DETAILED ACTION
Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Election/Restrictions
Applicant's election with traverse of Group I, claims 68 to 89, drawn to a method for quantitating a plurality of nucleic acid molecules, in the reply filed on 07/15/2025 is acknowledged. Claims 90-91 are withdrawn from further consideration pursuant to 37 CFR 1.142(b), as being drawn to a nonelected Group II, drawn to a mixture and a kit comprising a plurality of detectably labeled probes and extensions primers, there being no allowable generic or linking claim. Applicant timely traversed the restriction (election) requirement in the reply filed on 07/15/2015.
The traversal is on the ground(s) as recited “As support for the imposition of the unity of invention requirement, the Office cites to U.S. Pat. App. Pub. No. 2019/0225999 to Marras et al. (hereafter, "Marras et al."). In a very conclusory manner the Office merely states that Marras et al. also describes various labeled probes and extension primers. (Office Communication, at pages 3-4). The Office states that therefore the claimed invention "fails to make a special technical feature over the prior art." (Id., at page 4).
Barely any analysis is provided by the Office to support the above allegation of lack of special technical feature for the presently claimed invention. The present claims are directed to quantitating a plurality of nucleic acid molecules in a sample by measuring the rate at which certain detectable signals are released from the nucleic acids during processing, in real time. In contrast, Marras et al. is directed to amplifying target molecules using multi-part primers to detect rare mutations in a real-time monoplex PCR assay using plasmid DNA for detecting highly related alleles. (Marras et al., at col. 3, 11. 5-25). The Marras et al. assay is alleged to be capable of detecting 10 mutant alleles in a sample of 1,000,000 wild type alleles. (Id.). While it is true at the macro level that both systems include nucleic acids, it is clear that the design, use, and end products obtained by these nucleic acids are clearly very different. Thus, Marras et al. does not anticipate or make obvious the presently claimed invention. As such, neither does Marras et al. destroy the presently claimed special technical feature.
Further, according to M.P.E.P. § 803, if the search and examination of an entire application can be made without a serious burden, the Examiner must examine it on the merits, even though it includes claims to independent or distinct inventions. Applicant believes that the search of a few product claims (Group II) that include the same elements utilized in the method claims (Group I) would not amount to an undue burden since both sets of claims are centered on very similar recited elements.
As such, Applicant respectfully requests rejoinder of Groups I and II.”.
This is not found persuasive because: Groups I-II lack unity of invention because even though the inventions of these groups require the technical feature of a plurality of detectably labeled probes and extensions primers, this technical feature is not a special technical feature as it does not make a contribution over the prior art in view of Marras et al. (US Patent Application Number 2019/0225999 July 25, 2019). Marras et al. teaches of a plurality of detectably labeled probes and extensions primers (Para. 11) and therefore fails to make a special technical feature over the art.
The requirement is still deemed proper and is therefore made FINAL.
Claims Status
Claims 68-91 are pending.
Claims 90-91 are withdrawn, with traverse.
Claims 68-89 are currently under examination
Priority
This application is a 371 of PCT/US21/15859, filed on January 29,2021 which claims priority to U.S. Provisional Patent Application No. 62/968,898, filed on. January 31, 2020. The priority date of claim set filed on April 01, 2023, is determined to be January 31, 2020.
Specification
The abstract of the disclosure is objected to because it is not presented on a separate sheet, apart from any other text. A corrected abstract of the disclosure is required and must be presented on a separate sheet, apart from any other text. See MPEP § 608.01(b).
Claim Objections
Claim 71 and 89 are objected to because of the following informalities:
The claim is missing a period at the end of claim. (claim 89 ln 8)
The comma needs to be removed after wherein. (claim 71 ln 1)
Appropriate correction is required.
Claim Rejections - 35 USC § 101
35 U.S.C. 101 reads as follows:
Whoever invents or discovers any new and useful process, machine, manufacture, or composition of matter, or any new and useful improvement thereof, may obtain a patent therefor, subject to the conditions and requirements of this title.
Claims 68-89 are rejected under 35 U.S.C. 101 because the claimed invention is directed towards abstract ideas of calculating a number, determining a correlation factor and estimating the average size of a plurality of target nucleic acid fragments according to an equation and routine and conventional method for comprising contacting a plurality of detectably-labeled probes and a plurality of extension primers with the plurality of nucleic acid molecules; producing an extension product of each of the plurality of target nucleic acid fragments; hydrolyzing each of the plurality of detectably-labeled probes hybridized to the respective one of the target nucleic acid fragments; and detecting a first signal, without significantly more. The claim(s) recite(s) abstract ideas and routine and conventional methods. This judicial exception is not integrated into a practical application because no additional elements integrate the judicial exceptions into a practical application. The claims do not include additional elements that are sufficient to amount to significantly more than the judicial exception because no additional elements are considered significantly more than the judicial exceptions.
