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 1-2, 7, 9, 12, 14, 16-17, and 21) in the reply filed on 12/15/2025 is acknowledged. The traversal is on the ground(s) that searching and examination of all the pending claims would not cause a serious or undue burden. This is not found persuasive because the claims were restricted under 35 USC 371 (i.e. the unity of invention analysis), which does not consider search or examination burden. Such a burden is examined for national applications filed under 35 USC 111(a). See MPEP 823 and its associated resources.
The requirement is still deemed proper and is therefore made FINAL.
Claims 22-24, 41-46, and 56 are withdrawn from further consideration pursuant to 37 CFR 1.142(b), as being drawn to a nonelected inventions, there being no allowable generic or linking claim.
It is noted that Applicant has also added claims 62-73. Thus, claims 1-2, 7, 9, 12, 14, 16-17, 21, and 62-73 are pending and are examined on the merits herein.
Information Disclosure Statement
The information disclosure statement (IDS) submitted on 4/1/2025 is in compliance with the provisions of 37 CFR 1.97. Accordingly, the information disclosure statement is being considered by the examiner.
Nucleotide and/or Amino Acid Sequence Disclosures
Summary of Requirements for Patent Applications Filed On Or After July 1, 2022, That Have Sequence Disclosures
37 CFR 1.831(a) requires that patent applications which contain disclosures of nucleotide and/or amino acid sequences that fall within the definitions of 37 CFR 1.831(b) must contain a “Sequence Listing XML”, as a separate part of the disclosure, which presents the nucleotide and/or amino acid sequences and associated information using the symbols and format in accordance with the requirements of 37 CFR 1.831-1.835. This “Sequence Listing XML” part of the disclosure may be submitted:
1. In accordance with 37 CFR 1.831(a) using the symbols and format requirements of 37 CFR 1.832 through 1.834 via the USPTO patent electronic filing system (see Section I.1 of the Legal Framework for Patent Electronic System (https://www.uspto.gov/PatentLegalFramework), hereinafter “Legal Framework”) in XML format, together with an incorporation by reference statement of the material in the XML file in a separate paragraph of the specification (an incorporation by reference paragraph) as required by 37 CFR 1.835(a)(2) or 1.835(b)(2) identifying:
a. the name of the XML file
b. the date of creation; and
c. the size of the XML file in bytes; or
2. In accordance with 37 CFR 1.831(a) using the symbols and format requirements of 37 CFR 1.832 through 1.834 on read-only optical disc(s) as permitted by 37 CFR 1.52(e)(1)(ii), labeled according to 37 CFR 1.52(e)(5), with an incorporation by reference statement of the material in the XML format according to 37 CFR 1.52(e)(8) and 37 CFR 1.835(a)(2) or 1.835(b)(2) in a separate paragraph of the specification identifying:
a. the name of the XML file;
b. the date of creation; and
c. the size of the XML file in bytes.
SPECIFIC DEFICIENCIES AND THE REQUIRED RESPONSE TO THIS NOTICE ARE AS FOLLOWS:
This application contains sequence disclosures in accordance with the definitions for nucleotide and/or amino acid sequences set forth in 37 CFR 1.831(a) and 1.831(b). However, this application fails to comply with the requirements of 37 CFR 1.831-1.834. The examiner has noted that based on Applicant’s Sequence Incorporation by Reference paragraph (para. 3 of the instant specification), the provided Sequence Listing is in ASCII format. The present application was filed on April 11, 2023, and so must comply with WIPO Standard ST.26 guidelines. These guidelines require that the Sequence Listing be provided in XML format. See 37 CFR 1.8333. Applicant must provide:
• A replacement “Sequence Listing XML” part of the disclosure, as described above in item 1. or 2., as well as
• A statement that identifies the location of all additions, deletions, or replacements of sequence information in the “Sequence Listing XML” as required by 1.835(b)(3);
• A statement that indicates support for the amendment in the application, as filed, as required by 37 CFR 1.835(b)(4);
• A statement that the “Sequence Listing XML” includes no new matter in accordance with 1.835(b)(5); and
• A substitute specification in compliance with 37 CFR 1.52, 1.121(b)(3), and 1.125 inserting the required incorporation by reference paragraph as required by 37 CFR 1.835(b)(2), consisting of:
o A copy of the previously-submitted specification, with deletions shown with strikethrough or brackets and insertions shown with underlining (marked-up version);
o A copy of the amended specification without markings (clean version); and
A statement that the substitute specification contains no new matter.
Specific deficiency – Nucleotide and/or amino acid sequences appearing in the drawings are not identified by sequence identifiers in accordance with 37 CFR 1.821(d). Sequence identifiers for nucleotide and/or amino acid sequences must appear either in the drawings or in the Brief Description of the Drawings. Specifically, Figures 7 and 8 contain sequences without sequence identifiers.
Required response – Applicant must provide:
Replacement and annotated drawings in accordance with 37 CFR 1.121(d) inserting the required sequence identifiers;
AND/OR
A substitute specification in compliance with 37 CFR 1.52, 1.121(b)(3) and 1.125 inserting the required sequence identifiers into the Brief Description of the Drawings, consisting of:
A copy of the previously-submitted specification, with deletions shown with strikethrough or brackets and insertions shown with underlining (marked-up version);
A copy of the amended specification without markings (clean version); and
A statement that the substitute specification contains no new matter.
