs [0023]-[0025]).
In view of the above, Haff neither teaches or suggests the methods of the pending claims, nor cures the deficiencies of Sorge. Accordingly, Applicants submit that the Office
has failed to present aprimafacie case of obviousness, and, therefore, submit that the pending claims are patentable over Sorge, alone or in combination with Haff.
2. So
m
e, Haff, and Tyagi and Kramer
Reconsideration is respectfully requested of the rejection of claims 9-11 and 20 under 35 U.S.C. 103(a) as being unpatentable over Sorge in view of Haff as applied to claims 1 and 19 above and in further view of Tyagi and Kramer, Nature Biotechnology, 14, pp. 303-308, March 1996.
The Office admits that neither Sorge nor Haff specifically teach that the preparation and isolation of the molecular probe and the detectable components or the reprobing of said sample with a second detectable component or plurality of second detectable components.
The Office contends that Tyagi teaches a method of preparing a molecular probe with a detectable component, comprising providing the oligonucleotide sequence capable of binding a target nucleic acid and the fluorophore 5-(2'-aminoethyl)aminonapthalene-1- sulfonic acid (EDANS). The Office argues that upon incubation of the mixture of the oligonucleotide sequence with the fluorophore EDANS, the reaction mixture was precipitated with sodium acetate and ethanol, thereby removing unreacted EDANS. The Office argues that the resulting oligonucleotides which were then covalently bound to EDANS were then dissolved in triethylammonium acetate and fractionated using a C-18 reverse-phase column, wherein fractions absorbed at 260, 336, and 490 nm were isolated and assessed for characteristic fluorescence of EDANS. The Office argues that Tyagi goes on to point out that their method of preparing a molecular probe with a detectable component allows for the detection of probe-target hybrids without having to separate them from unhybridized probes. The Office notes that Tyagi does not specifically teach a 90% efficiency of the formation of the oligonucleotide conjugates. The Office argues that Tyagi also teaches that a single sample can be probed for several different targets using a method which comprises probing with a plurality of molecular probes, which are specific for different target sequences in the sample, wherein the probes can comprise a plurality of detectable components which can emit or can be excited by light at different wavelengths.
The Office argues that it would have been obvious to one skilled in the art that the precipitation of an oligonucleotide conjugate followed by the reverse-phase chromatography isolation of said conjugate, and a subsequent assessment of the isolated conjugate for characteristic EDANS fluorescence would ensure the isolation of a highly- efficient oligonucleotide conjugate. The Office argues that it would have also been obvious to one having ordinary skill in the art that said isolation steps could be repeated until a desired efficiency of 90% was reached. The Office further argues that it would have been obvious to one skilled in the art to combine the teachings of Sorge, Hall, and Tyagi to produce an oligonucleotide probe with the capability of binding a target nucleic acid and/or with a detectable component, and a person having ordinary skill in the art would have been motivated to do so in order to produce highly-efficient probes which are extraordinarily target-specific, wherein Tyagi discloses that the probes produced from their disclosure ignore nucleic acid target sequences that differ by as little as a single nucleotide.
Applicants maintain the above arguments concerning Sorge and Haff.
Further, Tyagi fails to cure the above-noted deficiencies of either Sorge or Haff, so Applicants submit that the pending claims are patentable over Sorge and Haff, alone or in combination with Tyagi.
CONCLUSION

NEXT OFFICE ACTION: DETAILED ACTION
Notice of Pre-AIA  or AIA  Status
1.	The present application is being examined under the pre-AIA  first to invent provisions.
Continued Examination Under 37 CFR 1.114
2.	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 20 June 2023 has been entered.
Status of the Claims
3.	This action is in response to papers filed 20 June 2023 in which claims 1-3, 7-11, 13-14, and 16-19 were amended, claims 4-6 and 12 and 20 were canceled, and new claims 21-31 were added.  All of the amendments have been thoroughly reviewed and entered.
Any previous rejections under 35 U.S.C. 103(a) not reiterated below are withdrawn in view of the amendments.
Applicant’s arguments have been thoroughly reviewed and are addressed following the rejections necessitated by the amendments.
4.	Claims 1-3, 7-11, 13-19, and 21-31 are under prosecution.
5.	This Office Action includes new rejections necessitated by the amendments.
Claim Interpretation
6.	 For the purposes of searching for and applying prior art under 35 U.S.C. 102 and 103, absent a clear indication in the specification or claims of what the basic and novel characteristics actually are, consisting essentially of, as found in claims 30 and 31, will be construed as equivalent to comprising. See, e.g., PPG, 156 F.3d at 1355, 48 USPQ2d at 1355 (PPG could have defined the scope of the phrase consisting essentially of' for purposes of its patent by making clear in its specification what it regarded as constituting a material change in the basic and novel characteristics of the invention.). See also AK Steel Corp. v. Sollac, 344 F3.d 1234, 1239-1240, 68 USPQ2d 1280, 1283-84 (Fed. Cir. 2003); In re Janakirama-Rao, 317 F.2d 951, 954, 137 USPQ 893, 895-96 (CCPA 1963).  See MPEP 2163.
Claim Rejections - 35 USC § 103
7.	The following is a quotation of pre-AIA  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, 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.
8.	This application currently names joint inventors. In considering patentability of the claims under pre-AIA  35 U.S.C. 103(a), the examiner presumes that the subject matter of the various claims was commonly owned at the time any inventions covered therein were made absent any evidence to the contrary.  Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and invention dates of each claim that was not commonly owned at the time a later invention was made in order for the examiner to consider the applicability of pre-AIA  35 U.S.C. 103(c) and potential pre-AIA  35 U.S.C. 102(e), (f) or (g) prior art under pre-AIA  35 U.S.C. 103(a).