Claim analysis
The instant claim 68 is directed towards: A method for quantitating a plurality of nucleic acid molecules comprising a plurality of target nucleic acid fragments, which comprises: contacting a plurality of detectably-labeled probes and a plurality of extension primers with the plurality of nucleic acid molecules, wherein each of the plurality of detectably- labeled probes comprises a first labeled nucleic acid domain comprising a first label; producing an extension product of each of the plurality of target nucleic acid fragments by extending a respective one of the plurality of extension primers with a polymerase; hydrolyzing each of the plurality of detectably-labeled probes hybridized to the respective one of the target nucleic acid fragments during extending the respective one of the plurality of extension primers with the polymerase; detecting a first signal produced as a result of hydrolyzing the plurality of detectably-labeled probes; and calculating a number of the plurality of target nucleic acid fragments based on signals detected upon extension reactions.
The “calculating a number of the plurality of target nucleic acid fragments” is an abstract idea.
The “contacting a plurality of detectably-labeled probes and a plurality of extension primers with the plurality of nucleic acid molecules, wherein each of the plurality of detectably- labeled probes comprises a first labeled nucleic acid domain comprising a first label; producing an extension product of each of the plurality of target nucleic acid fragments by extending a respective one of the plurality of extension primers with a polymerase; hydrolyzing each of the plurality of detectably-labeled probes hybridized to the respective one of the target nucleic acid fragments during extending the respective one of the plurality of extension primers with the polymerase; detecting a first signal produced as a result of hydrolyzing the plurality of detectably-labeled probes” are considered to be active steps requiring the analysis of a sample. The active step is routine and conventional as demonstrated by the 35 USC § 103 rejections stated below.
Dependent claims set forth further limitations about the calculating the number, contacting a plurality of detectably-labeled probes and a plurality of extension primers with the plurality of nucleic acid molecules, plurality of target nucleic acid fragments, plurality of nucleic acid molecules, first signal, labels, signals, plurality of adapter molecules, a second, third and/or fourth labeled nucleic acid domain, detecting a second, third, and/or fourth signal, plurality of nucleic acid binding dye molecules, determining correlation factor, estimating an average size of the plurality of target nucleic acid fragments according to the following equation: Average size= S / correlation factor x N.
The instant claim 87 is directed towards: The method of claim 68, further comprising: contacting a plurality of extension primers and a plurality of nucleic acid binding dye molecules with the plurality of nucleic acid molecules; subsequent to producing the extension product of each of the plurality of target nucleic acid fragments, measuring a signal produced by the plurality of nucleic acid binding dye molecules; and estimating an average size of the plurality of target nucleic acid fragments based on a number of the plurality of target nucleic acid fragments and the signal produced by the plurality of nucleic acid binding dye molecules.
The “estimating an average size of the plurality of target nucleic acid fragments based on a number of the plurality of target nucleic acid fragments and the signal produced by the plurality of nucleic acid binding dye molecules” is an abstract idea. See MPEP 2106.04(a)(2).
The remaining method steps of claim 87 are routine and conventional.
The instant claim 88 is directed towards The method of claim 87, wherein estimating an average size of the plurality of target nucleic acid fragments comprises: determining a correlation factor using signals respectively produced by a plurality of reference nucleic acid libraries; estimating the average size of the plurality of target nucleic acid fragments according to the following Equation:
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Average size= wherein S stands for the signal produced by the plurality of nucleic acid binding dye molecules, and N stands for the number of the plurality of target nucleic acid fragments.
The “determining a correlation factor using signals respectively produced by a plurality of reference nucleic acid libraries; estimating the average size of the plurality of target nucleic acid fragments according to the following Equation:
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”are abstract ideas. See MPEP 2106.04(a)(2).
According to the 2019 Patent Eligibility Guidance an initial two step analysis is required for determining statutory eligibility.
Step 1. Is the claim directed to a process, machine, manufacture, or composition of matter? In the instant case, the Step 1 requirement is satisfied as the claims are directed towards a process.
Step 2A Prong one. Does the claim recite a law of nature, a natural phenomenon or an abstract idea? Yes, abstract ideas.
With regard to claim 68, the claim recites “A method for quantitating a plurality of nucleic acid molecules comprising a plurality of target nucleic acid fragments, which comprises: contacting a plurality of detectably-labeled probes and a plurality of extension primers with the plurality of nucleic acid molecules, wherein each of the plurality of detectably- labeled probes comprises a first labeled nucleic acid domain comprising a first label; producing an extension product of each of the plurality of target nucleic acid fragments by extending a respective one of the plurality of extension primers with a polymerase; hydrolyzing each of the plurality of detectably-labeled probes hybridized to the respective one of the target nucleic acid fragments during extending the respective one of the plurality of extension primers with the polymerase; detecting a first signal produced as a result of hydrolyzing the plurality of detectably-labeled probes; and calculating a number of the plurality of target nucleic acid fragments based on signals detected upon extension reactions.” The method of calculating a number of the plurality of target nucleic acid fragments based on signals detected upon extension reactions is an abstract idea. The remaining method steps of claim 68 are routine and conventional.
