DETAILED ACTION
Notice of Pre-AIA or AIA Status
1. The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
2. Claims 1-2, 5-6, 8, 10-11, 15-20, 22, 24-25, 27-29, and 31 are currently pending and have been examined herein.
Claim Rejections - 35 USC § 112(b)
3. The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION. —The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claims 2, 19, 20, 22, 24, and 26 are rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
Claims 2, 19, 20, 22, 24, and 26 are rejected over the recitation “Sybr goldTM” in claims 2 and 19. It is noted that SYBR is a registered trademark. Where a trademark or trade name is used in a claim as a limitation to identify or describe a particular material or product, the claim does not comply with the requirements of 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph. See Ex parte Simpson, 218 USPQ 1020 (Bd. App. 1982). The claim scope is uncertain since the trademark or trade name cannot be used properly to identify any particular material or product. A trademark or trade name is used to identify a source of goods, and not the goods themselves. Thus, a trademark or trade name does not identify or describe the goods associated with the trademark or trade name. In the present case, the trademark/trade name is used to identify/describe fluorescent chemicals for use in scientific research sold by Molecular Probes and, accordingly, the identification/description is indefinite.
Claim Rejections - 35 USC § 103
4. The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
5. Claims 1, 2, 5-6, 8, 10-11, 17, 19-20, 22, 24, 27-29, and 31 are rejected under 35 U.S.C. 103 as being unpatentable over Zahringer (Journal of Molecular and Cellular Cardiology 1982 14 pages 539-550) in view of Zabzdyr (Journal of Chromatography A 911 (2001) 269-276) and Roos (US 2018/0142275 Pub 5/24/2018).
Regarding Claim 1 Zahringer teaches determination of Poly(A) tract length of cardiac mRNA by analysis of polyacrylamide gels. Zahringer teaches the average length of Poly(A) in purified cardiac mRNA was determined. Zahringer teaches twenty micrograms of purified cardiac mRNA were lyophilized overnight and then dissolved in 50 µl 20 mM Hepes, pH 7.6, 2 mM EDTA, 0.3 M NaCl. After addition of 3 units RNAse A and 1 unit of RNAse T1 incubation was performed at 37°C/1 h. This digests all RNA-sequences with the exception of Poly(A)-sequences. The digest was subjected to electrophoresis on denaturing 7% polyacrylamide- formamide slab gels (page 541). Zahringer teaches that Poly(A) is eluted from gel slices and hybridized to excess (3H)Poly(U). The distribution of radioactivity indicates the distribution of Poly(A) and allowed a direct measurement of the length of the Poly(A)-tract of cardiac mRNA (page 545). Thus Zahringer teaches a method of measuring poly A tail length in an mRNA sample, the method comprising: contacting the mRNA sample with one or more ribonucleases (RNAse A and RNAse T1); and assaying the sample using gel electrophoresis to determine the poly A tail length of the mRNA.
Regarding Claim 5 Zahringer teaches a method wherein the RNases are RNaseA and RNase T1 (page 541).
Regarding Claim 8 Zahringer teaches a method wherein the mRNA sample is incubated with the ribonucleases for 60 minutes (page 541).
Regarding Claim 10 Zahringer teaches a method wherein the poly A tail is 62-130 nucleotides (page 546 Table 2). Thus Zahringer teaches a method wherein the poly A tail length is 25 nucleotides or more, 50 nucleotides or more, 100 nucleotides or more.
Regarding Claim 11 Zahringer teaches a method wherein the poly A tail is 62-130 nucleotides (page 546 Table 2). Thus Zahringer teaches a method wherein the poly A tail length is between 50 nucleotides and 5,000 nucleotides, between 100 nucleotides and 1,500 nucleotides.
Zahringer does not teach a method wherein the mRNA sample is contacted with a minor groove binding dye (clm 1). Zahringer does not teach assaying the sample by capillary electrophoresis to determine the poly A tail length of the mRNA (clm 1). Zahringer does not teach a method wherein the minor-groove binding dye is Sybr goldTM a Hoechst dye, or 4',6-diamidino-2-phenylindole (DAPI) (clm 2). Zahringer does not teach a method wherein the CE is coupled with a fluorescence based detection (clm 6). Zahringer does not teach a method wherein the minor-groove binding dye non-covalently binds to single-stranded RNA (ssRNA) (clm 17).