9.	Claims 1-3, 8-11, 13-19, and 21-30 are rejected under pre-AIA  35 U.S.C. 103(a) as being unpatentable over Winther (PCT International Patent Application Publication No. WO 2007/031874 A2, published 22 March 2007, listed in the Information Disclosure Statement filed 20 April 2012 [citations are from the National Stage Entry, U.S. Patent Application Publication No. US 2010/0105145 A1, published 29 April 2010; the National Stage is deemed identical to the PCT]), Schwartz (U.S. Patent Application Publication No. US 2003/0013857 A1, published 16 January 2003), and Coull et al (U.S. Patent Application Publication No. US 2003/0124521 A1, published 3 July 2003).
Regarding claim 1, Winther teaches a method of detecting a target in a sample using flow cytometry (Abstract), comprising providing to the sample a molecular probe, in the form of a recognition unit, having a first oligonucleotide sequence, in the form of a nucleic acid segment, conjugated through a linker to a binding moiety, in the form of an antibody (Figure 3 and paragraph 0013).  Winther further teaches a detectable component, in the form of a detection unit, which comprises a second oligonucleotide (i.e., nucleic acid analog), which hybridizes to the first oligonucleotide sequence via a complementary sequence (e.g., paragraphs 0014 and 0168), and which further comprises a signal generator, in the form of multiple detectable labels, which are attached to a polymer scaffold (Figures 3 and 6, and paragraphs 0013 and 0016).  The polymer comprises a polysaccharide, in the form of the polysaccharide dextran or a dendrimer (paragraphs 0174-0175).
It is noted that the specification states that oligonucleotides comprise nucleobases and analogs thereof (paragraphs 0144-0145); thus, the nucleic acid analogs of Winther are oligonucleotides, and the claim has been given the broadest reasonable interpretation consistent with the teachings of the specification regarding an “oligonucleotide” (In re Hyatt, 211 F.3d1367, 1372, 54 USPQ2d 1664, 1667 (Fed. Cir. 2000) (see MPEP 2111 [R-1]).
Winther also teaches the labels are fluorescent dyes (paragraph 0100); thus, none of the components are immobilized on solid supports.  Winther further teaches the target is detected by binding the probe, and that labeling is later than probe binding (paragraph 0004), thus, it would have been obvious to bind the target to the binding moiety, followed by hybridizing the first and second oligonucleotides by subsequently adding the detection unit.
Alternatively, the courts have held that selection of any order of performing process steps is prima facie obvious in the absence of new or unexpected results (In re Burhans, 154 F.2d 690, 69 USPQ 330 (CCPA 1946).  See MPEP 2144.04 IV.C.
In addition, the courts have found that “where the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation.” In re Aller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA 1955).  See MPEP 2144.05 II.
Thus, any order of combining the components represents an obvious variant and/or routine optimization of the steps of the prior art.
Applicant is advised that MPEP 716.01(c) makes clear that “[t]he arguments of counsel cannot take the place of evidence in the record” (In re Schulze, 346 F.2d 600, 602, 145 USPQ 716, 718 (CCPA 1965)). Thus, Applicant should not merely rely upon counsel’s arguments in place of evidence in the record.
Winther also teaches detection of the signals (i.e., labels) using flow cytometry (Abstract), and that the method has the added advantage of being compatible with many differently types of samples, probes, targets, and labels (Abstract).  Thus, Winther teaches the known techniques discussed above.
While Winther teaches the oligonucleotide within the recognition unit and the oligonucleotide within the detection units are each covalently attached (paragraph 0174), as well as the use of hydrazines (paragraph 0078) and aldehydes (paragraph 0186), which form hydrazones, Winther does not explicitly teach the use of the functionally equivalent hydrazone group.
However, Schwartz teaches a method wherein a biomolecule is attached (i.e., crosslinked) using a hydrazine moiety to link an amino group (Abstract) to form a hydrazine (paragraph 0028).  Schwartz teaches the linking of oligonucleotides and antibodies (paragraph 0016), and that the method has the added advantage of allowing immobilization in a single step (paragraph 0111).  Thus, Schwartz teaches the known techniques discussed above.
It would therefore have been obvious to a person of ordinary skill in the art to have combined the method of Winther with the teachings of Schwartz, which utilizes a functionally equivalent amine derived hydrazine, to arrive at the instantly claimed method with a reasonable expectation of success.  The ordinary artisan would have been motivated to make the combination because said combination would have resulted in a method having the added advantage of being compatible with many differently types of samples, probes, targets, and labels as explicitly taught by Winther (Abstract) as well as the added advantage of allowing immobilization in a single step as explicitly taught by Schwartz (paragraph 0111). In addition, it would have been obvious to the ordinary artisan that the known techniques of using the functionally equivalent hydrazones of Schwartz could have been combined with the method of Winther with predictable results because the known techniques of Winther and Schwartz predictably result in reliable immobilization of the amine-bearing molecule.
Neither Winther nor Schwartz teach reducing the potential for crosstalk by adding an unconjugated oligonucleotide.
However, Coull et al teach a method wherein addition of unconjugated (i.e., unlabeled) oligonucleotides having a sequence complementary to a probe (i.e., a mismatch; paragraph 0025; see also Figure 3). The unconjugated (i.e., unlabeled) is therefore complementary to the claimed detectable component (i.e., the labeled molecule of Yang in view of Schwartz).  Thus, because the purpose of the unconjugated (i.e., unlabeled) probe is to prevent nonspecific binding of two nucleotide sequences, the unlabeled probe could alternatively have been complementary to the molecular probe.  Coull et al also teach the addition of the unlabeled probe has the added advantage of reducing crosstalk (i.e., by increasing the signal to noise ratio by reducing non-specific binding) without substantially altering the detection limit of an assay (paragraph 0025).  Thus, Coull et al teach the known techniques discussed above.
