CN115029343A - Nucleic acid extraction and purification reagent - Google Patents
Nucleic acid extraction and purification reagent Download PDFInfo
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- CN115029343A CN115029343A CN202210633617.1A CN202210633617A CN115029343A CN 115029343 A CN115029343 A CN 115029343A CN 202210633617 A CN202210633617 A CN 202210633617A CN 115029343 A CN115029343 A CN 115029343A
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- nucleic acid
- reagent
- extracting
- fixed connection
- detergent
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- 102000039446 nucleic acids Human genes 0.000 title claims abstract description 39
- 108020004707 nucleic acids Proteins 0.000 title claims abstract description 39
- 150000007523 nucleic acids Chemical class 0.000 title claims abstract description 39
- 239000003153 chemical reaction reagent Substances 0.000 title claims abstract description 23
- 238000000605 extraction Methods 0.000 title claims abstract description 19
- 238000000746 purification Methods 0.000 title claims abstract description 12
- 239000003599 detergent Substances 0.000 claims abstract description 15
- 238000010828 elution Methods 0.000 claims abstract description 15
- 239000007790 solid phase Substances 0.000 claims abstract description 15
- 239000000463 material Substances 0.000 claims abstract description 13
- QGZKDVFQNNGYKY-UHFFFAOYSA-O Ammonium Chemical compound [NH4+] QGZKDVFQNNGYKY-UHFFFAOYSA-O 0.000 claims description 4
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims description 3
- LZZYPRNAOMGNLH-UHFFFAOYSA-M Cetrimonium bromide Chemical compound [Br-].CCCCCCCCCCCCCCCC[N+](C)(C)C LZZYPRNAOMGNLH-UHFFFAOYSA-M 0.000 claims description 3
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 claims description 3
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 3
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 claims description 3
- 229960002233 benzalkonium bromide Drugs 0.000 claims description 3
- 229960000686 benzalkonium chloride Drugs 0.000 claims description 3
- KHSLHYAUZSPBIU-UHFFFAOYSA-M benzododecinium bromide Chemical compound [Br-].CCCCCCCCCCCC[N+](C)(C)CC1=CC=CC=C1 KHSLHYAUZSPBIU-UHFFFAOYSA-M 0.000 claims description 3
- CADWTSSKOVRVJC-UHFFFAOYSA-N benzyl(dimethyl)azanium;chloride Chemical compound [Cl-].C[NH+](C)CC1=CC=CC=C1 CADWTSSKOVRVJC-UHFFFAOYSA-N 0.000 claims description 3
- 229960003964 deoxycholic acid Drugs 0.000 claims description 3
- KXGVEGMKQFWNSR-LLQZFEROSA-N deoxycholic acid Chemical compound C([C@H]1CC2)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(O)=O)C)[C@@]2(C)[C@@H](O)C1 KXGVEGMKQFWNSR-LLQZFEROSA-N 0.000 claims description 3
- UMWKZHPREXJQGR-UHFFFAOYSA-N n-methyl-n-(2,3,4,5,6-pentahydroxyhexyl)decanamide Chemical compound CCCCCCCCCC(=O)N(C)CC(O)C(O)C(O)C(O)CO UMWKZHPREXJQGR-UHFFFAOYSA-N 0.000 claims description 3
- -1 ammonium ions Chemical class 0.000 abstract description 5
- 230000009471 action Effects 0.000 abstract description 2
- 238000005336 cracking Methods 0.000 abstract description 2
- 239000011324 bead Substances 0.000 description 7
- 239000000243 solution Substances 0.000 description 6
- 238000004140 cleaning Methods 0.000 description 5
- 239000007788 liquid Substances 0.000 description 4
- 239000012491 analyte Substances 0.000 description 3
- 238000001179 sorption measurement Methods 0.000 description 3
- 210000000170 cell membrane Anatomy 0.000 description 2
- 239000003480 eluent Substances 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 230000006037 cell lysis Effects 0.000 description 1
- 230000009089 cytolysis Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 238000002414 normal-phase solid-phase extraction Methods 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 239000000741 silica gel Substances 0.000 description 1
- 229910002027 silica gel Inorganic materials 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 238000007864 suspending Methods 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
- C12N15/1006—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers
- C12N15/1013—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers by using magnetic beads
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- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Biomedical Technology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Crystallography & Structural Chemistry (AREA)
- Plant Pathology (AREA)
- Molecular Biology (AREA)
- Microbiology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Analytical Chemistry (AREA)
- Saccharide Compounds (AREA)
Abstract
The invention discloses a nucleic acid extraction and purification reagent, which comprises base solution, ammonium ions, a detergent and a solid phase material, wherein an elution device separates and obtains purified nucleic acid from the solid phase material. The invention leads the cells to be fully cracked through the combined action of the detergent and the ammonium ions and keeps the stability of the nucleic acid in the cracking mixed liquor; the extraction efficiency of nucleic acid is improved by a special elution device.
