WO2018195878A1 - Identificateur d'adn et utilisation correspondante - Google Patents

Identificateur d'adn et utilisation correspondante Download PDF

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WO2018195878A1
WO2018195878A1 PCT/CN2017/082281 CN2017082281W WO2018195878A1 WO 2018195878 A1 WO2018195878 A1 WO 2018195878A1 CN 2017082281 W CN2017082281 W CN 2017082281W WO 2018195878 A1 WO2018195878 A1 WO 2018195878A1
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dna
sequence
sequencing
nucleic acid
treatment
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PCT/CN2017/082281
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Chinese (zh)
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柴相花
甄贺富
袁玉英
张现东
张爱萍
张红云
刘娜
尹烨
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深圳华大基因股份有限公司
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Priority to CN202310265316.2A priority Critical patent/CN116121243A/zh
Priority to PCT/CN2017/082281 priority patent/WO2018195878A1/fr
Priority to CN201780083033.9A priority patent/CN110168087B/zh
Publication of WO2018195878A1 publication Critical patent/WO2018195878A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C07H21/04—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with deoxyribosyl as saccharide radical
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6869—Methods for sequencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B40/00—Libraries per se, e.g. arrays, mixtures
    • C40B40/04—Libraries containing only organic compounds
    • C40B40/06—Libraries containing nucleotides or polynucleotides, or derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B50/00—Methods of creating libraries, e.g. combinatorial synthesis
    • C40B50/06—Biochemical methods, e.g. using enzymes or whole viable microorganisms
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • the present invention relates to the field of biological sequencing, and in particular, to DNA tags, DNA linkers, methods of constructing sequencing libraries, sequencing libraries, and sequencing methods.
  • high-throughput sequencing technology has brought genomics research to a new era. It can not only perform large-scale genome sequencing, but also can be used for gene expression analysis, non-coding small analysis RNA identification.
  • high-throughput sequencing technology breaks the flux limitation in the disease research process, making it possible to conduct multi-faceted and comprehensive research on diseases, providing an effective means for disease prevention, diagnosis and treatment.
  • DNA sequencing, DNA molecular quantification, RNA abundance analysis, etc. are of great significance in genomic, gene expression research or medical genetic testing.
  • high-throughput sequencing technology requires PCR amplification of sample DNA/RNA before sequencing, PCR has widespread problems such as amplification bias and amplification errors, and is also based on a specific sequencing platform and sequencing environment. Sequencing errors can occur, resulting in approximately 1% of the bases being incorrectly identified, which limits the detection of rare and low frequency variations.
  • the Unique Molecular Identifiers (UMI) technique is used to record the original DNA/RNA of a sample by randomly adding a synthetic sequence (generally 5-12 bp) at the end of the DNA/RNA molecular fragment as a unique identifier for identifying the DNA fragment. information.
  • a synthetic sequence generally 5-12 bp
  • UMI Unique Identifier
  • Schmitt et al. used further UMI and Duplex Sequencing (DS) techniques to detect very rare mutations. Also in Scott R Kennedy, Michael W Schmitt et al. provided detailed protocols for effective DS link synthesis, library preparation, target enrichment, and data analysis process overview in 2014. Next, in 2015, Michael W Schmitt et al. used DS technology to detect rare mutations in the ABL1 gene.
  • the present invention aims to solve at least one of the technical problems existing in the prior art.
  • the inventors of the present application developed a set of genomic minimal trace detection and verification systems based on the original UMI sequence.
  • the mutation frequency that can be detected by the system can be as low as 0.01%, and early screening for cancer, neurodegenerative diseases and cardiovascular diseases induced by cumulative mutations such as somatic cells and stem cells can be realized.
  • the invention proposes a DNA tag.
  • the tag has a sequence selected from at least one of: (1) HHATHHHTCACCHHATHHH (SEQ ID NO: 10); and (2) HHHTAHHTAHHHTAHH (SEQ ID NO: 11), wherein H represents A , T or C.
  • the invention proposes a DNA linker.
  • the DAN linker comprises a DNA tag as described above.
  • the sequencing library is constructed by using the DNA linker according to the embodiment of the present invention, and then the sequencing library is sequenced, and a very small amount of variation can be detected, and the detection sensitivity of a micro mutation or a rare mutation having a mutation frequency as low as 0.01% is high.
  • the DNA linker according to the embodiment of the present invention is of great significance for early screening of cancers, neurodegenerative diseases, cardiovascular diseases, and the like which are caused by cumulative mutations such as somatic cells and stem cells.
  • the invention proposes the use of the DNA tag described above and the DNA linker described above for the detection of minor variations.
