CN106222164B - Methods, compositions and kits for unidirectional amplification of nucleic acids in vitro using transposase - Google Patents
Methods, compositions and kits for unidirectional amplification of nucleic acids in vitro using transposase Download PDFInfo
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Abstract
The invention provides a method, a composition and a kit for in vitro unidirectional nucleic acid amplification by using transposon enzyme. The invention can complete the amplification process of the target region by combining the transposon enzyme, the unidirectional specific amplification and the high-throughput sequencing method, and well reduce the original DNA mutation frequency. Can realize the detection of specific DNA sequence and achieve the aim of detecting the gene of tumor cell mutation or genetic disease. The invention overcomes the problem that the traditional method needs hybridization or has bias in a large amount of PCR amplification, simultaneously reserves mutation frequency, and overcomes the problem of false positive of samples caused by sequencing error and amplification error.
Description
Technical Field
The present invention relates to the field of molecular biology, in particular to methods, compositions and kits for unidirectional amplification of nucleic acid molecules using transposon enzymes.
Background
The human genome has three billion base pairs, and the selection of part of the bases, such as pathogenic parts, for analysis is the main clinical requirement at present, and how to capture a specific sequence in the whole genome is a worldwide problem.
Transposon enzyme, an enzyme that performs transposition function, is usually encoded by a transposon, recognizes specific sequences at both ends of the transposon, and can detach the transposon from adjacent sequences and insert it into a new DNA target site without homology requirement. There are techniques to engineer transposon enzymes to allow insertion of transposon enzymes into specific sequences (e.g. NEXTERA from Epicentre)TMSystem) is provided. Fragmentation of genomic DNA can be achieved using such techniques, but the techniques still do not allow capture of specific sequences across the entire genome.
Disclosure of Invention
In order to solve the above technical problems, according to one aspect of the present invention, there is provided a method for unidirectional amplification of nucleic acids in vitro using a transposon enzyme, comprising the steps of:
a step of providing a transposon enzyme and a nucleic acid;
a step of providing a first oligonucleotide sequence and a second oligonucleotide sequence;
a step of reacting the transposon enzyme with the nucleic acid, thereby altering the sequence of the nucleic acid;
a step of performing nucleic acid amplification using the first oligonucleotide sequence and the second oligonucleotide sequence;
wherein the transposon enzyme comprises at least one transposon enzyme binding sequence, at least a portion of the first oligonucleotide sequence binds to a specific region in the nucleic acid, and the second oligonucleotide sequence comprises at least a portion of the transposon enzyme binding sequence.
In another aspect of the present invention, there are provided a composition and a kit for in vitro unidirectional amplification of nucleic acids using a transposase, comprising a transposase, a first oligonucleotide sequence and a second oligonucleotide sequence, respectively; wherein the transposon enzyme comprises at least one transposon enzyme binding sequence, at least a portion of the first oligonucleotide sequence binds to a specific region in the nucleic acid, and the second oligonucleotide sequence comprises at least a portion of the transposon enzyme binding sequence.
In preferred embodiments, the compositions and kits of the invention may further comprise nucleic acids, e.g., whole genomic DNA or fragments thereof.
In a preferred embodiment, the first oligonucleotide sequence comprises a 5 'primer and the second oligonucleotide sequence comprises a 3' primer. In another preferred embodiment, the first oligonucleotide sequence comprises a 3 'end primer and the second oligonucleotide sequence comprises a 5' end primer.
In a further preferred embodiment, said first oligonucleotide sequence and/or said second oligonucleotide sequence further comprises an index sequence and/or a marker sequence.
In a further preferred embodiment, the transposon enzyme comprises a first transposon enzyme binding sequence and a second transposon enzyme binding sequence.
In preferred embodiments, the specific region in the nucleic acid is within an open reading frame (e.g., an EGFR open reading frame).
The invention achieves amplification of specific sites in nucleic acids, such as DNA, by adding specific sequences to nucleic acids, particularly genomic DNA molecules, using transposase, and by directional amplification with uniform primers and amplification with specific primers. Further, the identification of the specific site genotype is finally realized by a method such as high-throughput sequencing, and the amplified sites are qualitatively and quantitatively analyzed.