With regard to claim 87, the claim recites “The method of claim 68, further comprising: contacting a plurality of extension primers and a plurality of nucleic acid binding dye molecules with the plurality of nucleic acid molecules; subsequent to producing the extension product of each of the plurality of target nucleic acid fragments, measuring a signal produced by the plurality of nucleic acid binding dye molecules; and estimating an average size of the plurality of target nucleic acid fragments based on a number of the plurality of target nucleic acid fragments and the signal produced by the plurality of nucleic acid binding dye molecules”. The “estimating an average size of the plurality of target nucleic acid fragments based on a number of the plurality of target nucleic acid fragments and the signal produced by the plurality of nucleic acid binding dye molecules” is an abstract idea. See MPEP 2106.04(a)(2). The remaining method steps of claim 87 are routine and conventional.
With regard to claim 88, the claim recites “The method of claim 87, wherein estimating an average size of the plurality of target nucleic acid fragments comprises: determining a correlation factor using signals respectively produced by a plurality of reference nucleic acid libraries; estimating the average size of the plurality of target nucleic acid fragments according to the following Equation:
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Average size= wherein S stands for the signal produced by the plurality of nucleic acid binding dye molecules, and N stands for the number of the plurality of target nucleic acid fragments”. The “determining a correlation factor using signals respectively produced by a plurality of reference nucleic acid libraries; estimating the average size of the plurality of target nucleic acid fragments according to the following Equation:
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”are abstract ideas. See MPEP 2106.04(a)(2).
Step 2A prong two. Does the claim recite additional elements that integrate the judicial exception into a practical application? No, there are no additional steps that integrate the claims into a practical application.
Step 2B. Does the claim recite additional elements that are significantly more than the judicial exceptions? No, there are no additional elements that are significantly more than the judicial exceptions.
Regarding claim 68, the claim requires the routine and conventional active steps of quantitating a plurality of nucleic acid molecules similar to that of White et al. (“White”; Patent App. Pub. US 20100069250 A1, March 18, 2010).
White discloses “a method for accurately determining the number of template molecules in a library of nucleic acids (e.g., DNA) to be sequenced. The method does not require large amounts of the DNA sample, nor does it require the preparation of a standard curve. The method is especially applicable to methodologies for "sequencing by synthesis," where quantitation of the starting library is important. The method uses quantitative real time PCR, especially digital PCR, which measures the number of individual molecules in a sample. The present method particularly may use a microfluidic device for running large numbers of PCR reactions. Each PCR reaction is monitored in real time by a primer/probe combination. The forward primer is adapted to contain a sequence not on the adapter but which corresponds to a probe sequence. A short probe which generates fluorescence during the PCR process is used.” (Abstract).Thus, the claim does not provide additional steps which are significantly more.
Dependent claims require methods comprising calculating the number, contacting a plurality of detectably-labeled probes and a plurality of extension primers with the plurality of nucleic acid molecules, plurality of target nucleic acid fragments, plurality of nucleic acid molecules, first signal, labels, signals, plurality of adapter molecules, a second, third and/or fourth labeled nucleic acid domain, detecting a second, third, and/or fourth signal, plurality of nucleic acid binding dye molecules, determining correlation factor, estimating an average size of the plurality of target nucleic acid fragments which are all routine and conventional based on White et al. (“White”; Patent App. Pub. US 20100069250 A1, March 18, 2010) in view of Burns et al. (“Burns”; Patent App. Pub. US 20130178369 A1, July 11, 2013) and Solinas et al. (“Solinas”; (2001). Duplex Scorpion primers in SNP analysis and FRET applications. Nucleic acids research, 29(20), E96.).
Claim Rejections - 35 USC § 102
The following is a quotation of the appropriate paragraphs of 35 U.S.C. 102 that form the basis for the rejections under this section made in this Office action:
A person shall be entitled to a patent unless –
(a)(1) the claimed invention was patented, described in a printed publication, or in public use, on sale, or otherwise available to the public before the effective filing date of the claimed invention.
Claims 68-74, 76-79 and 85-89 are rejected under 35 U.S.C. 102(a)(1)/102(a)(2) as being anticipated by White et al. (“White”; Patent App. Pub. US 20100069250 A1, March 18, 2010).
White discloses “a method for accurately determining the number of template molecules in a library of nucleic acids (e.g., DNA) to be sequenced. The method does not require large amounts of the DNA sample, nor does it require the preparation of a standard curve. The method is especially applicable to methodologies for "sequencing by synthesis," where quantitation of the starting library is important. The method uses quantitative real time PCR, especially digital PCR, which measures the number of individual molecules in a sample. The present method particularly may use a microfluidic device for running large numbers of PCR reactions. Each PCR reaction is monitored in real time by a primer/probe combination. The forward primer is adapted to contain a sequence not on the adapter but which corresponds to a probe sequence. A short probe which generates fluorescence during the PCR process is used.” (Abstract).
Regarding claim 68, White teaches a method comprising “The amplified product may be detected by optical means, namely a fluorescent probe or other molecule which fluoresces as a result of the amplification process” (Para. 28). White also teaches a method comprising “for quantifying a population of nucleic acid strands, comprising 5' adapters and 3' adapters for the nucleic acid strands, each 5' adapter and 3' adapter having the same sequence; forward and reverse primers complementary to the 5' and 3' adapters” (Para. 36). Thus, White teaches a method for quantitating a plurality of nucleic acid molecules comprising a plurality of target nucleic acid fragments comprising: contacting a plurality of detectably-labeled probes and a plurality of extension primers with the plurality of nucleic acid molecules (Para. 28; Para. 36).
