DETAILED CORRESPONDENCE
Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
This action is in response to the papers filed December 22, 2025. Currently, claims 1-20, 22, 24, 26, 28-30, 32, 34-38 are pending.
All arguments have been thoroughly reviewed but are deemed non-persuasive for the reasons which follow.
This action is FINAL.
Any objections and rejections not reiterated below are hereby withdrawn.
The 112b rejections have been withdrawn in view of the amendments to the claims.
The 101 rejections have been withdrawn because the steps in addition to judicial exceptions are not well understood routine and conventional.
Priority
This application claim priority:
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The provisional application does not teach separating fragmented adapter-ligated nucleic acid based on fragment size.
The provisional application does not teach MLL2 gene.
The provisional application does not teach fragmentation by ultrasonic, restriction enzymes, for example.
The provisional application does not teach fragmentation for nucleic acids greater than 1,000 to fragments of 500-600bp or nucleic acids 5,000-10,000 to fragments of 500-600bp.
The provisional does not teach an 8-base pair sample bar code.
The provisional application does not teach separating fragmented nucleic acid using columns, electrophoresis or anion exchange, for example.
Thus, the instant claims receive the benefit of October 11, 2016 filing date.
Drawings
The drawings are acceptable.
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.
Claim(s) 11, 14-17, 19-20, 34 is/are rejected under 35 U.S.C. 103 as being unpatentable over Millholland et al. (Research and Reports in Urology, Vol. 4, pages 33-40, 2012) or Ward et al. (PLOS, DOI:10.1371, February 22, 2016) in view of Hovsep et al. (WO2006/089203, August 24, 2006) and van Dijk (Trends in Genetics, Vol. 30, No. 9, pages 418-426, September 14, 2014).
As noted above, the instant claims receive benefit of the October 11, 2016 priority date.
Millholland teaches preparing nucleic acid from urine. Urine samples were collected and stabilized with ethylenediamine tetra-acetic acid (EDTA), a preservation reagent. DNA is isolated using the QIAamp MinElute virus vacuum kit (page 34, col. 2). The isolated DNA is then amplified using chimeric primers that include a library key and 10 base pair barcode sequences (page 35, col. 2). The primers of Millholland contain specific adapters for unidirectional sequencing on the Roche GS junior platform (pages 35, col. 2). The amplicons are fixed to DNA capture beads and sequencing is carried out (i.e. a hybrid capture)(pages 36, col. 2). The ultradeep amplicon sequencing technology that relies upon DNA capture beads in a water in oil emulsion PCR detected low levels of mutant DNA (page 36, col. 2). Millholland teaches analysis of FGFR3 mutations for bladder cancer using next generation deep sequencing (see limitations of Claim 2).
Ward also teaches extracting DNA from urine cell pellets and amplifying the regions of genes that are frequently mutated in bladder cancer. Specifically, urine was collected and DNA was extracted from the pellets using urine DNA isolation kits. Ward teaches 10bp barcodes were added to the PCR products (limitations of Claim 19). Ward teaches using a Miseq 500 cycle flow-cell (page 3). Ward teaches PCR amplification to amplify 16 amplicons of genes (page 3). The PCR products were barcoded, pooled and sequenced on an Illumina MiSeq, a hybrid capture method (page 1). Ward teaches raw trace files were processed with cutadapt in paired end mode to remove adapter sequence (thus, adapters were added) prior to analysis and sequencing. Ward teaches the non-invasive surveillance of patients is cost-effective. Ward specifically suggests extra markers could be added to develop a highly sensitive diagnostic test for bladder cancer (page 2, para 1). Ward teaches deep sequencing should reliably detect even very low levels of tumor DNA in large excess of non-tumor DNA (page 2, para 5).Mutations with allele frequencies below 0.5% are detected with NGS.
Millholland and Ward do not teach contacting a urine sample with a preservation buffer at room temperatures comprising a microbial static agent, a nucleic acid hydration modifier, a DNAse/RNAse inhibitor and a buffering reagent. Millholland and Ward also do not teach performing nucleic acid fragmentation prior to ligation of sequencing adapters in the next generation methods.
However, Hovsep teaches adding agents to urine samples to inhibit the degradation of the nucleic acids (see claims 14-16 of Hovsep, for example). Hovsep teaches ion-chelating agents, denaturing agents and ionic detergents may be added (see Summary of Invention). These agents include EDTA, guanidine isothiocyanate and sodium dodecyl sulfate (SDS). Hovsep teaches pretreatment of the urine sample with these reagents is at room temperature (page 12, line 8). Hovsep teaches EDTA and Tris-HCl were added to inhibit the nucleases that might be present in urine samples (Example 1, page 17).
Therefore, it would have been prima facie obvious prior to the effective filing date of the claimed invention to have included a pretreatment of urine step, as taught by Hovsep. Hovsep teaches urine should be pretreated to protect nucleic acids. Although Millholland and Ward teach using urine and preservation, Millholland and Ward do not provides a preservation buffer comprising EDTA, SDS and Tris, for example.