It would therefore have been obvious to a person of ordinary skill in the art to have combined the method of Winther and Schwartz so as to include the unconjugated oligonucleotide of Coull et al to arrive at the instantly claimed method with a reasonable expectation of success.  The ordinary artisan would have been motivated to make the modification because said modification would have resulted in a method having the added advantage of reducing crosstalk by increasing the signal to noise ratio via reduction of non-specific binding without substantially altering the detection limit of an assay of as explicitly taught by Coull et al (paragraph 0025).  In addition, it would have been obvious to the ordinary artisan that the known techniques of Coull et al could have been applied to the method of Winther in view of Schwartz with predictable results because the known techniques of Coull et al predictably result in an improved signal to noise ratio.
Regarding claims 2-3, the method of claim 1 is discussed above.  Winther teaches the target is detected by binding the probe, and that labeling may be later than probe binding (paragraph 0004), thus, it would have been obvious to either bind the target to the binding moiety, followed by hybridizing the first and second oligonucleotides by subsequently adding the detection unit, or to bind probe and detectable component prior to target binding.
Alternatively, it is reiterated the courts have held that selection of any order of performing process steps is prima facie obvious in the absence of new or unexpected results, and that the courts have found that where the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation.  Thus, any order of combining the components represents an obvious variant and/or routine optimization of the steps of the prior art.
Applicant is again cautioned to avoid merely relying upon counsel’s arguments in place of evidence in the record.
Regarding claim 8, the method of claim 1 is discussed above.  Winther further teaches preparing each nucleic acid (e.g., PNA) probes that are purified by HPLC to give pure fractions (paragraph 0238).
With respect to the claimed purity, it is reiterated that the courts have found that where the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation.
Therefore, the claimed purity merely represents routine optimization of the purification taught by the prior art.
Applicant is again cautioned to avoid merely relying upon counsel’s arguments in place of evidence in the record.
Regarding claims 9-10, the method of claim 1 is discussed above.  Winther teaches purification of an oligonucleotide (e.g., PNA) using HPLC, which retards the oligonucleotide and allows separation from other reagents (Example 9).  Schwartz also teaches wash steps (paragraph 0204), as well as HPLC purification (paragraph 0219).  Coull et al teach conjugation of a binding moiety (i.e., enzyme) with an oligonucleotide followed by washing (paragraph 0176), as well as HPLC purification (paragraph 0159).  Thus, all of the claimed steps are taught by the prior art.
It is reiterated that the courts have held that selection of any order of performing process steps is prima facie obvious in the absence of new or unexpected results.  Thus, any order of combining the components is an obvious variant of the steps of the prior art.
With respect to the claimed purity, Applicant is reminded that the courts have found that where the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation.
Therefore, the claimed purity merely represents routine optimization of the purification taught by the prior art.
Applicant is again cautioned to avoid merely relying upon counsel’s arguments in place of evidence in the record.
Regarding claim 11, the method of claim 1 is discussed above.  Schwartz et al teach conjugating with molecules having carbonyl groups (paragraphs 0145-0148); Winther teaches aldehydes (paragraph 0186), which have a carbonyl group.  Schwartz also teaches wash steps (paragraph 0204), as well as HPLC purification (paragraph 0219).  Winther teaches purification of an oligonucleotide (e.g., PNA) using HPLC, which retards the oligonucleotide and allows separation from other reagents (Example 9).  Coull et al teach washing (paragraph 0176), as well as HPLC purification (paragraph 0159).  Thus, all of the claimed steps are taught by the prior art.
It is reiterated that the courts have held that selection of any order of performing process steps is prima facie obvious in the absence of new or unexpected results.  Thus, any order of combining the components is an obvious variant of the steps of the prior art.
With respect to the claimed purity, Schwartz teaches greater than 80% formation of the conjugate (paragraph 0216).
It is noted that the courts have stated where the claimed ranges “overlap or lie inside the ranges disclosed by the prior art” and even when the claimed ranges and prior art ranges do not overlap but are close enough that one skilled in the art would have expected them to have similar properties, a prima facie case of obviousness exists (see In re Wertheim, 541 F.2d 257, 191 USPQ 90 (CCPA 1976); In re Woodruff, 919 F.2d 1575, 16 USPQ2d 1934 (Fed. Cir. 1990); Titanium Metals Corp. of America v. Banner, 778 F2d 775. 227 USPQ 773 (Fed. Cir. 1985) (see MPEP 2144.05.01).
Applicant is also reminded that the courts have found that where the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation.
Therefore, the claimed purity merely represents routine optimization and/or an obvious variant of the purity taught by the prior art.
Applicant is again cautioned to avoid merely relying upon counsel’s arguments in place of evidence in the record.
Regarding claim 13, the method of claim 1 is discussed above.  Winther teaches the scaffold is a polymer scaffold (Figures 3 and 6, and paragraphs 0013 and 0016), which comprises a polysaccharide, in the form of the polysaccharide dextran or a dendrimer (paragraphs 0174-0175).
Regarding claim 14, the method of claim 1 is discussed above.  Winther teaches the antibody is a monoclonal antibody (paragraph 0123).
Regarding claims 15-16 and 21-22, the method of claim 1 is discussed above.  Winther teaches the target is an antigen or a protein (paragraph 0087), and that the antibody is a monoclonal antibody (paragraph 0123).
Regarding claim 17, the method of claim 11 is discussed above.  Winther teaches the labels are fluorescent dyes (paragraph 0017).