Description
Technical Field
The invention relates to the technical field of biochemistry, in particular to a nucleic acid extraction and purification reagent.
Background
Solid phase extraction is a physical extraction process involving a liquid phase and a solid phase, in which the solid phase has a greater adsorption force on the analyte than the sample base solution, and when the sample contacts the solid phase adsorption material, the analyte is adsorbed on the surface of the solid phase adsorption material, and then the analyte is eluted with a suitable solvent.
The existing extraction reagent is low in efficiency of extracting nucleic acid through a washing step, protein is denatured, then cell membranes are ruptured, released nucleic acid is adsorbed and combined with silica gel membranes or magnetic beads in a high-temperature environment.
Disclosure of Invention
The invention aims to solve the defects in the prior art and provides a nucleic acid extraction and purification reagent so as to solve the problems in the prior art.
In order to achieve the purpose, the invention adopts the following technical scheme:
a nucleic acid extraction and purification reagent comprises base solution, ammonium ion, detergent and solid phase material, wherein the base solution is 85-95%, the ammonium ion is 0.1-3%, and the detergent is 1-6%.
Preferably, the detergent is selected from one or more of sodium dodecyl sulfate, sodium deoxycholate, cetyl trimethyl ammonium bromide, and N-decanoyl-N-methylglucamine.
Preferably, the detergent is one or two of benzalkonium chloride and benzalkonium bromide.
Preferably, the base solution is 30-40mmol/L Tris-HCl, 170-180mmol/L NaCL, 1.5-3mmol/LPMSF and 1.5-3mmol/L EDTA.
Preferably, after the nucleic acid released from the extraction and purification reagent is bound to the solid phase material, the purified nucleic acid is separated from the solid phase material by the elution device.
Preferably, the elution device includes the box, the box lateral wall is equipped with the guide way, sliding connection guide block in the guide way, guide block one side fixed connection diaphragm, the first motor of diaphragm upside fixed connection, the main shaft fixed connection extension board of first motor, extension board bottom symmetry sets up two electromagnetic rods, two layer boards of bottom plate upside central symmetry formula sliding connection of box, the interval sets up a plurality of mount pads on every layer board.
Preferably, the opposite sides of the two supporting plates are fixedly connected with toothed plates respectively, the bottom of the box body is fixedly connected with a second motor, a main shaft of the second motor is fixedly connected with a gear, and the gear is located between the two toothed plates and meshed with the two toothed plates.
Preferably, the bottom plate of the box body is provided with a sliding groove, the bottom of the supporting plate is fixedly connected with a sliding block, and the sliding block is in sliding connection with the sliding groove.
Preferably, the length of the supporting plate is twice of the length of the sliding chute.
The invention has the advantages that: the nucleic acid extraction and purification reagent provided by the invention can fully lyse cells through the combined action of the detergent and ammonium ions, and keep the stability of nucleic acid in a lysis mixed solution; the extraction efficiency of nucleic acid is improved by a special elution device.
Drawings
FIG. 1 is a schematic diagram of the basic structure of the present invention;
FIG. 2 is an enlarged view of a portion of FIG. 1 at F;
fig. 3 is a schematic view of a connection structure of the pallet and the box body according to the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and do not limit the invention.
Example 1
The invention provides a nucleic acid extraction and purification reagent which comprises base liquid, ammonium ions, a detergent and a solid phase material, wherein the base liquid is 95%, the ammonium ions are 2%, and the detergent is 3%.
The detergent is selected from one or more of sodium dodecyl sulfate, sodium deoxycholate, cetyl trimethyl ammonium bromide, and N-decanoyl-N-methylglucamine, and can be used for cracking cytoplasmic membrane.