Drawings
The drawings are objected to because Figures 5, 9-10C, 12, 13A, 13B, 13D, 13E, 13G, and 14 are not legible. Additionally, Figures 7 and 8 contain sequences without corresponding sequence identifiers, as noted in the “Nucleotide and/or Amino Acid Sequence Disclosure” section above. Corrected drawing sheets in compliance with 37 CFR 1.121(d) are required in reply to the Office action to avoid abandonment of the application. Any amended replacement drawing sheet should include all of the figures appearing on the immediate prior version of the sheet, even if only one figure is being amended. The figure or figure number of an amended drawing should not be labeled as “amended.” If a drawing figure is to be canceled, the appropriate figure must be removed from the replacement sheet, and where necessary, the remaining figures must be renumbered and appropriate changes made to the brief description of the several views of the drawings for consistency. Additional replacement sheets may be necessary to show the renumbering of the remaining figures. Each drawing sheet submitted after the filing date of an application must be labeled in the top margin as either “Replacement Sheet” or “New Sheet” pursuant to 37 CFR 1.121(d). If the changes are not accepted by the examiner, the applicant will be notified and informed of any required corrective action in the next Office action. The objection to the drawings will not be held in abeyance.
Claim Objections
Claim 1 is objected to because of the following informality: for oligonucleotide (b)(ii), it is recommended to state “pathogen specific” rather than “pathogen-specific,” as the former format is used elsewhere throughout the claim set. Appropriate correction is required.
Claim 2 is objected to because of the following informality: in (viii)(4), “pathogenic specific consensus sequence” should read “pathogen specific consensus sequence.” Appropriate correction is required.
Claim 7 is objected to because of the following informalities: in line 3, the word “a” should appear before “whole saliva sample” and “cell-free saliva sample.” Additionally, in line 4, the large space between “sputum,” and “stool,” should be removed. Appropriate correction is required.
Claim 17 is objected to because of the following informalities: for each of the presented options, it is recommended to state one of “with single-pot, closed tube chemistry,” “with single-pot, open tube chemistry,” and “with single-pot, multi-tube chemistry using…” for grammatical clarity, as appropriate. Appropriate correction is required.
Claim 21 is objected to because of the following informality: in line 3, it is recommended that “from the subject’s library” be removed, as the “plurality of nucleic acid reads” are already described in claim 1 and clearly are derived from the library, and the phrase “subject’s library” is not used elsewhere in the claim set. Appropriate correction is required.
Claim 62 is objected to because of the following informalities: the oligonucleotides should be numbered (v) and (vi) based on the numbering used in claim 1, from which this claim depends. Also, in (viii)(4), “pathogenic specific consensus sequence” should read “pathogen specific consensus sequence.” Appropriate correction is required.
Claim 71 is objected to because of the following informality: it is recommended that “in a single-pot, open tube chemistry” read “with single-pot, open tube chemistry”. Appropriate correction is required.
Claim 72 is objected to because of the following informality: it is recommended that “in a split-pot, multi tube chemistry” read “with single-pot, multi-tube chemistry using…”. Appropriate correction is required.
Claim 73 is objected to because of the following informality: it is recommended that “in a split-pot, multi tube chemistry” read “with single-pot, multi-tube chemistry using…”. Appropriate correction is required.
Claim Rejections - 35 USC § 112(b)
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 1-2, 7, 9, 12, 14, 16-17, 21, and 62-73 rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
Claim 1 is rejected because the pathogen-specific oligonucleotide primer is unclear. Specifically, the primer requires a minimal and extended 3’ end cDNA splint, as well as a 3’ end cDNA UMI. It is unknown how all of these components can simultaneously exist at the 3’ end of a single sequence. These limitations for this oligonucleotide will be interpreted as though they are referring to the 3’ ends of each individual portion (i.e. the 3’ end of the UMI sequence and the 3’ end of the splint sequences).
Similarly, the adapters of claim 1 are unclear as they require multiple simultaneous 3’ or 5’ components. It is unknown how all of these components can simultaneously exist at the 3’ or 5’ end of a single sequence. These limitations for the adapter oligonucleotides will be interpreted as though they are referring to the 3’ or 5’ ends of each individual portion of the adapter, similar to the interpretation above for the pathogen-specific oligonucleotide primer.
Claims 1-2 and 62 are rejected due to the use of the phrases “cDNA splint” and “cDNA UMI.” Specifically, as cDNA is typically made from reverse transcription of RNA, it is unclear if these terms indicate that the splint and UMI sections must be made of cDNA, or if they are simply used on oligonucleotide sequences that will be extended to include cDNA. Particularly, as cDNA generally contains exon sequences of genomic DNA, and a UMI is defined in the instant specification as “an oligonucleotide sequence that can be used to identify an individual molecule or nucleic acid fragment present in the sample” (see para. 101), it is unclear how such a sequence would be comprised of cDNA. Therefore, these terms will be interpreted to indicate that the oligonucleotides they are a part of should be composed of DNA, and that these oligonucleotides should at least partially form cDNA when extended.
Claim 2 is also rejected for reasons similar to those of claim 1. Specifically, for oligonucleotides (vii) and (viii), multiple simultaneous 5’ or 3’ components are recited. It is unknown how all of these components can simultaneously exist at the 3’ or 5’ end of a single sequence. These limitations for oligonucleotides (vii) and (viii) will be interpreted as though they are referring to the 3’ or 5’ ends of each individual portion, similar to the interpretation above for the pathogen-specific oligonucleotide primer of instant claim 1.
Claims 2, 7, 9, 12, 14, 16-17, 21, and 62-73 are rejected due to their dependence on rejected claim 1.