  • detection and verification of a very small amount (mutation frequency as low as 0.01%) variation cancer induced by cumulative mutation of somatic cells, stem cells, neurodegenerative diseases, cardiovascular diseases can be realized. Early screening is important.
  • the invention proposes a method of constructing a sequencing library.
  • the method comprises enriching a nucleic acid molecule linked to a DNA linker as described above to obtain a sequencing library.
  • the sequencing library constructed by the method according to the embodiment of the present invention can be used for the detection of extremely small variations, and the mutation frequency of the extremely small variation can be as low as 0.01%.
  • the invention proposes a sequencing library.
  • the sequencing library is obtained by the method of constructing a sequencing library as described above.
  • the high-throughput sequencing of the sequencing library can detect a mutation frequency of at least 0.01%, and can realize early screening for cancer, neurodegenerative diseases and cardiovascular diseases induced by cumulative mutations such as somatic cells and stem cells.
  • the invention proposes a sequencing method.
  • the method comprises sequencing and data analysis processing of the sequencing library described above.
  • detection and verification of low frequency mutations can be realized, and mutation frequencies that can be detected according to different UMI techniques of sequencing depth can be achieved.
  • the rate can reach 0.01%, and can be effectively applied to early screening of cancers, neurodegenerative diseases, cardiovascular diseases, etc. induced by cumulative mutations such as somatic cells and stem cells.
  • FIG. 1 is a flow chart showing the overall analysis of a very small variation detecting system according to an embodiment of the present invention
  • FIG. 2 is a flow chart of data analysis processing according to an embodiment of the present invention.
  • Figure 3 is a diagram showing the purification quantification and Sanger sequencing verification of PCR products according to one embodiment of the present invention
  • FIG. 4 is a graph showing the results of a joint prepared by detecting 2100 detection plus "T" strategy, in accordance with one embodiment of the present invention.
  • FIG. 5 is a graph showing the results of a joint prepared by detecting 2100 detection plus an anchor strategy according to an embodiment of the present invention
  • FIG. 6 is a graph showing the results of a joint prepared by detecting a 2100 detection enzyme digestion strategy according to an embodiment of the present invention
  • FIG. 7 is a graph showing the results of detecting a sequencing library using the detection 2100, in accordance with one embodiment of the present invention.
  • Figure 8 is a cumulative depth profile of a sample in accordance with one embodiment of the present invention.
  • Figure 9 is a depth profile of a sample in accordance with one embodiment of the present invention.
  • FIG. 11 is a diagram of a result of constructing a duplex consistency sequence in accordance with an embodiment of the present invention.
  • first and second are used for descriptive purposes only, and are not to be construed as indicating or implying a relative importance or implicitly indicating the number of technical features indicated. Thus, features defining “first” and “second” may include one or more of the features either explicitly or implicitly. Further, in the description of the present invention, the meaning of "a plurality" is two or more unless otherwise specified.
  • the invention proposes a DNA tag for detecting minor variations.
  • the tag has a sequence selected from at least one of: (1) HHATHHHTCACCHHATHHH; and (2) HHHTAHHTAHHHTAHH, wherein H represents A, T or C.
  • the invention proposes a DNA linker.
  • the DAN linker comprises a DNA tag as described above.
  • the sequencing library is constructed by using the DNA linker according to the embodiment of the present invention, and then the sequencing library is sequenced, and a very small amount of variation can be detected, and the detection sensitivity of a micro mutation or a rare mutation having a mutation frequency as low as 0.01% is high.
  • the DNA linker according to the embodiment of the present invention is of great significance for early screening of cancers, neurodegenerative diseases, cardiovascular diseases, and the like which are caused by cumulative mutations such as somatic cells and stem cells.
  • the joint has a sticky end dT. Furthermore, an efficient and rapid linkage of the linker to the gene fragment to be sequenced can be achieved by a rapid T-A linkage.
  • the DNA linker further comprises: an anchor sequence formed between the sticky end dT and the tag sequence.
  • an anchor sequence formed between the sticky end dT and the tag sequence.
  • the stability of blunt-end ligation to form prominent bases is relatively poor, and there is a certain failure rate; while the annealing reaction is performed by two sequences (where the anchor sequence has more dT bases), and the complementary pairing of the two sequences Finally, the protruding dT end is formed, and the latter has no requirement of the ligation reaction, as long as the two primers are complementary and paired one by one, so the introduction of the anchoring sequence is more efficient and firmer than the usual 3' end flat end connection dT.
  • the anchor sequence has the nucleotide sequence set forth in SEQ ID NO: 1.
  • CTATGTCGATGC SEQ ID NO: 1.