The detergent is one or the combination of benzalkonium chloride and benzalkonium bromide.
The base solution is 40mmol/L Tris-HCl, 180mmol/L NaCL, 12mmol/LPMSF and 2mmol/L EDTA, and is used for cell lysis and convenient release of nucleic acid.
After the nucleic acid released from the extraction and purification reagent is combined with the solid phase material, the nucleic acid is separated from the solid phase material through an elution device to obtain purified nucleic acid.
Respectively extracting 3 swab rinsing liquid samples, wherein the volume of each reagent and sample is 260 mu L, placing the samples into a 2ml centrifuge tube, adding 40 mu L of plastic magnetic beads into each sample, mixing the samples in a vortex, and placing the mixture for 15min at room temperature.
② placing the centrifuge tube in an elution device for nucleic acid extraction, and suspending and mixing the magnetic beads in the liquid once every 2 min.
As shown in fig. 1-3, the elution device includes a box 1, a guide groove 11 is formed in a side wall of the box 1, a guide block 12 is slidably connected in the guide groove 11, the guide block 12 is driven by a driving part to slide in the guide groove 11, the driving part is an electric push rod, a transverse plate 2 is fixedly connected to one side of the guide block 12, a first motor 21 is fixedly connected to the upper side of the transverse plate 2, a main shaft of the first motor 21 is fixedly connected to a support plate 22, and two electromagnetic rods 23 are symmetrically arranged at the bottom of the support plate 22.
The inner wall fixed connection power of box 1, the power is connected through the spring cable with electromagnetic rod 23, and the spring cable has flexible adaptability, and the structure of terminal box, vibrating motor, closing plate and the water proof membrane that the electromagnetic rod 23 outside set up refers to the structure that the laboratory nucleic acid of bulletin number CN 213803797U draws the elution device of instrument and discloses.
The upper side of a bottom plate of the box body 1 is centrally and symmetrically connected with two supporting plates 3 in a sliding mode, a plurality of mounting seats 4 are arranged on each supporting plate 3 at intervals, mounting holes are formed in the upper sides of the mounting seats 4, anti-slip rubber layers are arranged on the inner walls of the mounting holes, one row of elution cups are clamped in the mounting seat 4 on one supporting plate 3, one row of cleaning cups are clamped in the mounting seat 4 on the other supporting plate 3, an electromagnetic rod 23 vibrates and accelerates the movement of eluent in the elution cups, so that magnetic beads and the eluent are fully and uniformly mixed, and the combined nucleic acid is separated from the magnetic beads, so that the purified nucleic acid is obtained; the electromagnetic rod 23 generates magnetic force to adsorb magnetic beads without nucleic acid after being electrified, the electric push rod lifts the electromagnetic rod 23, the electromagnetic rod 23 is put into the cleaning cup by rotating 180 degrees, the magnetic beads are released by power failure, and the electromagnetic rod 23 is cleaned; after cleaning, the device rotates 180 degrees and is matched with the opposite movement of a plurality of groups of elution cups and cleaning cups, so that the separation of nucleic acid can be completed in batches, and the extraction efficiency is improved.
Opposite sides of the two supporting plates 3 are respectively fixedly connected with toothed plates 31, the bottom of the box body 1 is fixedly connected with a second motor 32, a main shaft of the second motor 32 is fixedly connected with a gear 33, and the gear 33 is positioned between the two toothed plates 31 and meshed with the two toothed plates 31; the gear 33 rotates to make the two support plates 3 move oppositely, so that different elution cups are matched with the cleaning cups to form a group, thereby facilitating the batch completion of nucleic acid separation and improving the extraction efficiency.
The bottom plate of box 1 sets up spout 34, the bottom fixed connection slider 35 of layer board 3, slider 35 and spout 34 sliding connection, the length of layer board 3 is the twice of spout 34 length, can cover spout 34 all the time when layer board 3 removes, avoids debris to fall into and causes the obstacle in spout 34.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (9)
1. A nucleic acid extraction and purification reagent comprises base solution, ammonium ion, detergent and solid phase material, wherein the base solution is 85-95%, the ammonium ion is 0.1-3%, and the detergent is 1-6%.