The invention completes the process of target region amplification by combining transposon enzyme, unidirectional specific amplification and high-throughput sequencing, and well reduces the original DNA mutation frequency. Can realize the detection of specific DNA sequence and achieve the aim of detecting genes, in particular to the genes of tumor cell mutation or genetic diseases. The invention overcomes the problem that the traditional method needs hybridization or has bias in a large amount of PCR amplification, simultaneously reserves mutation frequency, and overcomes the problem of false positive of samples caused by sequencing error and amplification error.
Drawings
FIG. 1 is a diagram schematically showing the structure of a transposon enzyme in the present invention. Wherein 1 is a protein having transposon enzyme activity; 2 is a first transposase binding sequence; 3 is a first primer sequence; 4 is a second transposon enzyme binding sequence; and 5 is a second primer sequence.
FIG. 2 is a diagram schematically showing an amplification method according to the present invention. In fig. 2, 6 and 9 are index sequences (index sequences), respectively, where the index sequences denoted by 6 and 9 may be the same or different; 7 is a P5 sequence (e.g., SEQ ID NO: 4); 8 is a 3' end primer; 10 is a P7 sequence (e.g., SEQ ID NO: 5).
It is to be noted that the same reference numerals are used for the same or similar meanings in the present invention. For example, reference numeral 2 in both fig. 1 and fig. 2 denotes a first transposase binding sequence.
Detailed Description
Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but as a more detailed description of certain aspects, features and embodiments of the invention.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. Further, for numerical ranges in this disclosure, it is understood that each intervening value, between the upper and lower limit of that range, is also specifically disclosed. Every smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in a stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only the preferred methods and materials (e.g., compositions or kits) are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference herein for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.
As used herein, the term "comprises" includes both singular and plural forms, unless the context clearly dictates otherwise. For example, "transposase" includes a plurality of such transposases, "sample" includes one or more samples and equivalents thereof known to those skilled in the art, and so forth. The term "at least one" or "at least one" as used herein is intended to mean not only the case where "one" or "one" is included, but more importantly, the case where "a plurality" or "a plurality" is included.
In the present invention, the term "nucleic acid" refers to any type of DNA molecule, preferably genomic DNA, which is typically between thousands to millions of bases in length. In the present invention, "nucleic acid" also includes the meaning of a genomic DNA fragment.
In the present invention, the term "transposase", sometimes referred to as "transposase", is an enzyme that performs a transposition function, and is usually encoded by a transposon, recognizes specific sequences at both ends of the transposon, and can detach the transposon from adjacent sequences and insert it into a new DNA target site without homology requirement. The transposase in the present invention includes a naturally occurring transposase or a recombinant transposase. Preferably, the transposase is isolated or purified from its natural environment (i.e., the nucleus or cytoplasm) at least to some extent. For recombinantly produced transposases, preferably, the recombinant transposase is isolated or purified, at least to some extent, from the recombinant host environment (i.e., the nucleus or cytoplasm). Most preferably, the transposase is purified to greater than 90%, e.g., greater than about 95%, greater than about 98%, or greater than 99%.
Preferably, the transposase includes not only any transposase identified before the filing date of this application, but also transposases whose activity can be determined to be likely to have a transposase activity based on currently known genomic knowledge and identified after the filing date of this application.
Preferably, the present invention uses enzymes that have been engineered into native transposase enzymes to allow for the insertion of the transposase into a particular sequence. In an exemplary embodiment, the transposase of the present invention IS a member of the IS4 family of transposases, such as one found naturally in species Vibrio, including but not limited to Vibrio harveyi.
In exemplary embodiments, the invention uses engineered transposases that include one or more amino acid deletions, substitutions, or additions. It will be appreciated that while modifications may alter the specificity or activity in some way, such modifications to a naturally occurring transposase do not eliminate the transposase activity of the enzyme.
In exemplary embodiments, the invention can use a commercially available transposase, e.g., NEXTERA from EpicentreTMA transposase in the system; alternatively, transposases disclosed in the literature known at present, such as those described in CN102796728A, can be used. Preferably, the present invention uses a modified Tn5 transposase.