Claim 12 is also rejected because the scope is generally unclear. The claim recites that the pathogen “is selected from” a group of options, where the options are recited in non-alternative fashion (i.e. …and Zika virus). It is unclear if Applicant was intending to recite a group of alternatively useable pathogen types (i.e. a Markush group), in which case the language “is selected from the group consisting of” or acceptable alternatives should be used (see MPEP 2117). Applicant may also be intending to recite language where the pathogen may be one listed, but may also be other pathogens, including others not listed. Overall, as the claim currently stands, it is not clear if the group of pathogen options should be interpreted as open or closed. For the purposes of applying prior art, art will be considered to read on the claim if said art applies in either the open or closed interpretation.
Claims 17 and 71-73 all recite that the method of claim 1 must be performed with particular types of chemistries, without specifying that these chemistries apply to a particular step of the method. Thus, the recited chemistries are considered to apply to the entirety of the method of claim 1. However, the entirety of the method of claim 1 involves steps which cannot be performed in a tube, such as obtaining a sample from the subject. Additionally regarding claim 17, claim 1 recites the use of sequencing, which could not be performed if closed tube chemistry was used. Thus, the metes and bounds of the claims are unclear. It will be interpreted as though the chemistries recited in these claims are specifically referring to the library preparation step of claim 1.
Claim 62 is rejected because the preamble recites “preparing the library comprises further nucleic acid amplification,” when initial amplification is not explicitly recited in claim 1. It is therefore unclear where initial amplification during the library preparation is meant to occur relative to this further amplification. Prior art will be considered to read on this claim if it teaches the stated oligonucleotides within the context of library preparation generally.
Additionally, claim 62 is rejected because for oligonucleotide (viii), multiple simultaneous 5’ components are recited. It is unknown how all of these components can simultaneously exist at the 5’ end of a single sequence. These limitations for oligonucleotides (viii) will be interpreted as though they are referring to the 5’ ends of each individual portion, similar to the interpretation above for the pathogen-specific oligonucleotide primer of instant claim 1.
Claim Interpretation
For clarity, the interpretation of the required oligonucleotides of the instant claims is provided below. It is noted that the function of each individual oligonucleotide during library preparation is not specifically stated in the claims. For example, the particular uses of the primers, the anchored oligonucleotide, adapters, blocking oligonucleotide, and template switching oligonucleotides in the library preparation process of the claims are not stated. Thus, prior art will considered to read on the claimed oligonucleotides if it teaches use of oligonucleotides with the claimed required structural components at any point during library preparation.
The term “splint” has no specific definition in the instant specification. Based on its use in the claims, a “splint” sequence will be interpreted as a sequence capable of hybridizing to another sequence.
In claim 1:
Step (a) recites the use of a sample and “extracting nucleic acids” from said sample. However, “extracting” has no specific definition in the instant specification, and so will be interpreted to encompass any method that allows nucleic acids within the sample to be directly interacted with/manipulated. This would involve methods that include cell lysis to access nucleic acids, or methods involving the use of particular buffers that allow for access to nucleic acids.
For the anchored oligonucleotide, the term “anchored” has no specific definition in the instant specification. Para. 9 describes amplification as being “anchored” to consensus sequences, and the methods below para. 120 recite anchored multiplexing in a well plate, where it appears that anchoring involves attachment to the well plate. In considering the broadest reasonable interpretation of the term in light of the prior art, an “anchored oligonucleotide” will be considered one that is capable of being anchored to a substrate, without specifically requiring that the oligonucleotide be anchored to said substrate in the method of claim 1.
For the pathogen-specific oligonucleotide primer, a pathogen specific consensus sequence is defined in the instant specification in para. 103 as a conserved region in a pathogen’s genome that is well-conserved across a plurality of sequences belonging to the same pathogen species, and so any sequence in the prior art that contains such a sequence anywhere along its length will be considered to read on this limitation. As for the “minimal” and “extended” splint sequences, these are not defined by the instant specification. Given that this oligonucleotide is a primer, and given how primers are generally used in the prior art, these splints will be considered to be the 3’ end before extension in an amplification reaction (for the minimal end splint) and after extension in the same reaction (for the extended splint), though see the 35 USC 112(b) Rejections above.
As this sequence involves nucleotides that are not present on the primer until after extension occurs (e.g. the extended 3’ end cDNA splint), and the claimed method only states that the listed oligonucleotides must be used, and not that they cannot be manipulated or must contain each listed component at the outset of the method, the listed component limitations will be considered to be met if they are taught in the prior art at any point during a library preparation process, and not just when an oligonucleotide is introduced. For example, if a primer does not initially contain a pathogen specific consensus sequence upon binding to a target, but does contain such a sequence after extension, then the primer will be considered to contain the pathogen specific consensus sequence as claimed.
For the 3’ and 5’ adapters, the term “adapter” is stated in para. 102 to generally refer to linear oligonucleotides which can be ligated to a nucleic acid molecule, but this is not a requirement, as later in the same paragraph it is noted that adapters may be attached via PCR. The “coupling sequence” for each adapter is not specified in the instant specification, but will be interpreted as the specific portion of the overall adapter that is used for attachment to a nucleic acid molecule. Thus, the “a 3’ adapter” and “a 5’ adapter” terms will be interpreted as the entirety of the adapter sequence, where the barcode and coupling sequences are contained within the adapters.
In claim 2:
For the pathogen specific template switching oligonucleotide, a “template switching motif” will be considered the portion of the oligonucleotide that renders it capable of being used for template switching. This will also apply to the “template switching motif” of the generic template switching oligonucleotide and the generic cDNA coupler reverse primer oligonucleotide.