Regarding claim 18, the method of claim 1 is discussed above.  Winther teaches the method is automated (Abstract).
Regarding claim 19, the method of claim 1 is discussed above.  Winther teaches washing to remove hybridized components; i.e., use of stringent conditions (paragraph 0168).
Regarding claim 23, the method of claim 1 is discussed above.  Winther teaches lengths of 10 bases (Table 8), which is in the claimed range.
In addition, it is reiterated that the courts have stated that where the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation, and that the courts have stated where the claimed ranges overlap or lie inside the ranges disclosed by the prior art and even when the claimed ranges and prior art ranges do not overlap but are close enough that one skilled in the art would have expected them to have similar properties, a prima facie case of obviousness exists.
Therefore, the claimed ranges merely represent an obvious variant and/or routine optimization of the lengths taught by the prior art.
Applicant is again cautioned to avoid merely relying upon counsel’s arguments in place of evidence in the record.
Regarding claims 24-25, the method of claim 1 is discussed above.  Winther teaches the target is detected by binding the probe, and that labeling may be later than probe binding (paragraph 0004); thus, it would have been obvious to bind probe and detectable component, which bind to each other via hybridization, prior to target binding.
Alternatively, it is reiterated the courts have held that selection of any order of performing process steps is prima facie obvious in the absence of new or unexpected results, and that the courts have found that where the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation.  Thus, any order of combining the components represents an obvious variant and/or routine optimization of the steps of the prior art.
Applicant is again cautioned to avoid merely relying upon counsel’s arguments in place of evidence in the record.
Regarding claims 25-28, the method of claim 1 is discussed above.
It is reiterated the courts have held that selection of any order of performing process steps is prima facie obvious in the absence of new or unexpected results, and that the courts have found that where the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation.  Thus, any order of combining the components represents an obvious variant and/or routine optimization of the steps of the prior art.
Applicant is again cautioned to avoid merely relying upon counsel’s arguments in place of evidence in the record.
Regarding claims 29-30, the method of claim 1 is discussed above.  Winther teaches the signal generator comprises multiple detectable labels, which are attached via a polymer scaffold (Figures 3 and 6, and paragraphs 0013 and 0016), and that polymer comprises a polysaccharide, in the form of the polysaccharide dextran (paragraphs 0174-0175).
10.	Claims 1-3, 8-11, 13-19, and 21-30 are rejected under pre-AIA  35 U.S.C. 103(a) as being unpatentable over Winther (U.S. Patent Application Publication No. US 2010/0105145 A1, published 29 April 2010), Schwartz (U.S. Patent Application Publication No. US 2003/0013857 A1, published 16 January 2003), and Coull et al (U.S. Patent Application Publication No. US 2003/0124521 A1, published 3 July 2003) based on the citations provided above.
11.	Claim 7 is rejected under pre-AIA  35 U.S.C. 103(a) as being unpatentable over Winther (PCT International Patent Application Publication No. WO 2007/031874 A2, published 22 March 2007, listed in the Information Disclosure Statement filed 20 April 2012 [citations are from the National Stage Entry, U.S. Patent Application Publication No. US 2010/0105145 A1, published 29 April 2010; the National Stage is deemed identical to the PCT]), Schwartz (U.S. Patent Application Publication No. US 2003/0013857 A1, published 16 January 2003), and Coull et al (U.S. Patent Application Publication No. US 2003/0124521 A1, published 3 July 2003) as applied to claim 1 above, and further in view of Salazar et al (U.S. Patent Application Publication No. US 2009/0051912 A1, published 26 February 2009).
Regarding claim 7, the method of claim 1 is discussed above in Section 9.
While Winther also teaches multiplex assays (i.e., simultaneous detection of two conjugates; paragraph 0115), as well as binding detection using microscopy (paragraph 0091), neither Winther, Schwartz, nor Coull et al explicitly teach the combination of flow cytometry and microscopy.
However, Salazar et al teach methods wherein flow cytometry is combined with microscopy, which has the added advantage of allowing analysis of the sizes and attributes of cells without having to replace major components of a flow cytometer (paragraph 0051).  Thus, Salazar et al teach the known techniques discussed above.
It would therefore have been obvious to a person of ordinary skill in the art to have combined the method of Winther, Schwartz, and Coull et al with the teachings of Salazar et al to arrive at the instantly claimed method with a reasonable expectation of success.  The ordinary artisan would have been motivated to make the combination because said combination would have resulted in a method having the added advantage of allowing analysis of the sizes and attributes of cells without having to replace major components of a flow cytometer as explicitly taught by Salazar et al (paragraph 0051). In addition, it would have been obvious to the ordinary artisan that the known techniques of Salazar et al could have been combined with the method of Winther, Schwartz, and Coull et al with predictable results because the known techniques of Salazar et al predictably result in a reliable combination of components for cell detection.
12.	Claim 7 is rejected under pre-AIA  35 U.S.C. 103(a) as being unpatentable over Winther (U.S. Patent Application Publication No. US 2010/0105145 A1, published 29 April 2010), Schwartz (U.S. Patent Application Publication No. US 2003/0013857 A1, published 16 January 2003), and Coull et al (U.S. Patent Application Publication No. US 2003/0124521 A1, published 3 July 2003) as applied to claim 1 above, and further in view of Salazar et al (U.S. Patent Application Publication No. US 2009/0051912 A1, published 26 February 2009) based on the citations provided above.