Regarding claim 68, White teaches a method comprising “This single hydrolysis probe contains two labels in close proximity to each other: a fluorescent reporter dye at the 5'-end and a (fluorescent or dark) quencher label at or near the 3'-end. When the probe is intact, the fluorescent signal is almost completely suppressed by the quenching label” (Para. 56). Thus, White teaches a method wherein each of the plurality of detectably-labeled probes comprises a first labeled nucleic acid domain comprising a first label.
Regarding claim 68, White teaches a method comprising “in PCR, the DNA polymerase synthesizes a new DNA strand complementary to the DNA template strand by adding dNTPs that are complementary to the template” (Para. 72). Thus, White teaches a method comprising producing an extension product of each of the plurality of target nucleic acid fragments by extending a respective one of the plurality of extension primers with a polymerase.
Regarding claim 68, White teaches a method comprising “a hydrolysis probe having a sequence complementary to a portion of a PCR primer, said portion being non-complementary to the primer's template. Hydrolysis, or cleavage, of the probe by a polymerase removes a quencher” (Para. 35). Thus, White teaches a method comprising hydrolyzing each of the plurality of detectably-labeled probes hybridized to the respective one of the target nucleic acid fragments during extending the respective one of the plurality of extension primers with the polymerase.
Regarding claim 68, White teaches a method comprising “TaqMan probes...to generate the fluorescence signal via the coupling of a fluorogenic dye molecule and a quencher moiety to the same or different oligonucleotide Substrates, and are potentially useful in the present methods” (Para. 22) and “Hydrolysis, or cleavage, of the probe by a polymerase removes a quencher, allowing a fluorescent signal to be generated” Para. 35). Thus, White teaches a method comprising detecting a first signal produced as a result of hydrolyzing the plurality of detectably-labeled probes.
Regarding claim 68, White teaches a method comprising “generating a signal by means of a probe which binds to a sequence defined by a forward primer or a reverse primer, said signal being dependent upon amplification, whereby the number of reaction areas generating a signal is indicative of the quantity of DNA molecules in the sample” (Para. 29) and “Statistical methods may be used to calculate the expected total number of molecules in the sample, based on the number of different reaction areas and the number of positives. This will result in a calculated concentration of DNA molecules in the sample that was applied to the different reaction areas” (Para. 49). Thus, White teaches a method comprising calculating a number of the plurality of target nucleic acid fragments based on signals detected upon extension reactions.
The teachings of White are documented above in the rejection of claim 68 under 35 U.S.C. 102 (a)(1)/ 102(a)(2). Claims 69-73, 76-79, 87 and 89 depend on claim 68. Claims 74, 80 and 85-86 depends on claim 73, which depends on claim 68. Claim 88 depends on claim 87, which depends on claim 68. Rejection of dependent claims 69-74, 76-79 and 85-89 under 35 U.S.C. 102 (a)(1)/ 102(a)(2) are documented below.
Regarding claim 69, White teaches a method wherein “a first reaction such as shown in FIG. 8A-B is carried out in the real-time mode (with a calibration standard), without a digital analysis, to range the library concentration so that an appropriate dilution can be made for absolute quantitation by UT-digital PCR” (Para. 71; Fig. 8B). Thus, White teaches a method wherein calculating the number of the plurality of target nucleic acid fragments is based on the first signal detected upon a single cycle of extension reactions.
Regarding claim 70, White teaches a method wherein “In the next cycle, as shown at 72, the polymerase, which has an exonuclease activity, cleaves the UT probe 60, releasing the quencher 68 and the fluorescent label 70. The release of the fluorescent label 70 releases the inhibitory effect of the quencher 68, which is no longer close enough to inhibit fluorescence. Thus, as shown, fluorescence occurs. Fluorescence is inhibited by the quencher until it binds to a template strand and is digested by exonuclease activity during the amplification process” (Para. 69). White suggests a method wherein “TaqMan(R) detection chemistry has the advantage of yielding a fluorescence signal proportional to the number of molecules that have been amplified” (Para. 20). White teaches a method wherein “By analyzing the number of positive reactions, insight into the number of starting molecules is obtained” (Para.52). Thus, White teaches a method wherein a total number of hydrolyzed detectably-labeled probes is substantially same as a total number of the plurality of target nucleic acid fragments prior to contacting the plurality of detectably-labeled probes and the plurality of extension primers with the plurality of nucleic acid molecules.
Regarding claim 71, White teaches a method wherein “In the next cycle, as shown at 72, the polymerase, which has an exonuclease activity, cleaves the UT probe 60, releasing the quencher 68 and the fluorescent label 70. The release of the fluorescent label 70 releases the inhibitory effect of the quencher 68, which is no longer close enough to inhibit fluorescence. Thus, as shown, fluorescence occurs” (Para. 69). Thus, White teaches a method wherein subsequent to contacting the plurality of detectably-labeled probes and the plurality of extension primers with the plurality of nucleic acid molecules, and prior to detecting the first signal, no additional cycle of extension reactions is performed other than a single cycle of extension reactions.