For the “generic tailing motif” as recited in (vi) and (ix), no specific definition for tailing is given by the instant specification, and so this will be considered any non-complementary tail sequence present at either end of an oligonucleotide.
Generally in claim 2, the guidance provided in (c)(i) above is also followed here. For example, if in the prior art a pathogen specific template switching oligonucleotide exists that only contains a pathogen specific consensus sequence after extension, then the prior art will still be considered to read on the pathogen specific template switching oligonucleotide as claimed.
In (x), “a rDNA blocking duplex oligonucleotide” is stated. Such an oligonucleotide is not defined in the instant specification. However, in para. 100, it is stated “In some embodiments, the methods disclosed here comprise using a single DNA duplex matching homologous rRNA sequences from ITS genomic loci in mammalian species. In some embodiments, the single DNA duplex has 3' hexanediol-modified strands to block DNA polymerase processivity.” Such a description appears to describe a potential rDNA blocking duplex oligonucleotide, but is not considered limiting. Thus, the “rDNA blocking duplex oligonucleotide” is considered to generally encompass rDNA that can be used to block any function during library preparation, such as the blocking of hybridization or the blocking of oligonucleotide extension.
These interpretations apply to additional claims that may include these terms (e.g. claim 62).
It is also noted that claim 2 requires one or more of the listed oligonucleotides, as evidenced by the “and/or” between options (ix) and (x).
Claim Rejections - 35 USC § 103
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
Claims 1-2, 7, 9, 12, 14, 16, 21, 64-67, 69, and 70 are rejected under 35 U.S.C. 103 as being unpatentable over Chappleboim et al. (medRxiv, 2020; cited in Applicant’s IDS) in view of Lee (Dissertation, 2020).
Chappleboim teaches RNA-sequencing protocols for the detection of SARS-CoV-2 (Title and Abstract; instant claims 12 and 69). The basic workflow is shown in Figure 1, where mRNA capture on beads, reverse transcription and PCR, and then sequencing and subsequent analysis is done to detect particular target genes in sequence reads. Chappleboim utilizes sample-specific barcodes, because multiple samples may be pooled with their method (see Figure 1, Abstract, and “Barcoded RT primers added to lysed samples prime RT reactions” on page 4). Chappleboim utilized this method to analyze two targets in a multiplex setting – the N1 amplicon and the E amplicon (page 8, “Multiple Target Assay”). Figure 2D notes that these target sequences conform to reference genomes in 99.9% of reads, and so would qualify as “pathogen specific consensus sequences” under the claim interpretation described above (and also meet the limitations of instant claim 67, as this provides evidence that these target genes are in the viral genome). On page 11, Chappleboim utilizes this method on clinical samples that were both positive and negative for SARS-CoV-2 (“Robotic ApharSeq on clinical samples” and Figure 6). Page 14 shows how this method may be utilized to quickly, cheaply, and efficiently evaluate many samples with a nasopharyngeal swab, where samples are eventually sent to a sequencing site (Figure 7, and see page 2, para. 2 which specifically notes that nasopharyngeal swabs are used for SARS-CoV-2 testing (instant claim 7, also this testing is done on humans specifically, see Figure 1C, page 8, para. 3, Figure 5C, and Figure 7 for example, instant claims 14 and 70). On pages 16-18, Chappleboim describes their primer design. For both the RT-primers and the PCR-primers, target-specific portions were utilized (see the N1 and E portions of the primer design). It is noted that the reference also specifically states the method is “extraction-free” (see Title), but this refers to extraction from the sample lysis buffer in which the sample arrives (page 4, para. 1). The reference does later either isolate RNA from the rest of a sample via polyT capture (page 18, para. 4) or SPRI beads (page 18, para. 5). These methods are also listed on page 4, para. 1. However, under the interpretation of “extracting” described above in the “Claim Interpretation” section, the fact that viral and human RNA is directly accessible by capture beads upon their contact with one another indicates that this reference performs “extracting” as claimed.
However, the oligonucleotides of Chappleboim described in Figure 1 do not appear to meet all the limitations of the oligonucleotides of the instant claims.
Lee presents a new RNA sequencing technology while also providing methods for capturing, barcoding, reverse transcribing, and amplification (Abstract). Section 4.5 provides a molecular workflow for sequencing library preparation (starting on page 56). Section 4.5.2.1 and Figure 4.14 describe and show this workflow.
In section A, target mRNAs are captured using oligo-dT beads, where the oligo-dT binds to the mRNA, thus acting as a splint.
In sections B and C, target mRNAs are annealed to a PCR primer sequence comprising a PCR handle, a UMI, and an oligo-dT sequence. Reverse transcription is then performed to obtain cDNA. In section 4.5.3.1 on pages 59-60, it is noted that entirety of the UMI-dT sequence is composed of DNA. Under the interpretations provided in the 35 USC 112(b) Rejections section and the “Claim Interpretation” sections above, the dT portion (and the following two nucleotides) that initially binds to the mRNA can be considered analogous to the “minimal 3’ end cDNA splint,” and the cDNA portion that is then extended from this minimal portion is analogous to the “extended 3’end cDNA splint.”
In section H, sequencing adapters are added via PCR, where the adapters include complementary sequences to the fragments (the PCR handle and R2 in H, which act as coupling sequences). These adapters also include P5 and P7 sequences, respectively, as well as i5 and i7 sequences, which are index sequences (page 58, para. 1). As noted in para. 99 of the instant specification, the barcodes of the invention can be index sequences, and so these i5 and i7 sequences are analogous to the barcodes of the adapters. This workflow is then used with particular cell types on pages 61-65, but these are cancer cells, and pathogens are not described. It is noted that in this second example of Lee it is specified that the sequencing library is used to produce sequencing reads, where gene expression can be analyzed (see Figure 4.15 and section 4.6.3.7 on page 64).