In exemplary embodiments, the transposon enzyme can be the protein itself having transposon enzyme activity. In another embodiment, the transposon enzyme is a composition comprising a protein having transposon enzyme activity and a transposon enzyme binding sequence. In the case of transposon enzyme assemblies, any desired sequence may be added to the nucleic acid or fragment thereof by binding the sequence by the transposon enzyme.
In the present invention, the term "transposase binding sequence" refers to a recognition sequence that binds to a specific transposase. Each transposase can comprise one transposase binding sequence or two different transposase binding sequences. Preferably, each transposase of the present invention comprises two different transposase binding sequences, e.g., comprising the sequences shown in SEQ ID NO:1 and SEQ ID NO: 2.
In an exemplary embodiment, different ds or ss DNA sequences (tags) may be attached to the transposase recognition sequence to allow PCR amplification, resulting in DNA fragments attached to a sequencing chip, such as an Illumina chip, and allowing identification of the source of the target DNA library, such as the Index sequence (Index sequence). For purposes applicable to next generation sequencing, preferably about half of the nucleic acid fragment ends are labeled with one type of label and the other half with a different label, with one label attached to one end of the target nucleic acid fragment and the other type attached to the opposite end to allow the nucleic acid fragment to be read in both directions. Further improvement is achieved by combining two different transposase recognition sequences. The recognition sequence can be a naturally occurring sequence of a transposase, or can be an engineered sequence that provides additional or alternative function of the nucleic acid molecule.
In exemplary embodiments, a nucleic acid (e.g., whole genomic DNA) is fragmented by a transposase; or by inserting a specific sequence inside a nucleic acid (e.g., whole genomic DNA) by a transposase without fragmenting the nucleic acid.
In exemplary embodiments, the first oligonucleotide sequence comprises a 5' end primer. The 5' primer can be used as a specific primer for the nucleic acid amplification of the present invention, and is combined with a specific region in the nucleic acid as a target, and the target can be an open reading frame of a gene. The specific region of the target is selected according to various requirements, and is not particularly limited. For example, in the case of detecting a L858R mutation in the EGFR gene, the specific region may be selected from any sequence upstream (5' -end side) of the mutation in the gene corresponding to L858R as long as the primer corresponding to the selected region satisfies the basic conditions known to those skilled in the art as a primer. See, in particular, publications such as the fourth edition of molecular cloning, a laboratory Manual of molecular cloning, Cold spring harbor.
In exemplary embodiments, the second oligonucleotide sequence comprises at least a portion or all of a 3' primer and/or a transposase binding sequence. The 3' primer can be used as a unified primer for nucleic acid amplification, and the sequence can be selected, for example, the sequence shown in SEQ ID NO. 3. The order of ligation of the 3' -end primer and/or transposase binding sequence is not particularly limited, and for example, the primer may be ligated in the direction from the 5' -end to the 3' -end in the form of a transposase binding sequence or primer.
Although the first oligonucleotide sequence comprises a 5 'primer and the second oligonucleotide sequence comprises a 3' primer, it will be understood by those skilled in the art after reading this specification that there are also situations where the first oligonucleotide sequence comprises a 3 'primer and the second oligonucleotide sequence comprises a 5' primer, which are also included in the scope of the present invention.
In the present invention, the first oligonucleotide sequence and/or the second oligonucleotide sequence may comprise other sequences including, but not limited to, for example, an index sequence, a tag sequence, and the like. The connection form of these sequences is not particularly limited. For example, in the first oligonucleotide sequence, from the 5' end to the 3' end, a tag sequence (for example, P7 sequence), an index sequence, and a 5' primer can be ligated in this order.
The method for carrying out in-vitro nucleic acid unidirectional amplification by using the transposon enzyme comprises the following steps: (1) a step of providing a transposon enzyme and a nucleic acid; (2) a step of providing a first oligonucleotide sequence and a second oligonucleotide sequence; (3) a step of reacting the transposon enzyme with the nucleic acid, thereby altering the sequence of the nucleic acid; (4) a step of performing nucleic acid amplification using the first oligonucleotide sequence and the second oligonucleotide sequence.