Regarding claim 72, White teaches a method wherein “Library creation starts with conversion of the sample to appropriately sized fragments, ligation of adaptor sequences onto the ends of the sample molecules, and selection for molecules properly appended with adaptors. The presence of the adaptor sequences on the ends of the library molecules enables amplification of random-sequence inserts by PCR” (Para. 12). White teaches a method wherein “a hydrolysis probe having a sequence complementary to a portion of a PCR primer, said portion being non-complementary to the primer's template" and "Hydrolysis, or cleavage, of the probe by a polymerase removes a quencher, allowing a fluorescent signal to be generated” (Para. 35). White teaches a method wherein “the probe must be complimentary to one of the two product strands such that the extending polymerase will encounter it and separate the two labels by exonuclease activity, activating the probe's fluorescence” (Para. 20). Thus, White teaches a method wherein each of the plurality of target nucleic acid fragments comprises a first adapter sequence, a target insert sequence, and a second adapter sequence; wherein the first adapter sequence is linked to the second adapter sequence through the target insert sequence; wherein hydrolyzing each of the plurality of detectably-labeled probes comprises hydrolyzing one of the plurality of detectably-labeled probes hybridized to at least a portion of the first adapter sequence; and wherein extending the respective one of the plurality of extension primers with the polymerase comprises extending an extension primer hybridized to at least a portion of the second adapter sequence.
Regarding claim 73, White teaches a method wherein “Library creation starts with conversion of the sample to appropriately sized fragments, ligation of adaptor sequences onto the ends of the sample molecules, and selection for molecules properly appended with adaptors. The presence of the adaptor sequences on the ends of the library molecules enables amplification of random-sequence inserts by PCR” (Para.12). White teaches a method wherein “the probe must be complimentary to one of the two product strands such that the extending polymerase will encounter it and separate the two labels by exonuclease activity, activating the probe's fluorescence” (Para. 20). White teaches a method wherein “the present invention may involve a hydrolysis probe having a sequence complementary to a portion of a PCR primer, said portion being non-complementary to the primer's template” (Para. 35). Thus, White teaches a method wherein the plurality of nucleic acid molecules further comprise a plurality of adapter molecules; wherein each of the plurality of adapter molecules comprises a first adapter sequence directly linked to a second adapter sequence; wherein the method further comprises contacting a plurality of hybridizing oligonucleotides with the plurality of nucleic acid molecules; wherein each of the plurality of hybridizing oligonucleotides comprises a sequence complementary to a contiguous domain of a respective one of the plurality of adapter molecules.
Regarding claims 74 and 86, White teaches a method wherein “"It contains a PCR blocker at the start of the hairpin loop, and HEG monomers are typically added as blocking agents” (Para. 21). White teaches a method wherein “the UT primer has, in addition to a sequence binding to the universal adapter attached to the template DNA, a sequence (UT portion) which will hybridize to the signal generating probe” (Para. 65). White teaches a method wherein “a hydrolysis probe having a sequence complementary to a portion of a PCR primer, said portion being non-complementary to the primer's template” (Para. 35). White teaches a method wherein “The probe is a self-complementary stem sequence with a fluorophore at one end and a quencher at the other. The Scorpion primer sequence is modified at the 5' end. It contains a PCR blocker at the start of the hairpin loop, and HEG monomers are typically added as blocking agents.” (Para. 21). Thus, White teaches a method wherein each of the plurality of hybridizing oligonucleotides comprises a blocker nucleic acid domain; subsequent to contacting the plurality of hybridizing oligonucleotides with the plurality of nucleic acid molecules, each of the plurality of hybridizing oligonucleotides hybridizes to the contiguous domain of the respective one of the plurality of adapter molecules, but not to the plurality of target nucleic acid fragments; and hydrolyzation of each of the plurality of hybridizing oligonucleotides hybridized to the contiguous domain and hydrolyzation of each of the plurality of detectably-labeled probes hybridized to the first adapter sequence of a respective one of the plurality of adapter molecules are blocked by a blocking moiety in the blocker nucleic acid domain; and wherein each of the plurality of hybridizing oligonucleotides comprises a blocker nucleic acid domain.
Regarding claim 76, White teaches a method wherein “The amplified product may be detected by optical means, namely a fluorescent probe or other molecule which fluoresces as a result of the amplification process” (Para. 9). Thus, White teaches a method wherein the first signal is a first fluorescent signal, and the signals detected upon the extension reactions are fluorescent signals comprising the first fluorescent signal.