  • An anchor sequence according to an embodiment of the invention is strictly not complementary to sequences other than its complementary sequence, and is not susceptible to ligation itself.
  • dDTP does not contain dC bases, so the extension reaction is terminated, so that the complementary structure of the anchor sequence can be effectively protected from damage.
  • the sticky end dT is formed at the 3' end of the DNA tag. Further A fast, efficient T-A linkage was made to the fragment to be sequenced to which A was ligated to the 5' end.
  • the linker to which the anchor sequence is attached is obtained by a gradient annealing process, a dDTP extension process, and an alcohol purification nick process in sequence.
  • the specific steps are as follows:
  • the DNA linker further comprises: a cleavage sequence formed at an end of the DNA tag, wherein the cleavage sequence carries a restriction suitable for generating a sticky end dT
  • the enzyme recognizes the site.
  • the endonuclease cleaves the 8 bases after the recognition strand of the sense strand, and the 7 bases after the recognition strand of the antisense strand, forming a sticky end of the dT base at the 3' end.
  • the linker to which the restriction enzyme sequence is ligated forms a 3' end protruding T-terminal structure which is more stable.
  • the restriction enzyme sequence is an HphI-specific recognition site. After the HphI-specific recognition site is specifically recognized and digested by HphI, a sticky terminal dT can be generated at the 3' end of the DNA linker, thereby enabling rapid and efficient T-A ligation with the fragment to be sequenced.
  • a linker ligated with an HphI-specific recognition site is sequentially obtained by gradient annealing treatment, dDTP extension treatment, and Hph1 digestion. Specifically, it can be obtained by two methods of short PCR after enzyme digestion and long PF after enzyme digestion.
  • the invention proposes the use of the DNA tag described above and the DNA linker described above for the detection of minor variations.
  • detection and verification of very small amounts can be achieved, and in scientific research, very small amounts are detected using labels and joints according to embodiments of the present invention ( Mutations with mutation frequencies as low as 0.01%) provide reliable detection of scientific research on very small amounts of variation, such as detection of somatic mitochondrial mutation rates, detection of rare DNA mutations (such as detection of novel susceptible sites), and use of single molecule counting Accurate calculation of DNA/RNA copy number, genetic disease research, aging research (such as senescence-related methylation site detection).
  • early screening for cancers, neurodegenerative diseases, and cardiovascular diseases induced by cumulative mutations such as somatic cells and stem cells is of great significance.
  • the invention proposes a method of constructing a sequencing library.
  • the method comprises enriching a nucleic acid molecule linked to a DNA linker as described above to obtain a sequencing library.
  • the sequencing library constructed by the method according to the embodiment of the present invention can be used for the detection of extremely small variations, and the mutation frequency of the extremely small variation can be as low as 0.01%.
  • the nucleic acid molecule is obtained by (1) performing PCR amplification of a nucleic acid sample to be tested to obtain a nucleic acid sample fragment; (2) performing the nucleic acid sample fragment The 3' end is treated with A; (3) the DNA linker described above is ligated with the nucleic acid sample fragment obtained in the step (2) to obtain the nucleic acid molecule to which the DNA linker described above is ligated.
  • the enrichment process is rich by PCR.
  • the specific steps are as follows:
  • DNA samples were added to a 96-well PCR reaction plate according to the format of the PCR Reaction Table. The batch samples were 3 ⁇ L per well and re-expanded to 5 ⁇ L per well. Note whether the DNA information is consistent with the PCR reaction table, the bottom of the tube or the wall is sampled, and the sealing film is briefly centrifuged at 2000 rpm for 30 s after sealing, and the bottom of the tube is checked for use;
  • the product after PCR amplification was briefly centrifuged at 2,000 rpm for 30 seconds, and transferred to an electrophoresis chamber for detection. When the following products are not detected in time, they are stored at 4 °C.
  • the enrichment treatment can also be carried out by the above-mentioned PCR enrichment method.
  • the linker having the restriction enzyme sequence is obtained by the above-described PF (PCR-Free)
  • the linker having the restriction enzyme sequence is ligated to the sample to be tested, and is rich. The set processing can be omitted.
  • the purification treatment before the enriching treatment, further comprising purifying the nucleic acid molecule to which the DNA linker described above is linked.
  • the purification treatment can be carried out by magnetic bead purification. The purification process removes the relevant enzymes and related buffers during the ligation process, thereby eliminating interference with subsequent enrichment processes, and the integrated power and efficiency of the ligation products are significantly improved.
  • the invention proposes a sequencing library.
  • the sequencing library is obtained by the method of constructing a sequencing library as described above.