13.	Claims 29-31 are rejected under pre-AIA  35 U.S.C. 103(a) as being unpatentable over Winther (PCT International Patent Application Publication No. WO 2007/031874 A2, published 22 March 2007, listed in the Information Disclosure Statement filed 20 April 2012 [citations are from the National Stage Entry, U.S. Patent Application Publication No. US 2010/0105145 A1, published 29 April 2010; the National Stage is deemed identical to the PCT]), Schwartz (U.S. Patent Application Publication No. US 2003/0013857 A1, published 16 January 2003), and Coull et al (U.S. Patent Application Publication No. US 2003/0124521 A1, published 3 July 2003) as applied to claim 1 above, and further in view of Rowley et al (U.S. Patent No. 4,975,532, issued 4 December 1990).
It is noted that while claims 29-30 are rejected as described above, the claims are also obvious using the interpretation outlined below.
Regarding claims 29-31, the method of claim 1 is discussed above in Section 9.
As noted above, Winther teaches the oligonucleotide within the recognition unit and the oligonucleotide within the detection units are each covalently attached (paragraph 0174), as well as the use of hydrazines (paragraph 0078) and aldehydes (paragraph 0186), which form hydrazones, and that the polymer comprising the labels is dextran (paragraphs 0174-0175).
Schwartz teaches crosslinking using a hydrazine moiety to link an amino group (Abstract) to form a hydrazine (paragraph 0028).
Rowley et al teach methods wherein dextran is linked to fluorescent labels using hydrazone linkages (column 7, lines 1-20).  Rowley et al also teach the method has the added advantage of allowing direct labeling with multiple labeling entities while overcoming the problem of fluorescence quenching (column 2, lines 35-45).  Thus, Rowley et al teach the known techniques discussed above.
It would therefore have been obvious to a person of ordinary skill in the art to have combined the method of Winther, Schwartz, and Coull et al with the teachings of Rowley et al to arrive at the instantly claimed method with a reasonable expectation of success.  The ordinary artisan would have been motivated to make the combination because said combination would have resulted in a method having the added advantage of allowing direct labeling with multiple labeling entities while overcoming the problem of fluorescence quenching as explicitly taught by Rowley et al (column 2, lines 35-45). In addition, it would have been obvious to the ordinary artisan that the known techniques of Rowley et al could have been combined with the method of Winther, Schwartz, and Coull et al with predictable results because the known techniques of Rowley et al predictably result in a reliable labeling of dextran.
14.	Claims 29-31 are rejected under pre-AIA  35 U.S.C. 103(a) as being unpatentable over Winther (U.S. Patent Application Publication No. US 2010/0105145 A1, published 29 April 2010), Schwartz (U.S. Patent Application Publication No. US 2003/0013857 A1, published 16 January 2003), and Coull et al (U.S. Patent Application Publication No. US 2003/0124521 A1, published 3 July 2003) as applied to claim 1 above, and further in view of Rowley et al (U.S. Patent No. 4,975,532, issued 4 December 1990) based on the citations provided above.
Double Patenting
15.	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 §§ 706.02(l)(1) - 706.02(l)(3) 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 USPTO Internet website contains terminal disclaimer forms which may be used. Please visit www.uspto.gov/patent/patents-forms. The 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/process/file/efs/guidance/eTD-info-I.jsp.
16.	Claims 1-3, 8-11, 13-19, and 21-30 are rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1-20 of U.S. Patent No. 9,222,936 B2 in view of Winther (PCT International Patent Application Publication No. WO 2007/031874 A2, published 22 March 2007, listed in the Information Disclosure Statement filed 20 April 2012 [citations are from the National Stage Entry, U.S. Patent Application Publication No. US 2010/0105145 A1, published 29 April 2010; the National Stage is deemed identical to the PCT]), Schwartz (U.S. Patent Application Publication No. US 2003/0013857 A1, published 16 January 2003), and Coull et al (U.S. Patent Application Publication No. US 2003/0124521 A1, published 3 July 2003).
Both sets of claims are drawn to the same limitations; e.g., reducing cross talk using a molecular probe comprising a binding moiety having a specific binding affinity range, covalent linkages including hydrazones, hybridization, detection using flow cytometry, etc.  Any additional limitations of the ‘936 claims are encompassed by the open claim language “comprising” found in the instant claims.
Any missing limitations are found in the cited references as noted in the obviousness rejections under 35 U.S.C. 103(a) as detailed above.
17.	Claims 1-3, 8-11, 13-19, and 21-30 are rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1-20 of U.S. Patent No. 9,222,936 B2 in view of Winther (U.S. Patent Application Publication No. US 2010/0105145 A1, published 29 April 2010), Schwartz (U.S. Patent Application Publication No. US 2003/0013857 A1, published 16 January 2003), and Coull et al (U.S. Patent Application Publication No. US 2003/0124521 A1, published 3 July 2003) based on the citations presented above.
18.	Claim 7 is rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1-20 of U.S. Patent No. 9,222,936 B2 in view of Winther (PCT International Patent Application Publication No. WO 2007/031874 A2, published 22 March 2007, listed in the Information Disclosure Statement filed 20 April 2012 [citations are from the National Stage Entry, U.S. Patent Application Publication No. US 2010/0105145 A1, published 29 April 2010; the National Stage is deemed identical to the PCT]), Schwartz (U.S. Patent Application Publication No. US 2003/0013857 A1, published 16 January 2003), and Coull et al (U.S. Patent Application Publication No. US 2003/0124521 A1, published 3 July 2003) as applied to claim 1 above, and further in view of Salazar et al (U.S. Patent Application Publication No. US 2009/0051912 A1, published 26 February 2009) based on the citations provided above.
19.	Claim 7 is rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1-20 of U.S. Patent No. 9,222,936 B2 in view of Winther (U.S. Patent Application Publication No. US 2010/0105145 A1, published 29 April 2010), Schwartz (U.S. Patent Application Publication No. US 2003/0013857 A1, published 16 January 2003), and Coull et al (U.S. Patent Application Publication No. US 2003/0124521 A1, published 3 July 2003) as applied to claim 1 above, and further in view of Salazar et al (U.S. Patent Application Publication No. US 2009/0051912 A1, published 26 February 2009) based on the citations provided above based on the citations presented above.