Regarding claims 77-79, White teaches a method wherein “This single hydrolysis probe contains two labels in close proximity to each other: a fluorescent reporter dye at the 5'-end and a (fluorescent or dark) quencher label at or near the 3'-end. When the probe is intact, the fluorescent signal is almost completely suppressed by the quenching label” (Para. 56). White teaches a method wherein “Chemistries which allow detection of PCR products via the generation of a fluorescent signal can be adapted, given the teachings below, to the present method. TaqMan probes, Molecular Beacons and Scorpions depend on Forster Resonance Energy Transfer (FRET) to generate the fluorescence signal via the coupling of a fluorogenic dye molecule and a quencher moiety to the same or different oligonucleotide substrates, and are potentially useful in the present methods” (Para. 22). White teaches a method wherein “Hydrolysis, or cleavage, of the probe by a polymerase removes a quencher, allowing a fluorescent signal to be generated” (Para. 35). Thus, White teaches a method wherein each of the plurality of detectably-labeled probes further comprises a first and second labeled nucleic acid domain; wherein the first labeled nucleic acid domain is a first reporter domain; wherein the second labeled nucleic acid domain is a first quencher domain or spectrally similar or identical report; and wherein the plurality of detectably-labeled probes are hydrolyzed to release at least one of the first label or the second label; and wherein the first labeled nucleic acid domain is a first reporter domain; wherein each of the plurality of detectably-labeled probes comprises a quenching nucleotide that quenches an energy from the first label in an excited state; and wherein the plurality of detectably-labeled probes are hydrolyzed to release at least one of the quenching nucleotide or the first label.
Regarding claim 85, White teaches a method wherein “''While TaqMan hydrolysis probes were used here, a multiplicity of detection technologies, including molecular beacon and hybridization, AmpliFluor, Scorpion (including the three-oligo scorpions format) and LUX probes, are compatible with the universal template approach adopted here, as is the use of modified probe chemistries including LNA (used here), minor-groove binders, PNA, and hydrolysis-resistant and extension-blocking nucleotides” (Para. 83). Scorpion format reads on the inclusion of a third label. White teaches a method wherein “the UT primer has, in addition to a sequence binding to the universal adapter attached to the template DNA, a sequence (UT portion) which will hybridize to the signal generating probe” (Para. 65). White teaches a method wherein “TaqMan probes...generate the fluorescence signal via the coupling of a fluorogenic dye molecule and a quencher moiety to the same or different oligonucleotide Substrates, and are potentially useful in the present methods” (Para. 22). White teaches a method wherein “a large number of distinct amplified nucleic acid sequences can be monitored at a fast rate” (Para. 25). White teaches a method wherein “During each PCR cycle, more of the released fluorescent dye accumulates, boosting the fluorescent signal” (Para. 56-58). Thus, White teaches a method wherein each of the plurality of hybridizing oligonucleotides comprises a third labeled nucleic acid domain comprising a third label; and subsequent to contacting the plurality of hybridizing oligonucleotides with the plurality of nucleic acid molecules, each of the plurality of hybridizing oligonucleotides hybridizes to the contiguous domain of the respective one of the plurality of adapter molecules, but not to the plurality of target nucleic acid fragments; the method further comprising: producing an extension product of each of the plurality of adapter molecules by extending a respective one of the plurality of extension primers in the extension reactions with the polymerase; hydrolyzing each of the plurality of hybridizing oligonucleotides hybridized to the contiguous domain and hydrolyzing each of the plurality of detectably-labeled probes hybridized to the respective one of the adapter molecules, during producing an extension product of each of the plurality of adapter molecules; detecting a second signal produced as a result of hydrolyzing the plurality of hybridizing oligonucleotides, wherein the first signal and the second signal are distinguishably detected.
Regarding claim 87, White teaches a method wherein “a fluorescent probe or other molecule which fluoresces as a result of the amplification process” (Para. 28). White teaches a method wherein “This single hydrolysis probe contains two labels in close proximity to each other” (Para. 56). White teaches a method wherein “the DNA polymerase synthesizes a new DNA strand” (Para. 72). White teaches a method wherein “a hydrolysis probe having a sequence complementary to a portion of a PCR primer, said portion being non-complementary to the primer's template. Hydrolysis, or cleavage, of the probe by a polymerase removes a quencher” (Para. 35). White teaches a method wherein “TaqMan probes...to generate the fluorescence signal via the coupling of a fluorogenic dye molecule and a quencher moiety to the same or different oligonucleotide Substrates, and are potentially useful in the present methods” (Para. 22). White teaches a method wherein “generating a signal by means of a probe which binds to a sequence defined by a forward primer or a reverse primer, said signal being dependent upon amplification, whereby the number of reaction areas generating a signal is indicative of the quantity of DNA molecules in the sample” (Para. 29) and “Statistical methods may be used to calculate the expected total number of molecules in the sample, based on the number of different reaction areas and the number of positives. This will result in a calculated concentration of DNA molecules in the sample that was applied to the different reaction areas” (Para. 49). Thus, White teaches a method further comprising: contacting a plurality of extension primers and a plurality of nucleic acid binding dye molecules with the plurality of nucleic acid molecules; subsequent to producing the extension product of each of the plurality of target nucleic acid fragments, measuring a signal produced by the plurality of nucleic acid binding dye molecules; and estimating an average size of the plurality of target nucleic acid fragments based on a number of the plurality of target nucleic acid fragments and the signal produced by the plurality of nucleic acid binding dye molecules.