However, this section of Lee does not specify a target mRNA sequence, and so it is unclear if the primer shown in B and C contains a pathogen specific consensus sequence. Additionally, the composition of the oligo-dT bead is not clear, and so it is not clear if this sequence has a UMI or a sample-specific barcode. Finally, the types of samples used with this workflow are not specified on pages 56-60.
Prior to the effective filing date of the claimed invention, it would have been prima facie obvious for one of ordinary skill in the art to combine the methods of Chappleboim and Lee to arrive at the method of instant claim 1. Specifically, Lee teaches a method of creating a sequencing library from mRNA, and Chappleboim teaches a method of applying a sequencing library to effectively detect pathogens in samples. The methods of Lee involve mRNA released from single cells (see page 56, Section 4.5.2.1), and mRNA is the same starting material used by Chappleboim (e.g. Figure 1), thus demonstrating that the manipulations described by both experiments would work from the same starting sample containing target mRNA. Chappleboim teaches that their library preparation methods have low cross-contamination (page 3, para. 1), can analyze multiple genes (Figure 5), and are cheap and scalable for large amounts of samples (Figure 7), which are benefits that would be motivating to the ordinary artisan for detecting pathogens in a sample. In Chappleboim’s two-step PCR protocol, after reverse transcription, PCR is performed and then index primers are used (see pages 18-19). Thus, this version of their method corresponds well to steps A (bead capture), B-C (reverse transcription), E (initial PCR), and H (index adapter use) of Lee. Due to these similarities, it would be prima facie obvious to use the teachings regarding sequencing library oligonucleotide design of Lee in the method of Chappleboim to detect target SARS-Cov-2 genes. Because Chappleboim already teaches the use of these elements (with the UMI-annealing oligonucleotide of B of Lee being analogous to the RT-primer of Chappleboim), altering their design slightly to align with the guidance of Lee while still being used for the same purposes would provide a reasonable expectation of success.
Specifically regarding the oligo-dT bead, the teachings of Chappleboim already teach the use of a dT sequence attached to a bead (Figure 1A). Lee teaches a dt sequence and a 3’ splint in Figure 4.14. However, on page 9, Lee teaches the use of a barcoding primer bead, where said bead contains a handle, a cell barcode, a UMI sequence, and a dT sequence for tagging mRNA specifically (see Figure 2.2). The cell barcode is used to tag individual cells in a cell-specific method (see para. below the figure). In considering this in the method of Lee described in Figure 4.14, this bead oligonucleotide is nearly the same as the UMI annealing oligonucleotide, with the addition of the barcode. This UMI-annealing oligonucleotide is used for reverse transcription in this figure, and in Chappleboim, the RT-primer similarly contains a PCR handle, UMI, and sample barcode. In considering all of these teachings together, it would be prima facie obvious to combine target capture and reverse transcription by utilizing a bead oligonucleotide as described in Figure 2.2 of Lee. Chappleboim utilizes sample-specific barcodes and focuses on multiple samples rather than individual cells. By combining the capturing and reverse transcription steps, this would cut down on required time and resources to perform the overall method, as separate anchoring sequences and RT-primers would not need to be designed. Lee teaches that this type of oligonucleotide can specifically be used as a RT primer (page 10, para. 1) providing a reasonable expectation of success.
Thus, Chappleboim in view of Lee teaches oligonucleotide (b)(i).
Specifically regarding the pathogen specific oligonucleotide primer, Chappleboim teaches that during PCR, the forward primer hybridizes to the RT-primer region via the PCR handle, and upon extension, would then contain the UMI sequence and the pathogen specific consensus sequence. The minimal and extended cDNA splints would also naturally exist on this primer before and after extension, according to the guidance provided in the “Claim Interpretation” and 35 USC 112(B) Rejections above.
Thus, Chappleboim in view of Lee teaches oligonucleotide (b)(ii).
Specifically regarding the indexed adapters, as noted above, Lee teaches adapters with the claimed structure. These adapters are used with Illumina sequencing methods (see Figure 4.14). Chappleboim notes that separate primers can be provided that utilize sequencing adapters (see the top of page 18), and also teaches the use of Illumina sequencing (see page 5, para. 1, page 17, para. 1, and page 19, para. 2 for example). While the P5 and P7 sequences for the Illumina adapters are added during normal PCR in Chappleboim, this still leaves the index sequences and target complementary sequences as shown in Lee. Chappleboim teaches that their sequencing adapters contain index sequences (see the top of page 18), and so it would be prima facie obvious to use the remaining structure of the sequencing adapters shown in Lee (i.e. the i5 and i7 sequences and the sequences complementary to the amplification product) as the sequencing adapters in Chappleboim. The complementary sequences would allow for hybridization of the adapters and the amplification products, and as Lee notes in the caption of Figure 4.14, the adapter sequences as a whole are then added via additional PCR. Chappleboim also notes that these additional adapters are added via PCR, and not another method such as ligation (see the top of page 18). Therefore, the sequencing adapters of the references provide the same function (labeling the amplification products so they may be sequenced and more easily analyzed), and are added the same way. The use of the sequencing adapter designs of Lee in Chappleboim would therefore amount to simple substitution. MPEP 2143 I (B) states, “The rationale to support a conclusion that the claim would have been obvious is that the substitution of one known element for another yields predictable results to one of ordinary skill in the art.” As both adapter designs are known in the art, and their use is made clear by both Chappleboim and Lee, such substitution would be prima facie obvious.
