CN114277103A - Efficient rolling circle amplification method based on hexamer random primer - Google Patents
Efficient rolling circle amplification method based on hexamer random primer Download PDFInfo
- Publication number
- CN114277103A CN114277103A CN202210072066.6A CN202210072066A CN114277103A CN 114277103 A CN114277103 A CN 114277103A CN 202210072066 A CN202210072066 A CN 202210072066A CN 114277103 A CN114277103 A CN 114277103A
- Authority
- CN
- China
- Prior art keywords
- rolling circle
- dna
- amplification
- circle amplification
- hexamer
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Abstract
The invention discloses a high-efficiency rolling circle amplification method based on hexamer random primers, relates to the field of molecular biology, relates to the field of nucleic acid amplification and biological detection, and comprises the following steps; carrying out sulfo-modification on the single-stranded DNA, and then carrying out cyclization; contacting single-stranded circular DNA with a reaction solution comprising a DNA polymerase, a molecular crowding agent, deoxyribonucleoside triphosphates, and a primer having a 3 'end and a 5' end; the single-stranded circular DNA is amplified through rolling circle amplification to form an amplified DNA product, and the method comprises design of a hexamer random primer, preparation of a rapid detection system and a one-tube amplification detection method.
Description
Technical Field
The invention relates to the field of molecular biology, relates to the field of nucleic acid amplification and biological detection, and particularly relates to a high-efficiency rolling circle amplification method based on a hexamer random primer.
Background
Rolling circle amplification is a simple isothermal DNA amplification technique that produces thousands of repeats by amplifying closed circular templates catalyzed by DNA polymerase (Phi 29). Compared with temperature-variable nucleic acid amplification technologies such as polymerase chain reaction, RCA does not need expensive temperature-variable instruments, and is more suitable for field detection. RCA uses the mode of copying circular DNA of microbial species for reference. Amplification was performed with phi29DNA polymerase at about 30-37 ℃. The process of ordinary RCA is: the primer is bound to the circular DNA template and then extended to form a single DNA strand containing a large amount of the target gene. On the basis, various improved technologies such as multi-primer RCA for multi-site simultaneous amplification and exponential RCA for improving amplification efficiency by using two primers have been developed in recent years, and RCA is also very wide in application, and can be combined with complementary oligonucleotides through RCA amplification products, including fluorescent dyes, electrochemical tags, biotin, antibodies, enzymes and nanoparticles.
Although RCA amplification is widely applied in the field of DNA and RNA detection. But the amplification efficiency is still insufficient, a specific primer is often needed to trigger an amplification reaction, the requirement of a user cannot be met, the random primer is used for combining phi29DNA polymerase to amplify the circular DNA template, the visible amplification amount of gel can be achieved within 10-30 minutes, the amplification lasts for 2 hours, and the RCA amplification product can reach flocculent gel.
Disclosure of Invention
The invention aims to provide a high-efficiency rolling circle amplification method based on a hexamer random primer, which has the effects that the primer is designed into a non-specific primer, the hexamer random primer can be combined with any site of a circular template theoretically so as to initiate high-efficiency amplification reaction, the sensitivity is high, and the requirement on a sample is low, and the problems that although RCA amplification is widely applied in the field of DNA and RNA detection, the RCA amplification is still insufficient in the aspect of amplification efficiency, and the specific primer is often needed to trigger the amplification reaction, so that the requirement of a user cannot be met are solved.
In order to achieve the purpose, the invention provides the following technical scheme: a high-efficiency rolling circle amplification method based on hexamer random primers comprises the following steps:
step S1: carrying out sulfo modification on the single-stranded DNA, and then carrying out cyclization on the single-stranded DNA to obtain sulfo-modified single-stranded circular DNA;
step S2: contacting single-stranded circular DNA with a reaction solution comprising a DNA polymerase, a molecular crowding agent, deoxyribonucleoside triphosphates, and a primer having a 3 'end and a 5' end;
step S3: amplifying the single-stranded circular DNA via rolling circle amplification to form an amplified DNA product, the rolling circle amplification temperature being 30 ℃ and the amplification time being 1 hour.