20.	Claims 29-31 are rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1-20 of U.S. Patent No. 9,222,936 B2 in view of Winther (PCT International Patent Application Publication No. WO 2007/031874 A2, published 22 March 2007, listed in the Information Disclosure Statement filed 20 April 2012 [citations are from the National Stage Entry, U.S. Patent Application Publication No. US 2010/0105145 A1, published 29 April 2010; the National Stage is deemed identical to the PCT]), Schwartz (U.S. Patent Application Publication No. US 2003/0013857 A1, published 16 January 2003), and Coull et al (U.S. Patent Application Publication No. US 2003/0124521 A1, published 3 July 2003) as applied to claim 1 above, and further in view of Rowley et al (U.S. Patent No. 4,975,532, issued 4 December 1990) based on the citations provided above.
21.	Claims 29-31 are rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1-20 of U.S. Patent No. 9,222,936 B2 in view of Winther (U.S. Patent Application Publication No. US 2010/0105145 A1, published 29 April 2010), Schwartz (U.S. Patent Application Publication No. US 2003/0013857 A1, published 16 January 2003), and Coull et al (U.S. Patent Application Publication No. US 2003/0124521 A1, published 3 July 2003) as applied to claim 1 above, and further in view of Rowley et al (U.S. Patent No. 4,975,532, issued 4 December 1990) based on the citations provided above.
Response to Arguments
22.	Applicant’s arguments have been considered.  The previous indefiniteness rejections are withdrawn in view of the amendments.  The arguments regarding the previous prior art rejections have been considered but are moot in view of the new rejections necessitated by the amendments.
Conclusion
23.	No claim is allowed.
24.

CITED REFERENCE SUMMARIES: Ref US20100159446A1: Claims:
1. A method of determining the presence and/or amount of a biological target in a sample, the method comprising:
(a) combining the sample with (1) a first probe comprising (i) a first binding moiety with binding affinity to the biological target, (ii) a first oligonucleotide sequence, and (iii) a first product precursor associated with the first oligonucleotide sequence, and (2) a second probe comprising (i) a second binding moiety with binding affinity to the biological target, (ii) a second oligonucleotide sequence capable of hybridizing to the first oligonucleotide sequence, and (iii) a second product precursor associated with the second oligonucleotide sequence, under conditions to permit both the first and second binding moieties to bind to the biological target, if present in the sample, whereupon the first and second oligonucleotide sequences hybridize to one another to bring the first and second product precursors into reactive proximity with one another to produce a reaction product, wherein the reaction product is selected from the group consisting of an intact epitope, an enzyme substrate, an enzyme activator and a ligand; and (b) exposing the reaction product of step (a), if present, to a detection system comprising an amplification component and a detection component under conditions to permit the production of a plurality of detectable moieties; and (c) determining the presence and/or amount of the detectable moieties produced in step (b) thereby to determine the presence and/or amount of the biological target in the sample.
2. The method of claim 1, wherein, in step (a), the first binding moiety is covalently associated with the first oligonucleotide sequence.
3. The method of claim 2, wherein, in step (a), the second binding moiety is covalently associated with the second oligonucleotide sequence.
4. The method of claim 1, wherein, in step (a), the first binding moiety is non-covalently associated with the first oligonucleotide sequence.
5. The method of claim 4, wherein the first binding moiety is non-covalently associated with the first oligonucleotide sequence through a zipcode sequence hybridized to an anti-zipcode sequence.
6. The method of claim 4, wherein, in step (a), the second binding moiety is non-covalently associated with the second oligonucleotide sequence.
7. The method of claim 6, wherein the second binding moiety is non-covalently associated with the second oligonucleotide sequence through a zipcode sequence hybridized to an anti-zipcode sequence.
8. A method of determining the presence and/or amount of a biological target in a sample, the method comprising:
(a) providing a first target binding component comprising (i) a first binding moiety having binding affinity to the biological target, and (ii) a first oligonucleotide zipcode sequence; (b) providing a second target binding component comprising (i) a second binding moiety having binding affinity to the biological target, and (ii) a second oligonucleotide zipcode sequence; (c) providing a first reporter component comprising (i) a first oligonucleotide anti-zipcode sequence capable of hybridizing to the first oligonucleotide zipcode sequence, (ii) a first reporter oligonucleotide, and (iii) a first product precursor associated with the first reporter oligonucleotide; (d) providing a second reporter component comprising (i) a second oligonucleotide anti-zipcode sequence capable of hybridizing to the second oligonucleotide zipcode sequence, (ii) a second reporter oligonucleotide capable of hybridizing to the first reporter oligonucleotide sequence, and (iii) a second product precursor associated with the second reporter oligonucleotide sequence and capable of reacting with the first product precursor when brought into reactive proximity with the first product precursor; (e) combining the sample with the first target binding component, the second target binding component, the first reporter component, and the second reporter component under conditions so that the first and second binding moieties bind to the biological target, if present in the sample, whereupon (i) the first zipcode sequence hybridizes to the first anti-zipcode oligonucleotide sequence, (ii) the second oligonucleotide zipcode sequence hybridizes to the second oligonucleotide anti-zipcode sequence, and (iii) the second reporter oligonucleotide hybridizes to the first reporter oligonucleotide to bring the first and second product precursors into reactive proximity to produce a reaction product; (f) exposing the reaction product of step (e), if present, to a detection system comprising an amplification component and a detection component under conditions to permit the production of a plurality of detectable moieties; and (g) determining the presence and/or amount of the detectable moieties thereby to determine the presence and/or amount of the biological target in the sample.