However Zabzdyr teaches that they examined the sensitivity of SYBR Green I and SYBR Gold for LIF detection of dsDNA and RNA following separation by CE. Both UV (275 nm) and visible (488 nm) excitation wavelengths are used to detect the DNA and the RNA with each dye. SYBR Green I has been used extensively for the detection of dsDNA in CE and gel electrophoresis. However, limited use of SYBR Gold has been reported for the detection of dsDNA and RNA following gel electrophoresis. Zabzdyr exemplifies detection of RNA using both of these dyes and the use of SYBR Gold for LIF detection of DNA following CE. Zabzdyr demonstrates the use of these dyes with a UV excitation source for LIF detection of dsDNA and RNA separated by CE. It is noted that SYBR gold is a minor groove binding dye that non-covalently binds to ssRNA.
Additionally Roos teaches a method of producing a defined poly A tail length. Roos teaches that the end product was analyzed on a 15% TBE-UREA PAGE and subsequently stained with Sybr® Gold to assess the polyadenylation of the RNA. PolyA tail lengths were estimated in comparison to a low range ssRNA Ladder (New England BioLabs Inc.). The result of the experiment is shown in FIG. 11 (para 0392).
Accordingly, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of Zahringer by using capillary electrophoresis to determine the poly A tail length of the mRNA as suggested by Zabzdyr. In the instant case Zahringer teaches the use of gel electrophoresis to detect poly A tail length. Zabzdyr teaches nucleic acids can be detected by gel electrophoresis or capillary electrophoresis and exemplifies the detection of RNA using capillary electrophoresis. The claims would have been obvious because the substitution of one type of electrophoresis (gel) for another (capillary) would have yielded predictable results to one of ordinary skill in the art at the time of the invention. Further it would have been obvious to have modified the method of Zahringer by contacting the mRNA sample with a minor groove binding dye (i.e., SYBR gold) as suggested by Zabzdyr and Roos. In the instant case Zabzdyr discloses SYBR gold bound to RNA can be used for capillary electrophoresis (abstract). Additionally Roos discloses using SYBR gold to detect poly A tails. One of skill in the art would have been motivated to contact the mRNA sample with SYBR gold for the benefit of being able to use a dye with a high fluorescence quantum yield, low background fluorescence, and high nucleic acid binding affinity (Zabzdyr page 269) for the detection of poly A tails by capillary electrophoresis.
Regarding Claim 19 Zahringer teaches determination of Poly(A) tract length of cardiac mRNA by analysis of polyacrylamide gels. Zahringer teaches the average length of Poly(A) in purified cardiac mRNA was determined. Zahringer teaches twenty micrograms of purified cardiac mRNA were lyophilized overnight and then dissolved in 50 µl 20 mM Hepes, pH 7.6, 2 mM EDTA, 0.3 M NaCl. After addition of 3 units RNAse A and 1 unit of RNAse T1 incubation was performed at 37°C/1 h. This digests all RNA-sequences with the exception of Poly(A)-sequences. The digest was subjected to electrophoresis on denaturing 7% polyacrylamide- formamide slab gels (page 541). Zahringer teaches that Poly(A) is eluted from gel slices and hybridized to excess (3H)Poly(U). The distribution of radioactivity indicates the distribution of Poly(A) and allowed a direct measurement of the length of the Poly(A)-tract of cardiac mRNA (page 545). Thus Zahringer teaches a method of measuring poly A tail length in an mRNA sample, the method comprising: contacting the mRNA sample with RNAse A and RNAse T1; and assaying the sample using gel electrophoresis to determine the poly A tail length of the mRNA.
Regarding Claim 22 Zahringer teaches a method wherein the mRNA sample is incubated with the ribonucleases for 60 minutes (page 541).
Regarding Claim 24 Zahringer teaches a method wherein the poly A tail is 62-130 nucleotides (page 546 Table 2). Thus Zahringer teaches a method wherein the poly A tail length is 25 nucleotides or more, 50 nucleotides or more, 100 nucleotides or more.
Zahringer does not teach a method wherein the mRNA sample is contacted with SYBR gold minor groove binding dye (clm 19). Zahringer does not teach assaying the sample by capillary electrophoresis to determine the poly A tail length of the mRNA (clm 19). Zahringer does not teach a method wherein the CE is coupled with a fluorescence based detection (clm 20).
However Zabzdyr teaches that they examined the sensitivity of SYBR Green I and SYBR Gold for LIF detection of dsDNA and RNA following separation by CE. Both UV (275 nm) and visible (488 nm) excitation wavelengths are used to detect the DNA and the RNA with each dye. SYBR Green I has been used extensively for the detection of dsDNA in CE and gel electrophoresis. However, limited use of SYBR Gold has been reported for the detection of dsDNA and RNA following gel electrophoresis. Zabzdyr exemplifies detection of RNA using both of these dyes and the use of SYBR Gold for LIF detection of DNA following CE. Zabzdyr demonstrates the use of these dyes with a UV excitation source for LIF detection of dsDNA and RNA separated by CE. It is noted that SYBR gold is a minor groove binding dye that non-covalently binds to ssRNA.