Optionally, the primer in step S2 is a hexamer having the general structure of (atN) (atN) (atN) (atN) (atN) × N, wherein (atN) is the 5 'terminal nucleotide and × N is the 3' terminal nucleotide of the hexamer, and wherein "N" represents deoxyadenosine (dA), deoxycytidine (dC), deoxyguanosine (dG) or deoxythymidine (dT), (atN) represents a random mixture of 2-amino-dA, dC, dG and 2-thio-dT, and "×" represents a phosphorothioate bond.
Optionally, the molecular crowding reagent in step S2 is selected from PEG400, PEG2000, PEG6000, PEG8000 or their combination, and the DNA polymerase is Phi29DNA polymerase.
Optionally, in the step S1, the concentration of the single-stranded circular DNA is 5 to 50nM, and the length of the single-stranded DNA is 30 to 90 nt.
Optionally, the rolling circle amplification in step S3 includes a sulfur-containing modified hexamer random primer of seq id No.1, RCA reaction buffer composition: 10mM Tris-HCl, 2mM MgCl2, 2mM (NH4)2SO4, 0.8mM DTT, 2mM dNTP, 4mg/ml BSA, 2uM6Ns (pH7.5@25 ℃ C.).
Optionally, the method also comprises a one-tube efficient detection method for cyclization, amplification and detection, which comprises preparation of different buffer components, pH, and time for cyclization, amplification and detection.
Optionally, the method includes an integrated bufferMix system 2 × oneptofbuffermix: 10mM Tris-HCl, 2mM MgCl2, 2mM (NH4)2SO4, 0.8mM DTT, 2mM dNTP, 4mg/ml BSA, 2uM6Ns, 120. mu.l MATP (pH7.5@25 ℃); the integrated detection time is 1.5 h; the procedure for amplification was step 1: 1min at 37 ℃; step 2: 30s at 37 ℃; step 3: 30s (one fluorescence signal was collected) at 37 ℃ and gotostep2 for 90 cycles, and the negative control was sterile ultradian water.
Optionally, in step S2, the single-stranded circular DNA is denatured, and the denatured system contains EDTA at a concentration of 0.05 mM; the denaturation treatment consists of heating the single-stranded circular DNA to 95 ℃ for 3 minutes, and then cooling to 4 ℃ and holding.
Optionally, in the step S3, after the rolling circle amplification is finished, the amplification product is treated with BamH1-HF endonuclease at 37 ℃ for 1 hour.
Compared with the prior art, the invention has the following beneficial effects:
compared with the traditional rolling circle amplification technology, the method has the advantages of high sensitivity, high amplification efficiency and low requirements on samples, and is particularly significant to low copy number and trace samples.
Secondly, the primers are designed to be non-specific primers, and the hexamer random primer can be theoretically combined with any site of the circular template, so that the high-efficiency amplification reaction is initiated.
And thirdly, the single-chain circular DNA is denatured, the high temperature in the denaturation process can ensure the full release of the initial template DNA, and the random primer and the circular DNA template can be annealed and combined in the cooling process.
Drawings
FIG. 1 is a schematic diagram of a hexamer rolling circle amplification of the present invention;
FIG. 2 is a probe sequence table of APACE detection primers of the present invention;
FIG. 3 is an alignment of MCRCA amplification and 6NRCA amplification products in an example of the invention; 1 indicates MCRCA, 2 indicates 6NRCA, 3 positive, 4 indicates ddH 2O;
FIG. 4 is a fluorescent detection scheme of the MCRCA and 6NRCA amplification products of the present invention;
FIG. 5 is a diagram of the detection of one tube type rolling circle amplification combined with CRISPR-FnCpf 1.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
In the following examples, the specific experimental conditions were performed according to the reference in the art or the instructions for the use of the kit, and the instruments and consumables used therein were obtained from the commercial sources according to the laboratory conditions of the manufacturers, unless otherwise specified.