9. The method of claim 8, wherein in step (e) the first target binding component, the second target binding component, the first reporter component, and the second reporter component are all combined with the sample at the same time.
10. The method of claim 8, wherein in step (e) the first target binding component and the second target binding component are added to the sample before the first reporter component and the second reporter component.
11. The method of claim 1, wherein the amplification component comprises an enzyme that catalyzes the production of the detectable moieties.
12. The method of claim 1, wherein the amplification component is capable of producing at least 10 molecules of the detectable moieties per molecule of the reaction product.
13. The method of claim 12, wherein the amplification component is capable of producing at least 100 molecules of the detectable moieties per molecule of the reaction product.
14. The method of claim 13, wherein the amplification component is capable of producing at least 1,000 molecules of the detectable moieties per molecule of the reaction product.
15. The method of claim 1, wherein reaction product is a peptide or protein.
16. The method of claim 15, wherein the reaction product comprises at peptidyl sequence selected from the peptides listed in FIG. 15.
17. The method of claim 1, wherein the reaction product is a small molecule.
18. (canceled)
19. The method of claim 1, wherein the biological target is a protein, peptide, immunoglobulin, growth factor receptor or enzyme.
20. The method of claim 1, wherein the biological target is a homodimeric protein.
21. The method of claim 1, wherein the biological target is a heterodimeric protein.
22. The method of claim 1, wherein the biological target is selected from the group consisting of a Bcr-Abl heterodimer, an ErbB family homodimer, an ErbB family heterodimer, and PDGF.
23-35. (canceled)
36. A method of determining the presence and/or amount of a biological target in a sample, the method comprising:
(a) combining the sample with (1) a first probe comprising (i) a first binding moiety with binding affinity to the biological target, (ii) a first oligonucleotide sequence, and (iii) a first masked product precursor associated with the first oligonucleotide sequence and (2) a second probe comprising (i) a second binding moiety with binding affinity to the biological target, (ii) a second oligonucleotide sequence capable of hybridizing to the first oligonucleotide sequence, and (iii) an unmasking group associated with the second oligonucleotide sequence, under conditions to permit the first and second binding moieties to bind to the biological target, if present in the sample, whereupon the first and second oligonucleotide sequences hybridize to one another to bring the unmasking group into reactive proximity with the masked product precursor to produce an unmasked reaction product; (b) exposing the reaction product of step (a), if present, to a detection system comprising an amplification component and a detection component under conditions to permit the production of a plurality of detectable moieties; and (c) determining the presence and/or amount of the detectable moieties thereby to determine the presence and/or amount of the biological target in the sample.
37-51. (canceled)
52. A kit comprising:
(a) a first probe comprising (i) a first binding moiety with binding affinity to a biological target, (ii) a first reporter oligonucleotide sequence associated with the first binding moiety, and (iii) a first product precursor associated with the first reporter oligonucleotide sequence; (b) a second probe comprising (i) a second binding moiety with binding affinity to the biological target, (ii) a second reporter oligonucleotide sequence associated with the second binding moiety, and (iii) a second product precursor associated with the second reporter oligonucleotide sequence, wherein upon the binding of the first and second binding moieties to the biological target the first and second reporter oligonucleotide sequences are capable of hybridizing to one another and the first and second product precursors are capable of reacting with one another to produce a reaction product selected from the group consisting of an intact epitope, an enzyme substrate, an enzyme activator, and a ligand; (c) a detection system comprising an amplification component and a detection component capable of producing a plurality of detectable moieties; and (d) instructions for using the kit for detecting the biological target.
53. (canceled)
54. A kit comprising:
(a) a first target binding component comprising (i) a first binding moiety having binding affinity to the biological target, and (ii) a first oligonucleotide zipcode sequence associated with the first binding moiety; (b) a second target binding component comprising (i) a second binding moiety having binding affinity to the biological target, and (ii) a second oligonucleotide zipcode sequence associated with the second binding moiety; (c) a first reporter component comprising (i) a first oligonucleotide anti-zipcode sequence capable of hybridizing to the first oligonucleotide zipcode sequence, (ii) a first reporter oligonucleotide associated with the first oligonucleotide zipcode sequence, and (iii) a first product precursor associated with the first reporter oligonucleotide sequence; (d) a second reporter component comprising (i) a second oligonucleotide anti-zipcode sequence capable of hybridizing to the second oligonucleotide zipcode sequence, (ii) a second reporter oligonucleotide associated with the second oligonucleotide zipcode sequence and capable of hybridizing to the first reporter oligonucleotide sequence, and (iii) a second product precursor associated with the second reporter oligonucleotide sequence, wherein upon the binding of the first and second binding moieties to the biological target, hybridization of the first zipcode and anti-zipcode oligonucleotide sequences and hybridization of the second zipcode and anti-zipcode oligonucleotide sequences, the first and second reporter oligonucleotide sequences hybridize to one another to bring the first and second product precursors into reactive proximity to produce a reaction product selected the group consisting of an intact epitope, an enzyme substrate, an enzyme activator, and a ligand; (e) a detection system comprising an amplification component and a detection component capable of producing a plurality of detectable moieties; and (f) instructions for using the kit for detecting the biological target.