Thus, Chappleboim in view of Lee teaches oligonucleotides (b)(iii) and (b)(iv), and thus teaches each element of instant claim 1.
Therefore, Chappleboim in view of Lee renders prima facie obvious claims 1, 7, 12, 14, 67, and 69-70.
Regarding claims 2 and 62, specifically regarding the pathogen specific enrichment coupler reverse primer, Chappleboim teaches that their PCR primers contain portions complementary to the target cDNA (which is a pathogen specific consensus sequence), and as they are primers, they have a minimal end at the outset, and extended ends once PCR extension begins. Chappleboim also notes that additional barcodes can be added via PCR primers (see Figure 1B and the “pool barcode”). Lee also teaches that their PCR primers have a UMI. As Chappleboim teaches that the goal of their method is to be performed on a large scale with a great deal of pooling, it would be prima facie obvious to make this pooled barcode an additional UMI. This would ensure that no two pooled barcodes are the same, and would also provide a check on the method, improving accuracy (i.e. sequences that have successfully gone through each pre-sequencing step would have two UMIs, and so if a read was missing one of these sequences, it could indicate an error in the method). As UMIs are known in the art, as evidenced by Chappleboim and Lee, and only the barcode of the reverse primer shown in Figure 1B would be altered with said UMI, there would be a reasonable expectation of success.
Specifically regarding the generic cDNA coupler reverse primer oligonucleotide, Lee teaches that during their library preparation methods, 2nd strand cDNA synthesis involves a template-switching oligonucleotide with a template switching motif (the rG motif) and a tailing motif (the TS oligo). In Figure 1, Chappleboim does not explicitly show 2nd strand cDNA synthesis. However, the resulting cDNA strands contain both the forward and reverse primer sequences (see Figure 1), thus providing evidence that 2nd cDNA strands may be generated during the PCR of Chappleboim. As Lee provides a way of generating a 2nd cDNA strand before PCR begins through the simple use of a template-switching oligonucleotide, it would be prima facie obvious to use such an oligonucleotide in the method of Chappleboim in view of Lee. This is because such synthesis would allow for both the forward and reverse PCR primers to both begin amplifying targets immediately upon the first cycle, thereby generating more amplification product. This would provide more product for sequencing and thereby increase the odds that SARS-CoV-2 would be successfully detected in the sample. As Lee teaches template-switching in the context of sequencing library preparation, there would be a reasonable expectation of success.
Regarding claims 9 and 66, as the samples of Chappleboim are from human nasopharyngeal swabs, and as shown in Figure 1C, the sequencing analysis notes that both human and viral DNA may be present in the samples. As the instant specification does not define a “plurality,” the presence of nucleic acids from two organisms (human and SARS-CoV-2) will be considered a plurality.
Regarding claim 16, Figure 7 of Chappleboim shows the desired application of their method – where many samples are collected in arrays, processed simultaneously, additionally pooled, and then sequenced. The reference states that this can be used with initially pooled samples (page 13, para. 6). Thus, this reference teaches that multiple pooled samples on plates (where each sample contains RNA from multiple subjects) can be processed and sequenced simultaneously.
Regarding claim 21, the purpose of the method of Chappleboim is to detect SARS-CoV-2 infection (Abstract). Throughout the reference, Chappleboim teaches that their assay can be used to detect viral loads (Figures 4-5), and clinical samples were specifically tested for viral molecules (with the data shown in Figure 6). Thus, it would be prima facie obvious to the ordinary artisan that the method of Chappleboim could be used to determine infection status based on the presence or absence of targeted viral genes in the analysis of the sequencing library.
Regarding claim 64, as Chappleboim in view of Lee teaches the analysis of RNA, their nucleic acid sample would naturally comprise RNA.
Regarding claim 65, Chappleboim teaches that their detection methods involved the use of clinical samples (Abstract, page 11, Figure 6, and page 19, para. 4).
Claims 17 and 72 are rejected under 35 U.S.C. 103 as being unpatentable over Chappleboim et al. (medRxiv, 2020; cited in Applicant’s IDS), in view of Lee (Dissertation, 2020), and further in view of Takara (2019).
Chappleboim in view of Lee teaches the methods of claims 1-2, 7, 9, 12, 14, 16, 21, 64-67, 69, and 70, as described above. Chappleboim generally teaches that their library preparation method is a multi-tube method – see pages 18-19, “Option 1: Purification and hybridization on PolyT beads” and “cDNA synthesis and Library preparation.” However, neither Chappleboim nor Lee teaches the use of PCR pre-amplification.
Takara teaches a particular master mix for performing PCR pre-amplification. The main benefit of said pre-amplification is stated to be being able to use low amounts of starting nucleic acids, and said pre-amplification can be used with cDNA (page 1, “Introduction”). Pre-amplification provides lower Ct values, meaning amplification itself would be more efficient, and can be successful with as little as 100 pg of starting material (Figures 1 and 2). Takara also teaches that their mix is highly unbiased (Table I).
Prior to the effective filing date of the claimed invention, it would have been prima facie obvious for one of ordinary skill in the art to use the teachings of Takara to add a PCR-preamplification step to the method of Chappleboim in view of Lee. Such a step would occur after reverse transcription, as the pre-amplification can effectively be used on cDNA. Takara teaches a commercially available kit for performing said pre-amplification, as well as many benefits associated with including such a step in amplification methods. The ordinary artisan would be motivated to specifically use pre-amplification in the context of Chappleboim in view of Lee because this would increase the detection rate of SARS-CoV-2 in a sample, even if the virus is present in a sample in very low amounts, which can lead to more accurate diagnostics and earlier patient intervention, potentially improving patient outcomes, particularly for patients who are at increased risk of disease complications. There would be a reasonable expectation of success because the resources and protocols for using such pre-amplification are readily available from Takara.
