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 .
DETAILED ACTION
Status of the Claims
Claims 1-5, 7, 11-13, 16-17, 19, 21-22, 24-26, 28-29 and 42 are pending and the subject of this NON-FINAL Office Action. This is the first action on the merits.
Claim Rejections - 35 USC § 102
The following is a quotation of the appropriate paragraphs of 35 U.S.C. § 102 that form the basis for the rejections under this section made in this Office action:
(A) A person shall be entitled to a patent unless –
(1)the claimed invention was patented, described in a printed publication, or in public use, on sale, or otherwise available to the public before the effective filing date of the claimed invention; or
(2) the claimed invention was described in a patent issued under section 151, or in an application for patent published or deemed published under section 122(b), in which the patent or application, as the case may be, names another inventor and was effectively filed before the effective filing date of the claimed invention.
Claims 1-5, 7, 11, 16-17, 19, 24-26, 29 and 42 are rejected under 35 U.S.C. § 102(a)(2) as being anticipated by HENIKOFF (US 20220214356).
As to claim 1, HENIKOFF teaches a method for multi-dimensional analysis of cell epigenomics, comprising the following steps: utilizing a ChiTag transposase (pA-Tn5; para. 1035) and a Tn5 transposase to respectively embed different adapter sequences in a cell (ATAC-Seq uses Tn5; para. 1035); and performing a co-analysis for information of an accessible-chromatin region and a target-protein binding sequence at the cellular level (“These results also suggest that by incubating with untethered Tn5 complexed with one set of adapters followed by pA-Tn5 complexed with a different set of adapters, and omitting high-salt treatments, both ITIS and ATAC-seq can be performed on the same sample”; para. 1035).
As to claim 2, HENIKOFF teaches wherein the method is a method A or a method B,
the method A is a method for multi-dimensional analysis of a single cell epigenomics (Abstract; Example 1, explaining the below protocols), comprising the following steps:
(A1) changing the permeability of a cell to be tested;
(A2) adding an antibody corresponding to the target protein to the cell after the treatment of the step (A1) for incubation;
(A3) adding a secondary antibody to the cell after the treatment of the step (A2) for incubation;
(A4) adding the ChiTag transposase to the cell after the treatment of the step (A3) for incubation;
(A5) adding a reaction reagent to the cell after the treatment of the step (A4) for incubation, and adding the Tn5 transposase after the incubation;
(A6) generating a droplet comprising a single cell with a water-in-oil structure and performing an amplification within the droplet after the reaction of the step (A5);
(A7) performing a demulsification, amplification and purification;
(A8) performing an enzyme digestion to obtain a final library; and
(A9) performing high-throughput sequencing on the final library obtained in the step (A8) to analyze the information of the accessible-chromatin region and the target-protein binding sequence at a single cellular level, and
the method B is a method for multi-dimensional analysis of multi-cell epigenomics, comprising the following steps:
(B1) changing the permeability of a cell to be tested;
(B2) adding an antibody corresponding to the target protein to the cell after the treatment of the step (B1) for incubation;
(B3) adding a secondary antibody to the cell after the treatment of the step (B2) for incubation;
(B4) adding the ChiTag transposase to the cell after the treatment of the step (B3) for incubation;
(B5) adding a reaction reagent to the cell after the treatment of the step (B4) for incubation, and adding the Tn5 transposase after the incubation;
(B6) performing an amplification and purification;
(B7) performing an enzyme digestion to obtaining a final library; and
(B8) performing high-throughput sequencing on the final library obtained in the step (B7) to analyze the information of the accessible-chromatin region and the target-protein binding sequence at a multiple cellular level.
As to claim 3, HENIKOFF teaches wherein in the steps (A1) and (B1), changing the permeability of the cell to be tested is achieved by resuspending the cell to be tested in a NP40-digoxin wash buffer, the NP40-digoxin wash buffer is obtained by adding 0.01% NP40 and 0.01% digoxin to a basic wash buffer, the basic wash buffer comprises: 20 mM HEPES in pH 7.5, 150 mM NaCl, 0.5 mM spermidine, and 1× protease inhibitor (Examples).