In exemplary embodiments, the transposase and the nucleic acid in step (1) may be provided separately, or both may be provided together as a mixture. The concentration or amount of transposase and nucleic acid, or the ratio between the two, is not particularly limited, and these parameters can be varied by those skilled in the art according to various needs. In the case where the transposon enzyme is the protein itself having transposon enzyme activity, the method of the invention further comprises the step of combining the transposon enzyme with a transposon enzyme binding sequence, thereby obtaining a combination.
In exemplary embodiments, the first oligonucleotide sequence and the second oligonucleotide sequence in step (2) may be provided separately, or as a mixture of both. The concentrations or amounts of the first and second oligonucleotide sequences are generally the same, and one skilled in the art can formulate the concentrations or amounts of the two to be different according to different needs.
In exemplary embodiments, the reacting of the transposase with the nucleic acid in step (3) may include establishing a reaction environment such that the transposase binds to the nucleic acid and cuts at a specific site of the nucleic acid, thereby fragmenting the nucleic acid and adding a specific sequence at a terminal, and may also include further repairing at the specific site to change the nucleic acid sequence or inserting a specific sequence. The reaction environment is typically an aqueous environment and the temperature is between 20-40 c, preferably around 37 c. The environmental conditions can be determined by one skilled in the art. The binding site between the nucleic acid and the transposon enzyme is at least one, preferably 5 or more, more preferably 10 or more, and still more preferably 50 or more, 100 or more, 500 or more, etc. In the case of fragmenting a nucleic acid such as a genomic DNA, the length of the resulting nucleic acid such as a DNA fragment is not particularly limited, and may be, for example, 50 to 5000bp, preferably 100 to 2000bp, more preferably 200 to 1000bp, further preferably 500 to 800bp, or 5000bp or more.
In exemplary embodiments, the nucleic acid amplification described in step (4) may employ methods commonly used in the art, such as various types of PCR methods.
It will be understood by those skilled in the art that the order of the above steps is not particularly limited as long as the object of the present invention can be achieved, and for example, the order of the steps may be (1), (2), (3), (4); further, (2), (1), (3), (4) and the like may be used. Further, two or more of the above steps may be combined and performed simultaneously, for example, steps (3) and (4) may be performed simultaneously. In addition, it will be understood by those skilled in the art that other steps or operations may be included before, after, or between any of the above steps (1) - (4), such as to further optimize and/or improve the methods of the present invention.
As a further step, a step of rendering the fragmented DNA molecules suitable for subsequent analysis is preferred. Any available technique may be used in accordance with the present invention for use in subsequent assays (e.g., next generation sequencing, microarray analysis, high throughput detection, etc.).
The composition and the kit for in vitro unidirectional nucleic acid amplification by using the transposon enzyme comprise the transposon enzyme, a first oligonucleotide sequence and a second oligonucleotide sequence. Preferably, further comprising a nucleic acid. In an exemplary embodiment, any one of the above substances may be present alone in a state separated from the other substances, for example, the transposase, the first oligonucleotide sequence and the second oligonucleotide sequence are stored in different containers (e.g., vials), respectively, as long as they can be brought into contact with each other at the time of use, thereby serving as a set or a set of components. In addition, preferably, any two or more of the above-mentioned substances may be mixed to exist as a mixture. For example, a state in which both the first oligonucleotide sequence and the second oligonucleotide sequence are present as a mixture, and the other substances are present alone; alternatively, a mixture of the nucleic acid, the first oligonucleotide sequence and the second oligonucleotide sequence may be present as one, and the transposon enzyme may be present as one. Alternatively, the three substances may be present mixed together as a mixture.
In an exemplary embodiment, the transposase, the first oligonucleotide sequence and the second oligonucleotide sequence are provided in the form of a dry powder, respectively. Alternatively, at least one of the three substances is present in the form of a solution, for example an aqueous solution. The concentrations or contents of these substances, in the case of their presence in aqueous solution, are readily determinable by the person skilled in the art as a function of the various requirements. For example, for storage purposes, the concentration of the substance, e.g., oligonucleotide sequence, may be present in a higher form, and when in the working state or in use, the concentration may be reduced to the working concentration, e.g., by diluting the higher concentration solution.