Thus, claims 17 and 72 are prima facie obvious over Chappleboim, in view of Lee, and further in view of Takara.
Claim 63 is rejected under 35 U.S.C. 103 as being unpatentable over Chappleboim et al. (medRxiv, 2020; cited in Applicant’s IDS), in view of Lee (Dissertation, 2020), and further in view of Liu et al. (Front. Microbiol., 2019).
Chappleboim in view of Lee teaches the methods of claims 1-2, 7, 9, 12, 14, 16, 21, 64-67, 69, and 70, as described above. However, neither reference teaches the use of a rDNA blocking oligonucleotide.
Liu focuses on analysis of gut microorganisms in marine animals, and specifically, how to effectively analyze said microorganisms in the presence of host DNA (Abstract). Liu focused on the 18S region of the microorganisms in question, and developed a blocking primer to “block the amplification of host DNA in a complex sample for the detection of eukaryotic species of interest,” as the blocking primer preferentially binds to host DNA (pages 2, columns 1-2 joining para.). The blocking primer was based on rDNA regions that corresponded to rRNA in the microorganisms (particularly eukaryotic bacteria; page 2, column 2, para. 3). Multiple blocking primers were developed (page 3, column 1, para. 4). These blocking primers were then used in methods involving amplification, library preparation, and sequencing (pages 3-4, “Amplification With Blocking Primers” and “Library Construction and Sequencing”). Liu teaches that the blocking primers were able to successfully suppress unwanted amplification of host sequences during PCR (page 10, columns 1-2 joining para.).
Prior to the effective filing date of the claimed invention, it would have been prima facie obvious for one of ordinary skill in the art to use the guidance provided by Liu in the context of Chappleboim in view of Lee. Specifically, Chappleboim in view of Lee deals with examining viruses from human nasopharyngeal samples, which, by nature of the sampling, would likely include human, bacterial, and viral nucleic acids. Liu teaches means by which to block undesired sequences from being amplified utilizing rDNA blocking primers. While Chappleboim does analyze host target genes in some of their embodiments (e.g. Figure 1C and Figure 5D) this is not a requirement for every embodiment (e.g. Figure 5B). Additionally, in determining if a particular patient sample has SARS-CoV-2, from a diagnostic perspective, it would not be necessary to also analyze the human DNA in a sample as well. In fact, the presence and amplification of such DNA may hinder virus amplification and detection by providing false or noisy results. And although the PCR primers used in Chappleboim in view of Lee do contain target specific portions, they also contain barcodes/UMIs/PCR handles that would allow for potential non-specific or non-desired amplification. As Lee teaches that utilizing rDNA primers specifically can block human amplification without interfering with PCR primer function, and Liu carefully lays out design guidance for creating an effective blocking primer (see the guidance at the top of page 3 of Liu, for instance), the design and inclusion of such blocking primers would not require a great deal of time and resources to include in Chappleboim in view of Lee, and their inclusion would potentially improve the analysis efficiency of sequencing results. Furthermore, as human DNA is double-stranded, it would be prima facie obvious to create duplex rDNA blocking primers to target potentially more interfering human sequences. There would be a reasonable expectation of success in the design and inclusion of the rDNA sequences as Liu teaches they can explicitly be used with amplification and library creation.
Thus, claim 63 is prima facie obvious over Chappleboim, in view of Lee, and further in view of Liu.
Claim 68 is rejected under 35 U.S.C. 103 as being unpatentable over Chappleboim et al. (medRxiv, 2020; cited in Applicant’s IDS), in view of Lee (Dissertation, 2020), and further in view of Kim et al. (Cell, 2020).
Chappleboim in view of Lee teaches the methods of claims 1-2, 7, 9, 12, 14, 16, 21, 64-67, 69, and 70, as described above. As noted above, Chappleboim teaches the use of pathogen specific consensus sequences (the N1 amplicon and the E amplicon), but these are not taught to be transcription-regulation sequence motifs.
Kim teaches the use of DNA and RNA sequencing to create a high-resolution map of the SARS-CoV-2 transcriptome (Summary). The reference teaches that coronaviral RNA contains motifs called transcription-regulatory sequences (TRSs) that are immediately adjacent to open reading frames (ORFs; page 914, column 2, para. 2 and Figure 1), and Kim was able to map TRSs (page 916, column 1, para. 1 and Figure 2A). The examination of these sequences allowed for the determination of fusion and discontinuous transcription sites (Figure 3D-G), and it was noted that TRSs can affect sgRNA production (page 917, columns 1-2 joining para., for example).
Prior to the effective filing date of the claimed invention, it would have been prima facie obvious for one of ordinary skill in the art to use the teachings of Kim to examine additional SARS-CoV-2 genes with the method of Chappleboim in view of Lee, and in particular to analyze TRSs. As TRSs are important for viral transcription, the ordinary artisan would recognize that they would be valuable to study, particularly as their manipulation may lead to changes in sgRNA production or transcription that would render a virus weaker or non-functional, which would have implications for disease control and treatment. Though Chappleboim in view of Lee is focused on diagnostics, the ordinary artisan would also recognize the general utility in using Chappleboim in view of Lee as a sequencing method to analyze particular viral variants in individual patients, and targeting TRSs could provide information about disease prognosis for particular viral strains, as TSRs are vital to viral function. As Kim teaches that they were capable of sequencing TRSs with RNA sequencing, and specifically utilizes reverse transcription and PCR methods (see e3, para. 1), the ordinary artisan would have a reasonable expectation of success in designing primers in the method of Chappleboim in view of Lee capable of targeting TRSs. In particular, this would only involve altering the existing primer structure of Chappleboim in view of Lee to contain TRS-specific sequences in the target region of the primer – the other features of the primers, such as UMIs, would remain the same. As Kim teaches the discovery of TRS region sequences, the ordinary artisan would be capable of then incorporating those sequences (or their complements) into a primer.
Thus, claim 68 is prima facie obvious over Chappleboim, in view of Lee, and further in view of Kim.
Claim 71 is rejected under 35 U.S.C. 103 as being unpatentable over Chappleboim et al. (medRxiv, 2020; cited in Applicant’s IDS), in view of Lee (Dissertation, 2020), and further in view of Bibillo et al. (US 2019/0071711 A1).
Chappleboim in view of Lee teaches the methods of claims 1-2, 7, 9, 12, 14, 16, 21, 64-67, 69, and 70, as described above. Chappleboim generally teaches that their library preparation method is a multi-tube method – see pages 18-19, “Option 1: Purification and hybridization on PolyT beads” and “cDNA synthesis and Library preparation.” Neither Chappleboim nor Lee teaches the use of single-pot, open-tube chemistry for library preparation.
Bibillo teaches methods for amplifying and analyzing nucleic acid samples, particularly for creating cDNA libraries (Abstract). Specifically, the reference teaches various methods for single-pot library preparation (e.g. paras. 72, 74-75, 165, 207, and 212). In paras. 338-339, Example 4 shows that an RNA molecule was captured, reverse transcribed, and amplified in an open tube reaction. Para. 341 and Example 5 show a similar single-pot reaction that is specifically used for library preparation. Bibillo also specifically states that their methods are compatible with capture on a solid support, such as a bead (para. 224). Paras. 53-54 note advantages of their invention, including quick library preparation (which is noted to be useful in clinical settings), efficiency and simplicity, and high quality library preparation.
Prior to the effective filing date of the claimed invention, it would have been prima facie obvious for one of ordinary skill in the art to use the guidance provided by Bibillo in the method of Chappleboim in view of Lee to arrive at the method of claim 71. Specifically, Bibillo teaches that library preparation, with reverse transcription and amplification, can occur in a single tube, and also teaches that initial RNA capture methods can be added to this. In Chappleboim in view of Lee, RNA capture, reverse transcription, and amplification are performed, followed by sequencing. Though Chappleboim notes that samples can be pooled, the purpose of the invention generally is to rapidly detect SARS-CoV-2. Thus, the ordinary artisan would recognize the value in preparing a single sample in a single reaction tube, as this can all be performed in a clinical setting without needing to ship the sample to another location, saving time, thereby more quickly providing accurate and important information to patients. Such benefits are also explicitly stated by Bibillo. There would be a reasonable expectation of success because the same general library preparation steps are taught by Bibillo and Chappleboim in view of Lee.
Thus, claim 71 is prima facie obvious in view of Chappleboim, in view of Lee, and further in view of Bibillo.
Claim 73 is rejected under 35 U.S.C. 103 as being unpatentable over Chappleboim et al. (medRxiv, 2020; cited in Applicant’s IDS), in view of Lee (Dissertation, 2020), and further in view of Parker et al. (Journal of Applied Microbiology, 2010).
Chappleboim in view of Lee teaches the methods of claims 1-2, 7, 9, 12, 14, 16, 21, 64-67, 69, and 70, as described above. Chappleboim generally teaches that their library preparation method is a multi-tube method – see pages 18-19, “Option 1: Purification and hybridization on PolyT beads” and “cDNA synthesis and Library preparation. However, neither Chappleboim nor Lee teaches the use of MDA pre-amplification.
Parker teaches amplification of viral RNA from environmental samples (Abstract). The goal of the reference was to perform whole transcriptome amplification (WTA) via an initial round of MDA followed by PCR, followed by targeted qPCR (page 216, “Introduction” and page 217, “WTA reactions” and “qRT-PCR”). Generally, the reference teaches that this process allows for successful amplification of environmentally relevant amounts of RNA, and thus small amounts of pathogens (page 221, “TransPlex WTA for small quantities of pathogens”).
Prior to the effective filing date of the claimed invention, it would have been prima facie obvious for one of ordinary skill in the art to use the guidance provided by Parker to add a WTA MDA-preamplification step to the method of Chappleboim in view of Lee. Parker teaches this pre-amplification is compatible with downstream targeted PCR methods, and also teaches that this method can allow for detection of small quantities of viruses in samples. The ordinary artisan would thus be motivated to specifically use this pre-amplification in the context of Chappleboim in view of Lee because this would increase the detection rate of SARS-CoV-2 in a sample, even if the virus is present in very low amounts, which can lead to more accurate diagnostics and earlier patient intervention, potentially improving patient outcomes, particularly for patients who are at increased risk of disease complications. There would be a reasonable expectation of success because the WTA method taught by Parker is commercially available, and so the resources and protocols for using such pre-amplification are readily available.
Thus, claim 73 is prima facie obvious over Chappleboim, in view of Lee, and further in view of Parker.
Conclusion
No claims are currently allowable.
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/FRANCESCA FILIPPA GIAMMONA/Examiner, Art Unit 1681