Regarding claim 88, White teaches a method wherein “First, mass-based quantitation also requires an accurate estimate of the length of the molecules to determine the molar concentration of DNA fragments” (Para. 16). White teaches a method wherein “The user can translate number of spots on chip (single molecules) into molecules per µL In order to run on a high throughput sequencer, the correct concentration (in molecules/ µL) is vital in order to ensure the throughput of the instrument and quality of the sequencing result. For example, one can take the number of positive spots in a panel (e.g., 200), divide by 4.6 µL which is the volume in the panel, and times that by the prepared reaction volume (e.g., 10 µL) and divide it by the input DNA volume (e.g., 1 µL). This is then multiplied by the dilution factor, that is, initial molecule count. The sample may then be diluted to a working concentration of e.g., 4 pM for a Solexa type sequencing, or 2.x105 molecules/µL, for a 454 Flx type of sequencing.” (Para. 39). White teaches a method wherein “a series of equations that may be used to estimate the concentration of molecules and statistical confidence interval based on the number of reaction areas used in a digital PCR array and the number of positive results” (Para. 49). Thus, White teaches a method wherein estimating an average size of the plurality of target nucleic acid fragments comprises: determining a correlation factor using signals respectively produced by a plurality of reference nucleic acid libraries; estimating the average size of the plurality of target nucleic acid fragments according to the following Equation: Average size = (S/ (correlation factor x N)); wherein S stands for the signal produced by the plurality of nucleic acid binding dye molecules, and N stands for the number of the plurality of target nucleic acid fragments.
Regarding claim 89, White teaches a method wherein “whole blood genomic DNA sample was sonicated to produce fragments between 100-400 bp” (Para. 85). White teaches a method wherein “The amplified product may be detected by optical means, namely a fluorescent probe or other molecule which fluoresces as a result of the amplification process” (Para. 28). White teaches a method wherein “the method may involve a kit for quantifying a population of nucleic acid strands, comprising 5' adapters and 3' adapters for the nucleic acid strands, each 5' adapter and 3' adapter having the same sequence; forward and reverse primers complementary to the 5' and 3' adapters” (Para. 36). White teaches a method wherein “This single hydrolysis probe contains two labels in close proximity to each other: a fluorescent reporter dye at the 5'-end and a (fluorescent or dark) quencher label at or near the 3'-end. When the probe is intact, the fluorescent signal is almost completely suppressed by the quenching label” (Para. 56). White teaches a method wherein “the DNA polymerase synthesizes a new DNA strand complementary to the DNA template strand by adding dNTPs that are complementary to the template” (Para. 72). White teaches a method wherein “a hydrolysis probe having a sequence complementary to a portion of a PCR primer, said portion being non-complementary to the primer's template. Hydrolysis, or cleavage, of the probe by a polymerase removes a quencher” (Para.35). White teaches a method wherein “TaqMan probes...to generate the fluorescence signal via the coupling of a fluorogenic dye molecule and a quencher moiety” (Para. 22). White teaches a method wherein “generating a signal by means of a probe which binds to a sequence defined by a forward primer or a reverse primer, said signal being dependent upon amplification, whereby the number of reaction areas generating a signal is indicative of the quantity of DNA molecules in the sample” (Para. 29) and “Statistical methods may be used to calculate the expected total number of molecules in the sample, based on the number of different reaction areas and the number of positives. This will result in a calculated concentration of DNA molecules in the sample that was applied to the different reaction areas” (Para. 49). White teaches a method wherein “During each PCR cycle, more of the released fluorescent dye accumulates” (Para. 56). White teaches a method wherein “The libraries were diluted to roughly 100-360 molecules per µl before running on the Fluidigm's Digital Array microfluidic chip” (Para. 88). White teaches a method wherein “Sequencing was carried out on the Genome Analyzer II” (Para. 89). Thus, White teaches a method further comprising: generating a plurality of nucleic acid molecules comprising a plurality of target nucleic acid fragments from a nucleic acid sample by incubation of the target nucleic acid fragments with a DNA polymerase under conditions in which the DNA polymerase catalyzes polymerization; diluting the plurality of target nucleic acid fragments to a predetermined concentration, and sequencing at least one portion of the plurality of target nucleic acid fragments.
Claim Rejections - 35 USC § 103
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
Claims 68, 73-75, 80 and 84 are rejected under 35 U.S.C. 103 as being unpatentable over White et al. (“White”; Patent App. Pub. US 20100069250 A1, March 18, 2010) in view of Burns et al. (“Burns”; Patent App. Pub. US 20130178369 A1, July 11, 2013).
The teachings of White are documented above in the rejection of claims 68-74, 76-79 and 85-89 under 35 U.S.C. 102 (a)(1)/102(a)(2). Claim 75 depends, depends on claim 74, which depends on claim 73, which depends on claim 68. Claim 84 depends on claim 80, which depends on claim 73, which depends on claim 68. White does not explicitly teach the limitations of claims 75, 80 and 84.