  • the sequencing library is obtained by the method of constructing a sequencing library as described above.
  • the high-throughput sequencing of the sequencing library can detect a mutation frequency of at least 0.01%, and can realize early screening for cancer, neurodegenerative diseases and cardiovascular diseases induced by cumulative mutations such as somatic cells and stem cells.
  • the invention proposes a sequencing method.
  • the method comprises sequencing and data analysis processing of the sequencing library described above.
  • the detection and verification of the low frequency mutation can be realized, and can be effectively applied to early screening of cancers, neurodegenerative diseases, cardiovascular diseases, and the like which are caused by cumulative mutations such as somatic cells and stem cells.
  • the sequencing is performed via the Hiseq 2500 platform.
  • High-throughput sequencing on the Hiseq2500 platform can significantly reduce costs, ensure the stability of experimental data and analysis results, and more importantly, the mutation frequency that can be detected by different UMI techniques can reach 0.01%.
  • the data analysis processing flow refers to FIG. 2, as follows:
  • the detection and verification of the low frequency mutation can be realized, and the mutation frequency that can be detected can be as low as 0.01%. Furthermore, it can be effectively applied to early screening of cancers, neurodegenerative diseases, cardiovascular diseases, and the like which are caused by cumulative mutations such as somatic cells and stem cells. Specifically, as follows: Since the present invention employs a special library preparation and analysis strategy, the prepared linker sequence is ligated to the sample DNA, although the linker sequence contains 10 degenerate bases, but specific to each molecule, Still have a specific sequence.
  • each template is obtained, and the end of each template is added with a 19-base molecular tag, and the left and right ends of each template add up to 38 bases.
  • Molecular label There are 3 choices for each annexed base, and 20 bases are 3 ⁇ 20, which is equivalent to nearly 350 million possibilities. This ensures that each original template is unique in the original library.
  • the original library is PCR amplified, and each template forms a family of molecules complementary to the two intermediate sequences of the original template: forward and reverse. Based on this library preparation and sequencing strategy, some of the false positive mutation sites can be excluded by specific strategies in the specific analysis. The specific strategies are as follows:
  • PCR is performed on the target region using two sets of DNA samples, and after determining the specific base points by Sanger sequencing, the molar ratios are 1:1, 1:100, 1:1000, 1:10000, respectively.
  • the four groups of products were finally tested in response to three UMI strategies, as shown in Table 8.
  • the target area is shown in Table 9.
  • the target sequence is represented by DRB1*01:01:01 (instruction: the sequence corresponding to this type is the reference sequence of the DRB1 gene, and the sequence shown below is the sequence of the type in the target region), and the sequence is as follows:
  • the PCR primer amplification region is as short as possible while satisfying the coverage target region.
  • the primer sequences are finally determined for the above target regions as shown in Table 10.
  • end repair take more than 200ng of the product for end repair, and purification
  • Alcohol purification nick the specific steps include:
  • ac-Adpater-1.T.1 45 ⁇ l 2x Rapid ligation buffer 50 ⁇ l T4DNA Ligase (600U/ ⁇ l) 5 ⁇ l In Total 50 ⁇ l
  • Enzyme digestion strategy ie, Hphl digestion, including short sequence scheme (S) and long sequence scheme (L), ie PCR scheme and PCR-Free scheme, the specific steps are as follows:
  • the primers for the short sequence scheme are shown in Table 15:
  • the detection ratio has a good correspondence with the sample mixing ratio.
  • the mixing ratio is 10000:1
  • the determined mutation sites can be correctly detected. Therefore, the UMI sequence designed by this system can detect mutations with a mutation rate of 0.01%.
  • the method of the invention can be effectively applied to the detection and verification of low-frequency mutations, and the detection mutation frequency can be as low as 0.01%, and can be effectively applied to cancers and neurodegenerative diseases induced by cumulative mutations such as somatic cells and stem cells. Early screening for diseases, cardiovascular diseases, etc.

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Abstract

L'invention concerne un identificateur d'ADN pour détecter une mutation dans une quantité en trace. Ledit identificateur a une séquence choisie parmi au moins l'une des séquences suivantes : (1) HHATHHHTCACCHHATHHH ; ou (2) HHHTAHHTAHHHTAHH, H représentant A, T ou C.
PCT/CN2017/082281 2017-04-27 2017-04-27 Identificateur d'adn et utilisation correspondante WO2018195878A1 (fr)

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CN202310265316.2A CN116121243A (zh) 2017-04-27 2017-04-27 Dna标签及其应用
PCT/CN2017/082281 WO2018195878A1 (fr) 2017-04-27 2017-04-27 Identificateur d'adn et utilisation correspondante
CN201780083033.9A CN110168087B (zh) 2017-04-27 2017-04-27 Dna标签及其应用

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DATABASE GenBank [O] 6 June 2016 (2016-06-06), XP055529036, Database accession no. XR-001757229 *

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