Additionally Roos teaches a method of producing a defined poly A tail length. Roos teaches that the end product was analyzed on a 15% TBE-UREA PAGE and subsequently stained with Sybr® Gold to assess the polyadenylation of the RNA. PolyA tail lengths were estimated in comparison to a low range ssRNA Ladder (New England BioLabs Inc.). The result of the experiment is shown in FIG. 11 (para 0392).
Accordingly, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of Zahringer by using capillary electrophoresis to determine the poly A tail length of the mRNA as suggested by Zabzdyr. In the instant case Zahringer teaches the use of gel electrophoresis to detect poly A tail length. Zabzdyr teaches nucleic acids can be detected by gel electrophoresis or capillary electrophoresis and exemplifies the detection of RNA using capillary electrophoresis. The claims would have been obvious because the substitution of one type of electrophoresis (gel) for another (capillary) would have yielded predictable results to one of ordinary skill in the art at the time of the invention. Further it would have been obvious to have modified the method of Zahringer by contacting the mRNA sample with a minor groove binding dye (i.e., SYBR gold) as suggested by Zabzdyr and Roos. In the instant case Zabzdyr discloses SYBR gold bound to RNA can be used for capillary electrophoresis (abstract). Additionally Roos discloses using SYBR gold to detect poly A tails. One of skill in the art would have been motivated to contact the mRNA sample with SYBR gold for the benefit of being able to use a dye with a high fluorescence quantum yield, low background fluorescence, and high nucleic acid binding affinity (Zabzdyr page 269) for the detection of poly A tails by capillary electrophoresis.
Regarding Claim 27 Zahringer teaches determination of Poly(A) tract length of cardiac mRNA by analysis of polyacrylamide gels. Zahringer teaches the average length of Poly(A) in purified cardiac mRNA was determined. Zahringer teaches twenty micrograms of purified cardiac mRNA were lyophilized overnight and then dissolved in 50 µl 20 mM Hepes, pH 7.6, 2 mM EDTA, 0.3 M NaCl. After addition of 3 units RNAse A and 1 unit of RNAse T1 incubation was performed at 37°C/1 h. This digests all RNA-sequences with the exception of Poly(A)-sequences. The digest was subjected to electrophoresis on denaturing 7% polyacrylamide- formamide slab gels (page 541). Zahringer teaches that Poly(A) is eluted from gel slices and hybridized to excess (3H)Poly(U). The distribution of radioactivity indicates the distribution of Poly(A) and allowed a direct measurement of the length of the Poly(A)-tract of cardiac mRNA (page 545). Thus Zahringer teaches a method of measuring homopolymeric nucleotide length (poly A tail length) in an mRNA sample, the method comprising: contacting the mRNA sample with one or more ribonucleases (RNAse A and RNAse T1); and assaying the sample using gel electrophoresis to determine the homopolymeric nucleotide length (poly A tail length) of the mRNA.
Regarding Claim 28 Zahringer teaches a method wherein the homopolymeric nucleotide (poly A tail) length is 62-130 nucleotides (page 546 Table 2). Thus Zahringer teaches a method wherein the homopolymeric nucleotide (poly A tail) length is 50 nucleotides or more, 100 nucleotides or more.
Regarding Claim 29 Zahringer teaches a method wherein the homopolymeric nucleotide (poly A tail) length is 62-130 nucleotides (page 546 Table 2). Thus Zahringer teaches a method wherein the homopolymeric nucleotide (poly A tail) length is between 50 nucleotides and 5,000 nucleotides.
Regarding Claim 31 Zahringer teaches a method wherein the nucleotide comprising the homopolymeric nucleotide is A (page 541).
Zahringer does not teach a method wherein the mRNA sample is contacted with a minor groove binding dye (clm 27). Zahringer does not teach assaying the sample by capillary electrophoresis to determine the homopolymeric nucleotide (poly A tail) length of the mRNA (clm 27).
However Zabzdyr teaches that they examined the sensitivity of SYBR Green I and SYBR Gold for LIF detection of dsDNA and RNA following separation by CE. Both UV (275 nm) and visible (488 nm) excitation wavelengths are used to detect the DNA and the RNA with each dye. SYBR Green I has been used extensively for the detection of dsDNA in CE and gel electrophoresis. However, limited use of SYBR Gold has been reported for the detection of dsDNA and RNA following gel electrophoresis. Zabzdyr exemplifies detection of RNA using both of these dyes and the use of SYBR Gold for LIF detection of DNA following CE. Zabzdyr demonstrates the use of these dyes with a UV excitation source for LIF detection of dsDNA and RNA separated by CE. It is noted that SYBR gold is a minor groove binding dye that non-covalently binds to ssRNA.
Additionally Roos teaches a method of producing a defined poly A tail length. Roos teaches that the end product was analyzed on a 15% TBE-UREA PAGE and subsequently stained with Sybr® Gold to assess the polyadenylation of the RNA. PolyA tail lengths were estimated in comparison to a low range ssRNA Ladder (New England BioLabs Inc.). The result of the experiment is shown in FIG. 11 (para 0392).
Accordingly, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of Zahringer by using capillary electrophoresis to determine the poly A tail length of the mRNA as suggested by Zabzdyr. In the instant case Zahringer teaches the use of gel electrophoresis to detect poly A tail length. Zabzdyr teaches nucleic acids can be detected by gel electrophoresis or capillary electrophoresis and exemplifies the detection of RNA using capillary electrophoresis. The claims would have been obvious because the substitution of one type of electrophoresis (gel) for another (capillary) would have yielded predictable results to one of ordinary skill in the art at the time of the invention. Further it would have been obvious to have modified the method of Zahringer by contacting the mRNA sample with a minor groove binding dye (i.e., SYBR gold) as suggested by Zabzdyr and Roos. In the instant case Zabzdyr discloses SYBR gold bound to RNA can be used for capillary electrophoresis (abstract). Additionally Roos discloses using SYBR gold to detect poly A tails. One of skill in the art would have been motivated to contact the mRNA sample with SYBR gold for the benefit of being able to use a dye with a high fluorescence quantum yield, low background fluorescence, and high nucleic acid binding affinity (Zabzdyr page 269) for the detection of poly A tails by capillary electrophoresis.
6. Claims 15, 16, and 25 are rejected under 35 U.S.C. 103 as being unpatentable over Zahringer (Journal of Molecular and Cellular Cardiology 1982 14 pages 539-550) in view of Zabzdyr (Journal of Chromatography A 911 (2001) 269-276) and Roos (US 2018/0142275 Pub 5/24/2018) as applied to claims 1 and 19 above and in further view of Boom (US 2005/0112590 Pub 5/26/2005).
The teachings of Zahringer, Zabzdyr, and Roos are presented above.
The combined references do not teach a method wherein one or more steps of the method is automated (clms 15 and 25). The combined references do not teach a method wherein incubating the mRNA sample from (a) with one or more ribonucleases (RNase) is automated (clm 16).
However Boom teaches methods for nucleic acid analysis that are automated. Boom further teaches an automated protocol for a one tube reaction in RNA is fragmented by cleavage with RNAse T1 (para 0363, 0379).
Accordingly, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of Zahringer, Zabzdyr, and Roos by automating the step of incubating the mRNA sample with the ribonuclease as suggested by Boom. One of skill in the art would have been motivated to automate this step for the benefit of reducing human error and the benefit of eliminating the accidental introduction of other nucleases into the sample which could cause degradation.
7. Claim 18 is rejected under 35 U.S.C. 103 as being unpatentable over Zahringer (Journal of Molecular and Cellular Cardiology 1982 14 pages 539-550) in view of Zabzdyr (Journal of Chromatography A 911 (2001) 269-276) and Roos (US 2018/0142275 Pub 5/24/2018) as applied to claim 1 above and in further view of Haines (Electrophoresis 2015 36, 941-944).
The teachings of Zahringer, Zabzdyr, and Roos are presented above.
The combined references do not teach a method wherein the minor-groove binding dye is not an intercalating dye.
However, Haines teaches that examples of minor groove binding dyes include DAPI and Hoechst dyes (page 941, col 1).
Accordingly, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of Zahringer, Zabzdyr, and Roos by using a minor-groove binding dye is not an intercalating dye as suggested by Haines. One of skill in the art would have been motivated to use a minor groove binding dye that does not intercalate particularly since Haines teaches that dyes that intercalate are known to be mutagenic and toxic (page 941, col 1-2).
8. Any inquiry concerning this communication or earlier communications from the examiner should be directed to AMANDA HANEY whose telephone number is (571)272-8668. The examiner can normally be reached Monday-Friday, 8:15am-4:45pm EST.
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If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, Wu-Cheng Shen can be reached at 571-272-3157. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
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/AMANDA HANEY/Primary Examiner, Art Unit 1682
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