Burns discloses “The present invention is directed to treatment of nucleic acid molecules that are attached or associated with solid supports for biochemical analysis, including nucleic acid sequencing. After loading on the solid support, the nucleic acid molecules are treated with a composition comprising a condensing agent, a volume excluding agent, or both, then treated with a composition comprising a protein.” (Abstract).
Regarding claim 75, Burns teaches a method wherein “pictured in FIG. 3, the "common" adaptor is added as two adaptor arms--one arm is blunt end ligated to the 5' end of the fragment and the other arm is blunt end ligated to the 3' end of the fragment. The second segment of the tagging adaptor is a "barcode" segment that is unique to each well…Thus, when the tagged fragments from all the wells are re-combined for sequencing applications, fragments from the same well can be identified through identification of the barcode adaptor. In the embodiment illustrated in FIG. 3, the barcode is ligated to the 5' end of the common adaptor arm.” (Para. 127). Thus, Burns and White teach a method wherein the contiguous domain comprises a portion of the first adapter sequence and a portion of the second adapter sequence directly adjacent to each other.
White and Burns are both considered to be analogous to the claimed invention because they are in the same field of nucleic acid analysis. Therefore, it would have been obvious to someone of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method for accurately determining the number of template molecules in a library of nucleic acids with 5' and 3' adaptors for probe binding as taught by White to incorporate a method wherein the contiguous domain comprises adjacent adaptors with a barcode as taught by Burns and provide method wherein the contiguous domain comprises a portion of the first adapter sequence and a portion of the second adapter sequence directly adjacent to each other. Doing so would allow for the design of a unique fused tag to be utilized in the identification of fragments.
Regarding claim 80, Burns teaches a method wherein “uracil incorporation and degradation may be used to render the Type IIS restriction endonuclease recognition site single-stranded; however, as described further herein, other methods may be employed to render these regions single-stranded including use of 3' or SU exonucleases in a limited digest” (Para. 225). Burns teaches a method wherein “e.g. the NNNANN-dye1 probe can be used alone in a reaction, and either a signal is detected or not, and the probes washed away; then a second pool, NNNTNN-dye 1 can be introduced” (Para. 298). Thus, Burns and White teach a method wherein each of the plurality of hybridizing oligonucleotides further comprises a third labeled nucleic acid domain comprising a third label; wherein subsequent to contacting the plurality of hybridizing oligonucleotides with the plurality of nucleic acid molecules, each of the plurality of hybridizing oligonucleotides hybridizes to the contiguous domain of the respective one of the plurality of adapter molecules, but not to the plurality of target nucleic acid fragments; and the method further comprising: detecting a second signal produced as a result of partially hydrolyzing the plurality of hybridizing oligonucleotides, wherein the first signal and the second signal are distinguishably detected.
Regarding claim 84, Burns teaches a method wherein “Alternatively, in some embodiments, if the reactions are done sequentially rather than simultaneously, the same dye can be done, just in different steps: e.g. the NNNANN-dye1 probe can be used alone in a reaction, and either a signal is detected or not, and the probes washed away; then a second pool, NNNTNN- dye 1 can be introduced” (Para. 298). Thus, Burns and White teach a method wherein the first signal is a first fluorescent signal, wherein the second signal is a second fluorescent signal, and wherein the signals detected upon the extension reactions are fluorescent signals comprising the first fluorescent signal and the second fluorescent signal.
Claims 81-83 are rejected under 35 U.S.C. 103 as being unpatentable over White et al. (“White”; Patent App. Pub. US 20100069250 A1, March 18, 2010) in view of Burns et al. (“Burns”; Patent App. Pub. US 20130178369 A1, July 11, 2013) as applied to claims 68, 73-75, 80 and 84 and further in view of Solinas et al. (“Solinas”; (2001). Duplex Scorpion primers in SNP analysis and FRET applications. Nucleic acids research, 29(20), E96.).
The teachings of White are documented above in the rejection of 68-74, 76-79 and 85-89 under 35 U.S.C. 102 (a)(1)/102(a)(2). The teaching of White and Burns are documented above in the rejection of claims 68, 73-75, 80 and 84. Claims 81-83 depends on claim 80, which depends on claim 73, which depends on claim 68. White and Burns do not explicitly teach the limitations of claims 81-83.
Solinas discloses “Scorpions are fluorogenic PCR primers with a probe element attached at the 5′-end via a PCR stopper. They are used in real-time amplicon-specific detection of PCR products in homogeneous solution. Two different formats are possible, the ‘stem–loop’ format and the ‘duplex’ format. In both cases the probing mechanism is intramolecular. We have shown that duplex Scorpions are efficient probes in real-time PCR. They give a greater fluorescent signal than stem–loop Scorpions due to the vastly increased separation between fluorophore and quencher in the active form. We have demonstrated their use in allelic discrimination at the W1282X locus of the ABCC7 gene and shown that they can be used in assays where fluorescence resonance energy transfer is required.” (Abstract).
Regarding claim 81, Solinas teaches a method wherein “the use of FRET in stem-loop Scorpions… optimizing the technique