Preferably, the compositions or agents of the inventionThe cartridge may further comprise other reagents or ingredients. For example, DNA polymerase, dNTPs of various types and ions such as Mg, required for carrying out PCR2+And the like. These additional agents or components are known to those skilled in the art and are readily known from publications such as molecular cloning, a laboratory manual, fourth edition, cold spring harbor, and the like.
Preferably, the kit of the present invention further comprises instructions for use, wherein instructions, directions or teaching for carrying out the method of the present invention or for using the composition or kit of the present invention are given or taught in the instructions for use.
Examples
Any reagent used in the present invention is a general reagent purchased from the market, unless otherwise specified. Blood samples were from samples collected in a collaborative hospital.
1. Sample extraction
Blood DNA was extracted using DNA extraction reagents. The method mainly comprises the following steps: the whole blood samples were stored temporarily in a 4 ℃ refrigerator. The blood DNA extraction kit of QIAgene company in USA is adopted for extraction, and the specific steps are referred to the instruction attached to the kit, which is not described herein. The extracted DNA was quantitatively analyzed to ensure that each extracted DNA exceeded 200 ng. Thus, 10 parts of DNA were obtained.
2. Transposon enzyme reaction
Unfreezing 5 × tag buffer L at room temperature, and reversing the upside down and mixing the mixture evenly for later use; it was confirmed that 5XNT Solution was at room temperature and no precipitate was confirmed by flicking the tube wall.
The following ingredients were added to a sterile PCR tube:
gently flick 20 times using a pipette. Placed in a PCR apparatus (Thermfisher StepOne Plus).
The reaction procedure is as follows:
hot cover 105 deg.C
55℃ 10min
4℃ Hold
2.5. mu.l of 5XNT Solution was added to the reaction Solution, and the mixture was gently pipetted 20 times and thoroughly mixed. Standing at room temperature for 5 min.
3. PCR amplification
dNTP Solution Set (10mM), MgSO was taken out from a reagent plate stored at-20 ℃ in advance4(50mM), PCR primers (10 pmol/. mu.l), and a set of adapters shown in Table 1 (i.e., adapters: PCR amplification primers corresponding to N7 (e.g., EGFR L858R).
TABLE 1
Placing on a centrifuge tube rack for dissolving at room temperature, mixing thoroughly, and centrifuging for a short time; preparing reaction mix in a 1.5mL centrifuge tube, carrying out short-time centrifugation after uniform acceleration, and subpackaging; MiX and mixing and centrifuging. The PCR reaction system is shown in Table 2 below:
TABLE 2
| Adaptor-ligated nucleic acid samples | 32.2μL |
| Platinum Pfx DNA polymerase | 0.8μL |
| Primer and method for producing the same | 4μL |
| MgSO4(50mM) | 2μL |
| dNTP Solution Set | 2μL |
| 10 XPfx buffer | 5μL |
| ddH2O | 4μL |
| Total volume | 50μL |
The PCR reaction program is:
after completion of the reaction, the reaction mixture was purified by the magnetic bead method and dissolved in 50. mu.L.
Thus, a PCR amplification product was obtained.
4. Miseq sequencing
10pmol of DNA was sequenced using Illumina Miseq PE-150 program, and the detailed procedures are described in Miseq instruction manual.
5. And (4) analyzing results:
the sequencing result produced by the Illumina Miseq is a series of DNA sequences, the sequencing sequences can be corresponding to each sample through a sequencing library label and an ID label of the sample, the sequence of the same sample is analyzed according to the flow of prenatal diagnosis, and a report is finally given: EGFR L858R site was detected in the positive samples and matched with the results obtained by ddPCR.
It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the present disclosure without departing from the scope or spirit of the disclosure. Other embodiments will be apparent to those skilled in the art from consideration of the specification. The specification and examples are exemplary only.