55-64. (canceled)
Ref US7183052B2: Claims:
1. A method of generating a signal indicative of the presence of a target nucleic acid sequence in a sample, comprising
forming a detection complex by incubating a sample comprising a target nucleic acid sequence and a probe, wherein said probe comprises:
a first and a second subunit and further comprises: a pair of interactive signal generating labeled moieties effectively positioned on said probe to quench the generation of a detectable signal when the probe is not bound to said target nucleic acid or when said first subunit of the probe is associated with said second subunit of the probe, and
wherein said first or second subunit of said probe comprises said pair of interactive signal generating moieties, and
binding said probe to said target nucleic acid sequence, wherein said first subunit of said probe dissociates from said second subunit of said probe and wherein said first or second subunit of said probe comprising said pair of interactive signal generating moieties undergoes a conformational change to generate a signal,
wherein said binding is performed at a binding temperature and said first subunit of said probe does not dissociate from said second subunit of said probe when not bound to said target nucleic acid sequence at or below said binding temperature, and
wherein generation of said signal is indicative of the presence of a target nucleic acid sequence in said sample.
2. A method of generating a signal indicative of the presence of a target nucleic acid sequence in a sample, comprising
a) forming a detection complex by incubating a sample comprising a target nucleic acid sequence comprising a first, second and third region, an upstream primer, and a downstream probe, wherein said downstream probe comprises a first and a second subunit, wherein said first and second subunit each comprise a first and second region, wherein said second region of said first subunit is complementary to said first region of said second subunit and said second region of said first subunit binds to said first region of said second subunit when not bound to said target nucleic acid sequence; and
wherein said first region of said first subunit is complementary to said first region of said target nucleic acid sequence, said second region of said first subunit is complementary to said second region of said target nucleic acid sequence, said first region of said second subunit is complementary to said second region of said target nucleic acid sequence and said second region of said second subunit is complementary to said third region of said target nucleic acid sequence;
and binding said probe to said target nucleic acid sequence, such that said first region of said first subunit binds to said first region of said target nucleic acid sequence, said first region of said second subunit binds to said second region of said target nucleic acid sequence and said second region of said second subunit binds to said third region of said target nucleic acid sequence, and said first and second subunits dissociate from each other;
b) extending said upstream primer by polymerization of a nucleic acid strand by a nucleic acid polymerization activity, and displacing said first subunit of said probe from said target nucleic acid sequence by said nucleic acid strand, such that said first subunit of said probe dissociates from said target nucleic acid sequence and is released to generate a signal,
wherein said binding is performed at a binding temperature and said first subunit of said probe does not dissociate from said second subunit of said probe when not bound to said target nucleic acid sequence at or below said binding temperature, and
wherein said polymerization is performed at a polymerization temperature and said first subunit of said probe does not dissociate from said second subunit of said probe when not displaced by said nucleic acid strand at or below said polymerization temperature, and
wherein generation of said signal is indicative of the presence of a target nucleic acid sequence in said sample.
3. The method of claim 1 or 2, wherein said signal is detected or measured, and wherein said detecting and/or measuring the signal comprises detecting and/or measuring the amount of said released first subunit.
4. A method of detecting or measuring a target nucleic acid sequence comprising the steps of:
a) forming a detection complex by incubating a sample comprising a target nucleic acid sequence with a probe, wherein said probe comprises:
a first and a second subunit, and further comprises:
a pair of interactive signal generating labeled moieties effectively positioned on said probe to quench the generation of a detectable signal when the probe is not bound to said target nucleic acid or when said first subunit of the probe is associated with said second subunit of the probe, and
wherein said first or second subunit of said probe comprises said pair of interactive signal generating moieties, and
binding said probe to said target nucleic acid sequence to dissociate said first subunit of said probe from said second subunit of said probe, and wherein said first or second subunit of said probe comprising said pair of interactive signal generating moieties undergoes a conformational change to generate a signal,
wherein said binding is performed at a binding temperature and said first subunit of said probe does not dissociate from said second subunit of said probe when not bound to said target nucleic acid sequence at or below said binding temperature; and
b) detecting and/or measuring the amount of said released first subunit as an indication of the presence of the target sequence in the sample.
5. A method of detecting or measuring a target nucleic acid sequence comprising a first, second and third region comprising the steps of:
a) forming a detection complex by incubating a sample comprising a target nucleic acid sequence, an upstream primer, and a downstream probe, wherein said probe comprises a first and a second subunit wherein said first and second subunit each comprise a first and second region, wherein said second region of said first subunit is complementary to said first region of said second subunit and said second region of said first subunit binds to said first region of said second subunit when not bound to said target nucleic acid sequence; and
wherein said first region of said first subunit is complementary to said first region of said target nucleic acid sequence, said second region of said first subunit is complementary to said second region of said target nucleic acid sequence, said first region of said second subunit is complementary to said second region of said target nucleic acid sequence and said second region of said second subunit is complementary to said third region of said target nucleic acid sequence; and
binding said probe to said target nucleic acid sequence such that said first region of said first subunit binds to said first region of said target nucleic acid sequence, said first region of said second subunit binds to said second region of said target nucleic acid sequence and said second region of said second subunit binds to said third region of said target nucleic acid sequence, and said first and second subunits dissociate from each other;
b) extending said upstream primer by polymerization of a nucleic acid strand by a nucleic acid polymerization activity, and displacing said first subunit of said probe from said target nucleic acid sequence by said nucleic acid strand, such that said first subunit of said probe dissociates from said target nucleic acid sequence and is released to generate a signal,
wherein said binding is performed at a binding temperature and said first subunit of said probe does not dissociate from said second subunit of said probe when not bound to said target nucleic acid sequence at or below said binding temperature,
wherein said polymerization is performed at a polymerization temperature and said first subunit of said probe does not dissociate from said second subunit of said probe when not displaced by said nucleic acid strand, at or below said polymerization temperature, and
detecting and/or measuring the amount of said released first subunit as an indication of the presence of the target sequence in the sample.
6. The method of claim 2 or 5 wherein said first subunit of said probe further comprise
[Truncated to input limit]