Referring to fig. 1 to 5, the present embodiment provides a hexamer random primer-based high efficiency rolling circle amplification method, which comprises the following steps:
step S1: carrying out sulfo modification on the single-stranded DNA, and then carrying out cyclization on the single-stranded DNA to obtain sulfo-modified single-stranded circular DNA;
step S2: contacting single-stranded circular DNA with a reaction solution comprising a DNA polymerase, a molecular crowding agent, deoxyribonucleoside triphosphates, and a primer having a 3 'end and a 5' end;
step S3: amplifying the single-stranded circular DNA via rolling circle amplification to form an amplified DNA product, the rolling circle amplification temperature being 30 ℃ and the amplification time being 1 hour.
Further, the primer in step S2 is a hexamer having a general structure of (atN) (atN) (atN) (atN) (atN) N, wherein (atN) is the 5 'nucleotide and N is the 3' nucleotide of the hexamer, and wherein "N" represents deoxyadenosine (dA), deoxycytidine (dC), deoxyguanosine (dG) or deoxythymidine (dT), (atN) represents a random mixture of 2-amino-dA, dC, dG and 2-thio-dT, and ". sup." represents a phosphorothioate linkage, wherein the incorporation of 2-amino-deoxyadenosine increases the melting temperature of the primer, and the nucleotide analogue 2-thio-deoxythymidine, wherein incorporation of the nucleotide analogue 2-amino-deoxyadenosine and the nucleotide analogue 2-thio-deoxythymidine in the primer prevents primer dimer formation.
Further, in step S2, the molecular crowding reagent is selected from PEG400, PEG2000, PEG6000, PEG8000 or their combination, the DNA polymerase is Phi29DNA polymerase, and the specific Phi29DNA polymerase can highly amplify DNA by using phage Phi29DNA polymerase, firstly, the random primer anneals with the template DNA at multiple sites, then the Phi29DNA polymerase simultaneously initiates replication at multiple sites of DNA, it synthesizes DNA along the DNA template, at the same time, replaces the complementary strand of the template, and the replaced complementary strand becomes a new template for amplification, thereby obtaining a large amount of high molecular weight DNA.
Further, in the step S1, the concentration of the single-stranded circular DNA is 5-50 nM, and the length of the single-stranded DNA is 30-90 nt.
Further, the rolling circle amplification in step S3 includes a sulfur-containing modified hexamer random primer of seq id No.1, RCA reaction buffer composition: 10mM Tris-HCl, 2mM MgCl2, 2mM (NH4)2SO4, 0.8mM DTT, 2mM dNTP, 4mg/ml BSA, 2uM6Ns (pH7.5@25 ℃ C.).
Further, the method also comprises a one-tube efficient detection method for cyclization, amplification and detection, which comprises the steps of preparation of different buffer components, pH, and time for cyclization, amplification and detection.
Further, the method comprises an integrated bufferMix system 2 × oneptofbuffermix: 10mM Tris-HCl, 2mM MgCl2, 2mM (NH4)2SO4, 0.8mM DTT, 2mM dNTP, 4mg/ml BSA, 2uM6Ns, 120. mu.l MATP (pH7.5@25 ℃); the integrated detection time is 1.5 h; the procedure for amplification was step 1: 1min at 37 ℃; step 2: 30s at 37 ℃; step 3: 30s (one fluorescence signal was collected) at 37 ℃ and gotostep2 for 90 cycles, and the negative control was sterile ultradian water.
Further, in step S2, the single-stranded circular DNA is denatured with EDTA at a concentration of 0.05 mM; the denaturation treatment comprises heating the single-chain circular DNA to 95 ℃ for 3 minutes, then cooling to 4 ℃ and keeping, specifically, the single-chain circular DNA is subjected to denaturation treatment, the high temperature in the denaturation treatment process can ensure the full release of the initial template DNA, and the cooling process can enable the random primer and the circular DNA template to be annealed and combined.