2. The reagent for extracting and purifying nucleic acid according to claim 1, wherein: the detergent is selected from one or more of sodium dodecyl sulfate, sodium deoxycholate, cetyl trimethyl ammonium bromide and N-decanoyl-N-methylglucamine.
3. The reagent for extracting and purifying nucleic acid according to claim 1, wherein: the detergent is one or the combination of benzalkonium chloride and benzalkonium bromide.
4. The reagent for extracting and purifying nucleic acid according to claim 1, wherein: the base solution is 30-40mmol/L Tris-HCl, 170-180mmol/L NaCL, 1.5-3mmol/LPMSF and 1.5-3mmol/L EDTA.
5. The reagent for extracting and purifying nucleic acid according to claim 1, wherein: and after the nucleic acid released from the extraction and purification reagent is combined with the solid phase material, separating the nucleic acid from the solid phase material through an elution device to obtain the purified nucleic acid.
6. The reagent for extracting and purifying nucleic acid according to claim 5, wherein: the elution device includes box (1), box (1) lateral wall is equipped with guide way (11), sliding connection guide block (12) in guide way (11), guide block (12) one side fixed connection diaphragm (2), the first motor of diaphragm (2) upside fixed connection (21), main shaft fixed connection extension board (22) of first motor (21), extension board (22) bottom symmetry sets up two electromagnetic rod (23), two layer board (3) of bottom plate upside central symmetry formula sliding connection of box (1), every layer board (3) go up the interval and set up a plurality of mount pads (4).
7. The reagent for extracting and purifying nucleic acid according to claim 6, wherein: two opposite sides of layer board (3) are fixed connection pinion rack (31) respectively, box (1) bottom fixed connection second motor (32), main shaft fixed connection gear (33) of second motor (32), gear (33) are located between two pinion racks (31) and mesh with two pinion racks (31).
8. The reagent of claim 7, wherein the reagent comprises: the bottom plate of box (1) sets up spout (34), the bottom fixed connection slider (35) of layer board (3), slider (35) and spout (34) sliding connection.
9. The reagent for extracting and purifying nucleic acid according to claim 8, wherein: the length of the supporting plate (3) is twice of that of the sliding chute (34).
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210633617.1A CN115029343A (en) | 2022-06-06 | 2022-06-06 | Nucleic acid extraction and purification reagent |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210633617.1A CN115029343A (en) | 2022-06-06 | 2022-06-06 | Nucleic acid extraction and purification reagent |
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| Publication Number | Publication Date |
|---|---|
| CN115029343A true CN115029343A (en) | 2022-09-09 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210633617.1A Pending CN115029343A (en) | 2022-06-06 | 2022-06-06 | Nucleic acid extraction and purification reagent |
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| Country | Link |
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| CN (1) | CN115029343A (en) |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6921817B1 (en) * | 1999-08-05 | 2005-07-26 | Ranjit Banerjee | Methods for simultaneous isolation of biologically active transcription factors and DNA |
| CN102533724A (en) * | 2010-12-30 | 2012-07-04 | 上海复星医学科技发展有限公司 | Cell lysis reagent for extracting and purifying nucleic acids in biological samples |
| US20210222229A1 (en) * | 2018-03-02 | 2021-07-22 | Quantumcyte, Inc. | Methods, compositions, and devices for isolation and expression analysis of regions of interest from a tissue |
| CN213803797U (en) * | 2020-10-12 | 2021-07-27 | 苏州盈玛精密机械有限公司 | Elution device of nucleic acid extraction instrument for laboratory |
-
2022
- 2022-06-06 CN CN202210633617.1A patent/CN115029343A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6921817B1 (en) * | 1999-08-05 | 2005-07-26 | Ranjit Banerjee | Methods for simultaneous isolation of biologically active transcription factors and DNA |
| CN102533724A (en) * | 2010-12-30 | 2012-07-04 | 上海复星医学科技发展有限公司 | Cell lysis reagent for extracting and purifying nucleic acids in biological samples |
| US20210222229A1 (en) * | 2018-03-02 | 2021-07-22 | Quantumcyte, Inc. | Methods, compositions, and devices for isolation and expression analysis of regions of interest from a tissue |
| CN213803797U (en) * | 2020-10-12 | 2021-07-27 | 苏州盈玛精密机械有限公司 | Elution device of nucleic acid extraction instrument for laboratory |
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