Even more, vanDijk teaches next generation sequencing technology relies upon fragmentation. Box 3 of VanDijk illustrates fragmentation of DNA molecules into a size of 50-500nucleotides followed by adapter addition. A size selection step is usually performed to enrich further for molecules of the desired size and to eliminate free adapters. PCR is then performed to select for molecules containing adapters at both ends and to generate sufficient quantities for sequencing. vanDijk teaches NGS fragments genomic DNA, i.e. nucleic acids great than 1,000bp into fragments of 200-400 (see Figure 1 A, read length)(limitations of Claims 14-17).
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Therefore, it would have been prima facie obvious prior to the effective filing date of the claimed invention to have included a fragmentation step and separation of fragmented adapter-ligated nucleic acid based on the fragment size because vanDijk teaches this was a hallmark of NGS library preparation. Although Millholland and Ward did not specifically teach performing the steps of fragmentation and separation, vanDijk teaches these are basic and elementary steps of NGS workflow to generate a sequencing library.
Response to Arguments
The response traverses the rejection. The response asserts that the limitations of Claim 33 are presented in newly amended Claim 11 and are therefore patentable. This argument has been reviewed but is not persuasive because not all of the limitations from previously presented Claim 33 are presented in Claim 11.
Further the response argues neither Millholland nor Ward disclose or suggest contacting the urine sample with a urine preservation buffer. This argument has been considered but is not convincing in view of the newly added reference Hovsep in response to the newly added limitations in Claim 11.
Thus, for the reasons above and those already of record, the rejection is maintained.
Claim(s) 1-2, 5-7, 9-10, 29-30, 32 is/are rejected under 35 U.S.C. 103 as being unpatentable over Millholland et al. (Research and Reports in Urology, Vol. 4, pages 33-40, 2012) or Ward et al. (PLOS, DOI:10.1371, February 22, 2016) in view of Hovsep et al. (WO2006/089203, August 24, 2006) and van Dijk (Trends in Genetics, Vol. 30, No. 9, pages 418-426, September 14, 2014) as applied to claims 11, 14-17, 19-20, 34 above and further in view of Steve et al. (US 2014/0303001).
Millholland and Ward, Hovsep and vanDijk are silent on detecting the presence or absence of at least one mutation or epigenetic alteration in the MLL2 gene.
However, Steve et al. disclose methods for detecting and monitoring bladder cancer by identifying diagnostic genes harboring bladder cancer specific mutations (para 0051). Steve et al. prepare a list of 10 diagnostic genes and teach the top 7th diagnostic gene bearing bladder cancer specific mutations is MLL2 (para 0051). Steve et al. teach that genomic DNA, including cell-free DNA, can be found in the urine of patients with bladder cancer and that a target nucleic acid molecule can be detected by amplifying a nucleic acid sample in or obtained from a sample obtained from a patient, using, e.g., oligonucleotide primers that are specifically designed to hybridize with a portion of the target nucleic acid sequence (para 0002, 0093) (limitations of claims 11, 13, 53). Alternatively, Steve et al. teach that a nucleic acid can be indirectly detected using target hybridization (para 0094-0095) (limitations of claim 2). Steve et al. teach that NGS techniques such as massively parallel sequencing can be used to detect the presence of signature mutations in nucleic acids derived from patient samples such as urine (para 0093)(limitations of Claim 10).
Therefore, it would have been prima facie obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to modify the methods taught by Millholland, Ward and vanDijk to analyze the MLL2 gene in the methods of Millholland, Ward and vanDijk. Ward specifically suggests extra markers could be added to develop a highly sensitive diagnostic test for bladder cancer (page 2, para 1). Steve et al. teach that MLL2 is a well-known diagnostic for bladder cancer. Thus, assaying for MLL2 mutations was known in the art at the time the invention and would have been obvious to detect bladder cancer.
Response to Arguments
The response traverses the rejection. The response asserts that the limitations of Claim 31 are presented in newly amended Claim 1 and are therefore patentable. This argument has been reviewed but is not persuasive because not all of the limitations from previously presented Claim 31 are presented in Claim 1.
Further the response argues neither Millholland nor Ward disclose or suggest contacting the urine sample with a urine preservation buffer. This argument has been considered but is not convincing in view of the newly added reference Hovsep in response to the newly added limitations in Claim 1.
Thus, for the reasons above and those already of record, the rejection is maintained.
Claim(s) 3, 12 is/are rejected under 35 U.S.C. 103 as being unpatentable over Millholland et al. (Research and Reports in Urology, Vol. 4, pages 33-40, 2012) or Ward et al. (PLOS, DOI:10.1371, February 22, 2016) ) in view of Hovsep et al. (WO2006/089203, August 24, 2006) and van Dijk (Trends in Genetics, Vol. 30, No. 9, pages 418-426, September 14, 2014) as applied to claims 11, 14-17, 19-20, 34 above and further in view of Fang et al. (WO2015/185427, December 10, 2015).
Millholland, Ward, Hovsep and vanDijk do not teach identifying molecular clonal families or distinguishing errors in the next generation sequencing method.
However, Fang et al. teaches optimization of DNA amplification reactions including next generation sequencing. Fang teaches the next generation sequencing process may include clonal amplification steps to secure DNA sequencing templates that are free of potential contamination PCR products. Fang teaches clonal amplification may be performed with either emulsion PCR or bridge amplification (see para 69). The methods efficiently avoid sequencing errors in NGS processes (see para 72).
Therefore, it would have been prima facie obvious prior to the effective filing date of the claimed invention to have performed clonal amplification in the next generation sequencing methods of Millholland, Ward, Hovsep and vanDijk to identify clonal families and distinguish and eliminate errors in sequencing. The ordinary artisan would have been motivated to have performed the additional steps to ensure sequencing errors were avoided.
Response to Arguments
The response argues neither Millholland nor Ward disclose or suggest contacting the urine sample with a urine preservation buffer. This argument has been considered but is not convincing in view of the newly added reference Hovsep in response to the newly added limitations in Claim 1.
Thus, for the reasons above and those already of record, the rejection is maintained.
Claim(s) 4, 13 is/are rejected under 35 U.S.C. 103 as being unpatentable over Millholland et al. (Research and Reports in Urology, Vol. 4, pages 33-40, 2012) or Ward et al. (PLOS, DOI:10.1371, February 22, 2016) in view of Hovsep et al. (WO2006/089203, August 24, 2006) and van Dijk (Trends in Genetics, Vol. 30, No. 9, pages 418-426, September 14, 2014) as applied to claims 11, 14-17, 19-20, 34 above and further in view of Knierim et al. (PLOS, Vol. 6, No. 11, e28240, November 2011).
Millholland, Ward, Hovsep and vanDijk do not teach fragmentation using ultrasonication or enzyme based fragmentation the next generation sequencing method.
However, Knierim teaches next generation sequencing (NGS) technologies rely on fragmentation of DNA for preparation of template libraries. Knierim teaches nebulization, sonication and random enzymatic digestion for producing high-quality sequencing libraries. Knierim teaches overall performance of all three methods was equal with only minor differences, a fragmentation method can be chosen solely according to lab facilities, feasibility and experimental design.
Therefore, it would have been prima facie obvious prior to the effective filing date of the claimed invention to have performed fragmentation in the next generation sequencing methods of Millholland, Ward, HOvsep and vanDijk to generate fragments for generation of the next generation library. The ordinary artisan would have been motivated to have performed fragmentation using known methods for fragmenting DNA using ultra-sonication or enzyme based methods. Knierim specifically teaches that the method for fragmentation generates the same results with only minor differences and the fragmentation method selected can be chose based upon experimental design, feasibility or lab facilities.
Response to Arguments
The response argues neither Millholland nor Ward disclose or suggest contacting the urine sample with a urine preservation buffer. This argument has been considered but is not convincing in view of the newly added reference Hovsep in response to the newly added limitations in Claim 1.
Thus, for the reasons above and those already of record, the rejection is maintained.
Claim(s) 8, 18 is/are rejected under 35 U.S.C. 103 as being unpatentable over Millholland et al. (Research and Reports in Urology, Vol. 4, pages 33-40, 2012) or Ward et al. (PLOS, DOI:10.1371, February 22, 2016) in view of Hovsep et al. (WO2006/089203, August 24, 2006) and van Dijk (Trends in Genetics, Vol. 30, No. 9, pages 418-426, September 14, 2014) as applied to claims 11, 14-17, 19-20, 34 above and further in view of Smith et al. (PLOS One, Vol. 7, No. 7, e40425, July 2012).
Millholland, Ward, Hovsep and vanDijk do not teach adapters of 8pb in length.
However, Smith teaches barcodes of 8-bp and 10-bp depending on the application. Smith teaches regular (non-encoded) 10bp barcodes and hamming barcode (8bp in length)(see Figure 1). Smith teaches unique DNA barcodes were introduced to the PCR amplicons. The barcodes were 8bp or 10bp in length.
Therefore, it would have been prima facie obvious prior to the effective filing date of the claimed invention to have substituted the 10bp adapters taught by Ward with 8bp adapters taught by Smith to function in next generation analysis. The simple substitution of 8bp adapters for the 10bp adapters would yield predictable results for next generation sequencing. The prior art teaches both the 8bp and 10bp barcodes may be used, thus the substitution of one adapter for another would have been obvious absent unexpected results.
Response to Arguments
The response argues neither Millholland nor Ward disclose or suggest contacting the urine sample with a urine preservation buffer. This argument has been considered but is not convincing in view of the newly added reference Hovsep in response to the newly added limitations in Claim 1.
Thus, for the reasons above and those already of record, the rejection is maintained.
Conclusion
No claims allowable.
Applicant's amendment necessitated the new ground(s) of rejection presented in this Office action. Accordingly, THIS ACTION IS MADE FINAL. See MPEP § 706.07(a). Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
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 nonprovisional extension fee (37 CFR 1.17(a)) pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
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/JEANINE A GOLDBERG/Primary Examiner, Art Unit 1682 February 17, 2026