Further, after the rolling circle amplification is finished, the amplification product is treated with BamH1-HF endonuclease at 37 ℃ for 1 hour in step S3, and the BamH1-HF endonuclease is used for the construction of the DNA genome physical map by treating the amplification product with BamH1-HF endonuclease; positioning and separating genes; the DNA molecule base sequence analysis can improve the defense capability of the amplification product.
The experimental reagents and instruments used in this example were as follows:
the instrument comprises the following steps: 7900 HTFastclean-TimeRCR system.
Consumable material: CRISPR-FnCpf1 nuclease, T4DNAligase, Phi29polymerase, 6N(s), Probe1, Pobe2, quantitative PCR sealing membrane (purchased from BIO-RAD), MCRCA commercial kit (iCloud publication).
1. T4DNA cyclization
The ssDNA circularization target template and the lock probe were diluted with TEbuffer to concentrations of 10. mu.M each, and the specific and non-specific probes were diluted with DNAse/RNAse-free deionized water to concentrations of 10. mu.M. The amount of the DNA template used for the lock-key probe was 20pmol, the ssDNA target was 20pmol, T4 DNAlagase 20U, 2. mu.l 1 XT 4 DNAlagassebuffer, the circularization temperature was 37 ℃ and the time was 30 min.
2. Phi29DNA random primer amplification
The amplification system contains a sulfur-modified hexamer random primer SEQIDNO.1, the primer can be combined with any site in a cyclization product to carry out exponential amplification, the RCA amplification temperature is 30 ℃, the time is 1h, and the electrophoresis detection result of the amplified product is shown in figure 1.
3. Fluorescence detection
Taking 1. mu.l of the amplified product to be used for CRISPR-Cpf1 in-vitro shearing reaction, wherein the system is as follows: mu.l FnCpf1(1000 ng/. mu.l), 1. mu.l rRNA (100 ng/. mu.l), 1. mu.l amplification product, 2. mu.l NEBbufferr2.1, 1. mu.l Probe1, 1. mu.l Probe2, 13. mu.lddH 2O, fluorescence detection using the 7900 HTFastFastRal-TimeRCR system, the program settings are as follows: step 1: 1min at 37 ℃; step 2: 30s at 37 ℃; step 3: 30s (one fluorescence signal was collected) at 37 ℃ and gotostep2 for 90 cycles, and the negative control was sterile ultradian water.
4. Analyzing the results of the detection
In the process of result determination, if there is an amplification curve and the fluorescence yield >10000 belongs to the detection of the substrate containing the template, if the fluorescence yield >5000 and less than 10000, this is considered to be the reason that the template concentration is too low, or the probe concentration is low, etc., the experiment should be repeated by increasing the template or the concentration and increasing the sample to a proper concentration, and if the fluorescence yield of the negative control >5000 indicates that the negative background value is too high, the negative should be prepared newly.
The invention combines rolling circle amplification with CRISPR-FnCpf1 detection, realizes DNA cyclization by taking RNA as a template through testing the RNA template, and obtains a specific probe for detecting SARS-CoV-2E gene target.
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 high-efficiency rolling circle amplification method based on hexamer random primers comprises the following steps:
step S1: carrying out sulfo modification on the single-stranded DNA, and then carrying out cyclization on the single-stranded DNA to obtain sulfo-modified single-stranded circular DNA;
step S2: contacting single-stranded circular DNA with a reaction solution comprising a DNA polymerase, a molecular crowding agent, deoxyribonucleoside triphosphates, and a primer having a 3 'end and a 5' end;
step S3: amplifying the single-stranded circular DNA via rolling circle amplification to form an amplified DNA product, the rolling circle amplification temperature being 30 ℃ and the amplification time being 1 hour.
2. The hexamer random primer based high efficiency rolling circle amplification method of claim 1, wherein: the primer in step S2 is a hexamer having the general structure of (atN) (atN) (atN) (atN) (atN) × N, wherein (atN) is the 5 'terminal nucleotide of the hexamer and × N is the 3' terminal nucleotide of the hexamer, and wherein "N" represents deoxyadenosine (dA), deoxycytidine (dC), deoxyguanosine (dG) or deoxythymidine (dT), (atN) represents a random mixture of 2-amino-dA, dC, dG and 2-thio-dT, and "×" represents a phosphorothioate bond.