Claims (11)
1. A method for unidirectional amplification of nucleic acids in vitro using a transposase enzyme comprising the steps of:
a step of providing a transposon enzyme and a nucleic acid;
a step of providing a first oligonucleotide sequence and a second oligonucleotide sequence;
a step of reacting the transposon enzyme with the nucleic acid, thereby altering the sequence of the nucleic acid;
a step of performing nucleic acid amplification using the first oligonucleotide sequence and the second oligonucleotide sequence;
wherein the transposase is a recombinant transposon enzyme comprising a protein having transposon enzyme activity and a first transposase binding sequence and a second transposase binding sequence, and
at least a portion of the first oligonucleotide sequence binds to a specific region of the nucleic acid that is within an open reading frame, the second oligonucleotide sequence comprising at least a portion of a transposase-binding sequence;
wherein the first oligonucleotide sequence comprises a 5 'primer and the second oligonucleotide sequence comprises a 3' primer; or the first oligonucleotide sequence comprises a 3 'end primer and the second oligonucleotide sequence comprises a 5' end primer;
wherein the first oligonucleotide sequence comprises a primer that is a specific primer and the second oligonucleotide sequence comprises a primer that is a uniform primer for nucleic acid amplification.
2. The method of claim 1, wherein the first oligonucleotide sequence and the second oligonucleotide sequence further comprise an index sequence or a tag sequence, respectively.
3. The method of claim 1, wherein the second oligonucleotide sequence comprises the sequence set forth in SEQ ID NO 1 or SEQ ID NO 2.
4. The method of claim 2, wherein the marker sequence is as set forth in SEQ ID NO 4 or SEQ ID NO 5.
5. The method of claim 2, wherein the second oligonucleotide sequence comprises a 3' primer as set forth in SEQ id No. 3.
6. A composition for unidirectional amplification of nucleic acids in vitro using a transposase, comprising a transposase, a first oligonucleotide sequence, and a second oligonucleotide sequence;
wherein the transposase is a recombinant transposon enzyme comprising a protein having transposon enzyme activity and a first transposase binding sequence and a second transposase binding sequence, and
at least a portion of the first oligonucleotide sequence binds to a specific region of the nucleic acid that is within an open reading frame, the second oligonucleotide sequence comprising at least a portion of a transposase-binding sequence;
wherein the first oligonucleotide sequence comprises a 5 'primer and the second oligonucleotide sequence comprises a 3' primer or the first oligonucleotide sequence comprises a 3 'primer and the second oligonucleotide sequence comprises a 5' primer.
7. The composition of claim 6, wherein the first oligonucleotide sequence and the second oligonucleotide sequence further comprise an index sequence or a marker sequence, respectively.
8. The composition of claim 6, wherein the second oligonucleotide sequence comprises the sequence set forth in SEQ ID NO 1 or SEQ ID NO 2.
9. The composition of claim 7, wherein the marker sequence is as set forth in SEQ ID NO 4 or SEQ ID NO 5.
10. The composition of claim 7, wherein said second oligonucleotide sequence comprises a 3' primer as set forth in SEQ ID NO. 3.
11. A kit for unidirectional amplification of nucleic acids in vitro using a transposase comprising a composition according to any one of claims 6-10.
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| CN1319135A (en) * | 1998-09-23 | 2001-10-24 | 威斯康星校友研究基金会 | Method for making insertional mutations |
| CN102703426A (en) * | 2012-05-21 | 2012-10-03 | 吴江汇杰生物科技有限公司 | Method for constructing a nucleic acid library, reagent and kit |
| CN103710323A (en) * | 2012-10-01 | 2014-04-09 | 安捷伦科技有限公司 | Immobilized transposase complexes for DNA fragmentation and tagging |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1319135A (en) * | 1998-09-23 | 2001-10-24 | 威斯康星校友研究基金会 | Method for making insertional mutations |
| CN102703426A (en) * | 2012-05-21 | 2012-10-03 | 吴江汇杰生物科技有限公司 | Method for constructing a nucleic acid library, reagent and kit |
| CN103710323A (en) * | 2012-10-01 | 2014-04-09 | 安捷伦科技有限公司 | Immobilized transposase complexes for DNA fragmentation and tagging |
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