As to claim 4, HENIKOFF teaches each of the steps (A1) and (B1) comprises: resuspending 500,000 to 1,000,000 cells in 1 ml of the NP40-digoxin wash buffer; centrifuging the buffer and discarding a supernatant; and resuspending the cells by adding 49 μl of the NP40-digoxin wash buffer comprising 1.5-2.5 mM EDTA, and
in the steps (A2) and (B2), the antibody corresponding to the target protein is directly added to a cell resuspension at the second resuspension obtained in the steps (A1) and (B1) (Examples).
As to claim 5, HENIKOFF teaches in the steps (A2) and (B2), the antibody corresponding to the target protein is H3K27me3 antibody (Examples).
As to claim 7, HENIKOFF teaches each of the steps (A3) and (B3) further comprises steps of washing and centrifugation at at least one of the following: before adding the secondary antibody, or after the incubation, wherein the washing is performed with the NP40-digoxin wash buffer, and the centrifugation is performed at 600 g for 3 min (Examples).
As to claim 11, HENIKOFF teaches each of the steps (A4) and (B4) further comprise steps of centrifugation and cell resuspension sequentially before adding the ChiTag transposase, wherein the centrifugation is performed at 600 g for 3 min, and the cell resuspension is performed by resuspending the cell with a chitag enzyme incubation buffer,
the chitag enzyme incubation buffer comprises 0.01% (volume percentage) NP40; 0.01% digoxin, 20 mM HEPES in pH 7.5; 300 mM NaCl; 0.5 mM spermidine and 1× protease inhibitor (Examples).
As to claim 16, HENIKOFF teaches each of the steps (A3) and (B3) further comprises steps of centrifugation and washing after the incubation, wherein the centrifugation is performed at 600 g for 3 min, and the washing is performed with a chitag enzyme incubation buffer (Examples).
As to claim 17, HENIKOFF teaches in the steps (A5) and (B5), the reaction reagent is directly added to a cell precipitation obtained with the centrifugation in the step (A4),
wherein the reaction reagent is a chitag enzyme breaking buffer, the chitag enzyme breaking buffer is obtained by adding 10 mM of MgCl2 to a chitag enzyme incubation buffer (Examples).
As to claim 19, HENIKOFF teaches in the steps (A5) and (B5), the incubation is performed at 37° C. for 60 min,
wherein a centrifugation at 300 g for 3 min after the incubation is performed, and the Tn5 transposase is added and reacted at 37° C. with 500 rpm for 30 min (Examples).
As to claim 24, HENIKOFF teaches in the step (A6), primers for the amplification within the droplet comprise a Tn Primer and a 183+C Primer, wherein the Tn Primer is a single strand DNA as shown in SEQ ID No. 18, and the 183+C Primer is a single strand DNA as shown in SEQ ID No. 19 (Examples).
As to claim 25, HENIKOFF teaches in the step (A7), primers for the amplification comprise the Tn Primer and a 183-pho Primer, wherein the Tn Primer is the single strand DNA as shown in SEQ ID No. 18, and the 183-pho Primer is a single strand DNA modified with a phosphate group at the 5′ end as shown in SEQ ID No. 20 (Examples).
As to claim 26, HENIKOFF teaches in the step (A7), the purification is performed by adding 1.2 volumes of magnetic beads to a product of the amplification (Examples).
As to claim 29, HENIKOFF teaches each of the steps (A8) and (B7) further comprises a cyclization before the enzyme digestion,
the cyclization is performed with a 153+181 Splint oligo, wherein the 153+181 splint oligo is a single strand DNA as shown in SEQ ID No. 21 (Examples).
As to claim 42, HENIKOFF teaches method for any one of:
(C1) researching cell population heterogeneity related to a development and/or a disease (para. 0117, 0155);
(C2) drawing a cell atlas (Example 7);
(C3) analyzing tumor cells with different clinical features (para. 0117, 0155); or
(C4) studying an evolution and/or metastasis of a tumor cell clinically,
wherein the method comprising the following steps:
utilizing a ChiTag transposase and a Tn5 transposase to respectively embed different adapter sequences in a cell; and performing a co-analysis for information of an accessible-chromatin region and a target-protein binding sequence at the cellular level (para. 1035).
Allowable Subject Matter
Claims 12-13, 21-22 and 28 are objected to as being dependent upon a rejected base claim, but would be allowable if rewritten in independent form including all of the limitations of the base claim and any intervening claims. The Examiner cannot find the specific sequences claimed.
Conclusion
No claims are allowed.
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/YUNG-SHENG M TSUI/ Primary Examiner, Art Unit 1743