3. The hexamer random primer based high efficiency rolling circle amplification method of claim 1, wherein: the molecular crowding reagent in the step S2 is selected from PEG400, PEG2000, PEG6000, PEG8000 or their combination, and the DNA polymerase is Phi29DNA polymerase.
4. The hexamer random primer based high efficiency rolling circle amplification method of claim 1, wherein: the concentration of the single-stranded circular DNA in the step S1 is 5-50 nM, and the length of the single-stranded DNA is 30-90 nt.
5. The hexamer random primer based high efficiency rolling circle amplification method of claim 1, wherein: the rolling circle amplification in the step S3 comprises a sulfur-containing modified hexamer random primer SEQ ID NO.1, and the RCA reaction buffer composition: 10mM Tris-HCl, 2mM MgCl2, 2mM (NH4)2SO4, 0.8mM DTT, 2mM dNTP, 4mg/ml BSA, 2uM6Ns (pH7.5@25 ℃ C.).
6. The hexamer random primer based high efficiency rolling circle amplification method of claim 1, wherein: also comprises a one-tube efficient detection method for cyclization, amplification and detection, which comprises the steps of preparation of different buffer components, pH, and time for cyclization, amplification and detection.
7. The method for circularization, amplification, and detection of one-tube high efficiency according to claim 6, wherein: the method comprises an integrated bufferMix system 2 multiplied by oneptofbuffermix: 10mM Tris-HCl, 2mM MgCl2, 2mM (NH4)2SO4, 0.8mM DTT, 2mM dNTP, 4mg/ml BSA, 2uM6Ns, 120. mu.l MATP (pH7.5@25 ℃); the integrated detection time is 1.5 h; the procedure for amplification was step 1: 1min at 37 ℃; step 2: 30s at 37 ℃; step 3: 30s (one fluorescence signal was collected) at 37 ℃ and gotostep2 for 90 cycles, and the negative control was sterile ultradian water.
8. The hexamer random primer based high efficiency rolling circle amplification method of claim 1, wherein: in step S2, the single-stranded circular DNA is denatured with EDTA at a concentration of 0.05 mM; the denaturation treatment consists of heating the single-stranded circular DNA to 95 ℃ for 3 minutes, and then cooling to 4 ℃ and holding.
9. The hexamer random primer based high efficiency rolling circle amplification method of claim 1, wherein: after the rolling circle amplification was completed in step S3, the amplification product was treated with BamH1-HF endonuclease at 37 ℃ for 1 hour.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210072066.6A CN114277103A (en) | 2022-01-21 | 2022-01-21 | Efficient rolling circle amplification method based on hexamer random primer |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210072066.6A CN114277103A (en) | 2022-01-21 | 2022-01-21 | Efficient rolling circle amplification method based on hexamer random primer |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN114277103A true CN114277103A (en) | 2022-04-05 |
Family
ID=80881381
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210072066.6A Pending CN114277103A (en) | 2022-01-21 | 2022-01-21 | Efficient rolling circle amplification method based on hexamer random primer |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114277103A (en) |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104093850A (en) * | 2012-02-15 | 2014-10-08 | 通用电气公司 | Methods and kits for reducing non-specific nucleic acid amplification |
| CN106319032A (en) * | 2015-06-24 | 2017-01-11 | 深圳华大基因研究院 | Method for measuring activity of Phi29 DNA polymerase and rolling circle sequencing method |
| CN106460040A (en) * | 2014-03-26 | 2017-02-22 | 通用电气公司 | Isothermal amplification under low salt condition |
| CN110484606A (en) * | 2019-08-15 | 2019-11-22 | 中国海洋大学 | A kind of primer self-generating Rolling Circle Amplification methods that restriction enzyme mediates |
| CN111334562A (en) * | 2018-12-19 | 2020-06-26 | 合肥铼科生物科技有限公司 | Nucleic acid amplification method and kit containing modified primer |
| CN114958978A (en) * | 2022-06-21 | 2022-08-30 | 上海交通大学 | One-pot single-stranded DNA circularization amplification and CRISPR/Cas-mediated nucleic acid molecule detection method |
-
2022
- 2022-01-21 CN CN202210072066.6A patent/CN114277103A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104093850A (en) * | 2012-02-15 | 2014-10-08 | 通用电气公司 | Methods and kits for reducing non-specific nucleic acid amplification |
| CN106460040A (en) * | 2014-03-26 | 2017-02-22 | 通用电气公司 | Isothermal amplification under low salt condition |
| CN106319032A (en) * | 2015-06-24 | 2017-01-11 | 深圳华大基因研究院 | Method for measuring activity of Phi29 DNA polymerase and rolling circle sequencing method |
| CN111334562A (en) * | 2018-12-19 | 2020-06-26 | 合肥铼科生物科技有限公司 | Nucleic acid amplification method and kit containing modified primer |
| CN110484606A (en) * | 2019-08-15 | 2019-11-22 | 中国海洋大学 | A kind of primer self-generating Rolling Circle Amplification methods that restriction enzyme mediates |
| CN114958978A (en) * | 2022-06-21 | 2022-08-30 | 上海交通大学 | One-pot single-stranded DNA circularization amplification and CRISPR/Cas-mediated nucleic acid molecule detection method |
Non-Patent Citations (1)
| Title |
|---|
| 张俊: "滚环扩增技术的原理及其应用的研究", 《生物信息学》, vol. 10, no. 1, pages 12 - 14 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN109055499B (en) | Isothermal nucleic acid detection method and kit based on CRISPR-Cas | |
| CN107541546B (en) | Compositions, methods, systems, and kits for target nucleic acid enrichment | |
| US9790540B2 (en) | Methods and kits for 3′-end-tagging of RNA | |
| KR20210039989A (en) | Use of high temperature resistant Cas protein, detection method and reagent kit of target nucleic acid molecule | |
| EP3985122A1 (en) | Methods of producing amplified double stranded deoxyribonucleic acids and compositions and kits for use therein | |
| JP2008525038A (en) | RNA amplification by ligation | |
| EP3378948B1 (en) | Method for quantifying target nucleic acid and kit therefor | |
| KR102445790B1 (en) | Methods for amplifying mRNA and methods for preparing full-length mRNA libraries | |
| JP2018183065A (en) | RNA detection method | |
| CN114250276B (en) | MicroRNA detection system and method based on exponential amplification reaction and Argonaute nuclease | |
| WO2011101744A2 (en) | Region of interest extraction and normalization methods | |
| CN114277103A (en) | Efficient rolling circle amplification method based on hexamer random primer | |
| EP4330424A1 (en) | Amplification of single stranded dna | |
| CN110551794B (en) | Method and kit for processing RNA molecules and complex | |
| JP2012040004A (en) | SYNTHETIC siRNA DETECTION METHOD | |
| US20230122979A1 (en) | Methods of sample normalization | |
| WO2012009464A2 (en) | Detection of nucleic acids by agglutination | |
| CN113073133A (en) | Method for amplifying trace amount of DNA and detecting multiple nucleic acids, and nucleic acid detecting apparatus | |
| CN117043355A (en) | Protective isothermal nucleic acid amplification (PINA) method for on-demand diagnosis of new-born infectious diseases | |
| CN117965687A (en) | Amplification method of single-stranded DNA product and application thereof | |
| CN116411049A (en) | Enzyme combination product for nucleic acid isothermal amplification reaction and application thereof | |
| CN115803433A (en) | Thermostable ligases with reduced sequence bias | |
| CN116064750A (en) | Reaction system for multiplex PCR amplification and amplification kit thereof | |
| CN115992206A (en) | Argonaute-mediated one-pot method microRNA detection system and detection method | |
| CN114414636A (en) | Electrochemical biosensor composition, working solution, electrochemical biosensor and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |