CN113249796A - Single molecule label for marking DNA fragment - Google Patents
Single molecule label for marking DNA fragment Download PDFInfo
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- CN113249796A CN113249796A CN202110547139.8A CN202110547139A CN113249796A CN 113249796 A CN113249796 A CN 113249796A CN 202110547139 A CN202110547139 A CN 202110547139A CN 113249796 A CN113249796 A CN 113249796A
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- 239000012634 fragment Substances 0.000 title claims abstract description 27
- ISAKRJDGNUQOIC-UHFFFAOYSA-N Uracil Chemical compound O=C1C=CNC(=O)N1 ISAKRJDGNUQOIC-UHFFFAOYSA-N 0.000 claims abstract description 20
- 238000000137 annealing Methods 0.000 claims abstract description 12
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- 238000012986 modification Methods 0.000 claims abstract description 7
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Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C40—COMBINATORIAL TECHNOLOGY
- C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
- C40B70/00—Tags or labels specially adapted for combinatorial chemistry or libraries, e.g. fluorescent tags or bar codes
-
- 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
- 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
Abstract
The invention provides a single molecular label for marking a DNA fragment, wherein the single molecular label is any one of a stem-loop structure formed by annealing one oligo, a structure with two complementary pairs formed by annealing two oligos or a structure with one complementary pair formed by annealing two oligos; the oligo is composed of a first nucleotide sequence, uracil, poly dNTP, a second nucleotide sequence, and 3 'overhanging base in sequence from 5' end to 3 'end, the first sequence and the second sequence are complementarily paired, wherein the first unpaired base near the 5' end has uracil modification on the non-complementarily paired circular structure. According to the invention, by selecting a specific structure and a modified label and combining a simple and efficient introduction method, the DNA double-end single-molecule coding marker is realized, the initial source of sequencing reading is more effectively positioned, manpower and material resources are saved, and the method has popularization and application prospects and values.
Description
The application is filed as2018, 06 and 20 monthsApplication No. is201810638123.6The invention is named asA kind of Single molecule label and application thereofDivisional application of the patent application.
Technical Field
The invention relates to the field of molecular biology, in particular to a single-molecule label for marking a DNA fragment.
Background
DNA sequencing is a very important experimental technique and has wide application in the field of biological research, especially in the gene detection industry. From the initial end-termination method to the now more mature second-Generation Sequencing technology (Next-Generation Sequencing). The current second generation sequencing has lower cost, higher flux and higher speed compared with the first generation sequencing.
The basic principle of Illumina sequencing is sequencing-by-synthesis. In the library construction process, genomic DNA is randomly fragmented, specific sequencing adapters (adapters) are added to two ends of the fragmented genome, and then library PCR amplification is carried out. Due to the randomness of the final PCR amplification and fragmentation, there is a problem with each read length of the final library sequencing, i.e. it is uncertain from which template the resulting read length was originally amplified. CN106834503A provides a primer group for amplifying cancer genes from peripheral blood free DNA by a primer label combined deep sequencing method, a primer label design method and application, the invention attaches molecular labels to each DNA molecule in a template by labeling two sides of each initial sample DNA molecule template, after PCR and sequencing, the original DNA molecules in a sample can be identified and classified by the molecular labels, and further, the systematic errors introduced in the PCR amplification and sequencing stages in NGS sequencing can be removed or reduced by the labels. CN107254514A discloses an SNP molecular marker for detecting heterologous cfDNA, a detection probe and a chip designed based on the SNP molecular marker, and a detection device, a kit and a detection method for the heterologous cfDNA; the SNP molecular marker comprises SNP loci of drug metabolism genes, provides mutation information of drug genes for patients with kidney transplantation rejection, and realizes individualized drug administration; the invention also provides a single molecular marker joint which is used for constructing a sequencing library to effectively remove repeated data and errors randomly introduced in the sequencing and PCR processes, thereby reducing the background noise of sequencing and improving the accuracy of detection. However, the prior art can not effectively trace the source of the sequencing read length, and has the disadvantages of complex operation steps, high requirement on equipment and difficult application and popularization.
In conclusion, a single-molecule label and application thereof are developed, and each initial DNA fragment is marked, so that each reading length specific source is presented in a final sequencing result, and the single-molecule label has a wide application prospect and a huge market value.
Disclosure of Invention
Aiming at the defects and practical requirements of the prior art, the invention provides a single-molecule label and application thereof, through designing a specific label and selecting an introduction method matched with the label, a DNA sample is processed at the early stage of constructing a library to realize the marking of each initial DNA fragment, so that the specific source of each read length is presented in the final sequencing result,
in order to achieve the purpose, the invention adopts the following technical scheme:
in a first aspect, the present invention provides a single molecule tag, wherein the tag is any one of a stem-loop structure formed by annealing one oligo, a structure formed by annealing two oligos and complementarily pairing two ends, or a structure formed by annealing two oligos and complementarily pairing one end, wherein the tag has a protruding 3 'end and a phosphorylated 5' end, and uracil is modified on an annular structure which is not complementarily paired.
Preferably, the number of uracil modifications of the tag is at least 1.
Preferably, the 3' end of the tag is overhanging thymine.
In the invention, based on the deep research of a DNA sequencing technology, the inventor analyzes the advantages and the disadvantages of Illumina sequencing and finds that the source template of the read length obtained by a final sequencing library cannot be known due to the randomness of PCR amplification and fragmentation, so that in order to realize single-molecule coding sequencing of a sequencing sample, the inventor researches a specific label by a large amount of experiments and combining with creative ideas, and finally realizes the marking of each DNA fragment, thereby presenting the specific source of each read length in the final DNA library sequencing result.
In the invention, the tag has three conditions, and when one chain is annealed, a stem-loop structure is formed; in the second case: when two strands are subjected to two-end complementary pairing, a structure is formed; in the third case: when two strands are complementarily paired at one end to form a structure, the three cases are schematically shown in FIG. 1.
In a second aspect, the present invention provides a label introducing method, comprising the steps of:
(1) ligating the tag of the first aspect to a DNA sample;
(2) processing the product obtained in the step (1) to form a structure with a protruding 5' end;
(3) dephosphorizing the DNA tail end of the product obtained in the step (2) to form a structure of terminal hydroxyl;
(4) and (4) carrying out extension reaction on the product obtained in the step (3) to form a DNA fragment with two blunt ends and a tag.
In the present invention, if the DNA sample in step (1) is gDNA, the method further comprises a step of first fragmenting the DNA sample, and if the processed sample is cfDNA, the sample can be directly used in the subsequent experiment process.
Preferably, the fragmentation step is as follows:
(1') fragmenting gDNA using a fragmenting enzyme;
(2 ') screening the product of step (1').
Preferably, the screening method comprises magnetic bead double screening or gel cutting.
Preferably, the selected fragment is 100-250bp in length.
Preferably, the DNA sample in step (1) needs to be subjected to end repair and adenine addition treatment at the 3' end before the tag is connected.
Preferably, the enzyme used for the treatment of step (2) comprises uracil-specific excisionase, preferably USER enzyme (NEB M5505V/S/L).
Preferably, the dephosphorylated enzyme of step (3) comprises a phosphatase, preferably recombinant shrimp alkaline phosphatase (rSAP NEB M0371V/S/L).
Preferably, the method further comprises subjecting the blunt-ended and tagged DNA fragments obtained in step (4) to DNA end repair, and treating the 3' ends with A in preparation for subsequent adaptor ligation. .
In a third aspect, the present invention also provides a kit comprising a label according to the first aspect.
In a fourth aspect, the present invention also provides a tag according to the first aspect and/or a kit according to the third aspect for use in constructing a gene sequencing library, for use in DNA paired-end single molecule coding markers or for locating a useful source of sequencing reads for subsequent analytical interpretation of sequencing results.
Compared with the prior art, the invention has the following beneficial effects:
(1) the label provided by the invention has the advantages of ingenious conception, simple structure and obvious function, realizes the single molecule coding marking of the two ends of the DNA by selecting the specific structure and the modified label, more effectively positions the initial source of the sequencing read length, and solves the problem that the read length cannot be determined from which template the initial read length is amplified due to the randomness of PCR amplification and fragmentation in the prior art.
(2) The label introduction method provided by the invention is simple and efficient, is matched with a specific label, is convenient to operate, plays a role in assistance, realizes the label marking function, saves manpower and material resources, and has popularization and application prospects and values.
Drawings
FIG. 1 is a schematic diagram of three aspects of a tag of the present invention;
FIG. 2 is a schematic diagram of random tag (Barcode) of the present invention introduced into both ends of a DNA fragment.
Detailed Description
To further illustrate the technical means and effects of the present invention, the following further describes the technical solutions of the present invention by way of specific embodiments with reference to the drawings, but the present invention is not limited to the scope of the embodiments.
In the examples, the relevant terms explain:
(1) methylation modified primers: in the primer sequence, cytosine (C) is methylated to 5-methylcytosine (5-mdC).
(2) Barcode: and (4) random labeling.
(3) And (3) purification: in the examples, "purification" refers to magnetic bead purification, unless otherwise specified.
(4) Annealing: two or one DNA oligos with reverse complementary all or part are combined into double-stranded DNA under certain reaction condition.
(5) P5/P7: and (3) universal primers for final library amplification in the construction process of Illumina second generation library.
(6) Adding A: an adenine base (A) was added to the 3' -end of the DNA fragment.
Example 1
(1) A tag, the nucleotide sequence of which is shown in SEQ ID NO. 1:
SEQ ID NO.1, Oligo:
-CATAG/dU/NNNNNNNNCTATGT 3’
modification of 5' phosphorylation of Oligo: (
And five bases at the 5 'end are reversely complementary with the 3' end, and form a stem-loop structure after annealing, and the tag Barcode (namely random sequence Poly dNTP) of thymine is overhung at the 3 'end, and the first unpaired base close to the 5' end in the formed stem-loop structure is uracil (U).
(2) Sample treatment:
if the processed sample is gDNA, fragmenting 2 mu g of gDNA of the sample by utilizing a fragmenting enzyme of NEB company, selecting a DNA fragment of 100-250bp by a magnetic bead double-screen or gel cutting method, and treating the conditions of the fragmenting enzyme: dissolving the DNA after fragment selection with 30 mu L deionized water at 37 ℃ for 43 min;
if the processed sample is cfDNA, the sample can be directly used in the subsequent experimental process.
(3) Fragmented DNA end repair, 3' end addition a:
DNA end repair was performed using NEBNext Ultra II DNA Library Prep Kit for Illumina Kit, and A was added to the 3' end.
(4) And (3) label Barcode connection: the above products were reacted without purification according to the system shown in Table 1, and the schematic diagram of random tag (Barcode) introduced into both ends of DNA fragment is shown in FIG. 2:
TABLE 1
| Components | Volume of μ L |
| DNA after the last step | 60 |
| Barcode | 2.5 |
| Ultra connection II Master Mix | 30 |
| Ligation enhancing agent | 1 |
| In total | 93.5 |
(5) mu.L of USER enzyme and 3. mu.L of recombinant shrimp alkaline phosphatase (rSAP) were added to the above product, and reacted at 37 ℃ for 30min, see Table 2;
TABLE 2
| Components | Volume of μ L |
| DNA after the last step | 39 |
| rSAP | 2 |
| USER enzyme | 4 |
| |
5 |
| In total | 50 |
(6) Extending the label: the above product was purified and reacted according to the system shown in table 3:
TABLE 3
| Components | Volume of μ L |
| Barcode-ligated DNA | 30 |
| dNTP(mdCTP instead ofdCTP)(10mM) | 1 |
| 5 × EpiMark hot start Taq reaction buffer | 10 |
| EpiMark Hot Start Taq (2 units/. mu.L) | 0.25 |
| ddH2O | To 50 of |
PCR cycling conditions are shown in table 4:
TABLE 4
(7) And (3) purifying the product, repairing the DNA tail end by using a NEBNext Ultra II DNA Library Prep Kit for Illumina, adding A to the 3' end, connecting by a connector, and purifying.
(8) Final library amplification: the purified product was subjected to the reaction shown in table 5:
TABLE 5
| Components | Volume of μ L |
| Adaptor-ligated DNA | 30 |
| P7 | 2.5 |
| P5 | 2.5 |
| dNTP(10mM) | 1 |
| 5 × EpiMark hot start Taq reaction buffer | 10 |
| EpiMark H Hot Start Taq (2 units/. mu.L) | 0.25 |
| ddH2O | To 50 of |
PCR cycling conditions are shown in table 6:
TABLE 6
(11) The product was purified and stored at-20 ℃.
Example 2
(1) A tag, the nucleotide sequence of which is shown in SEQ ID NO. 1:
SEQ ID NO.1, Oligo:
-CATAG/dU/NNNNNNNNCTATGT 3’
modification of 5' phosphorylation of Oligo: (
And five bases at the 5 'end are reversely complementary with the 3' end, a stem-loop structure is formed after annealing, a tag Barcode of thymine is overhung at the 3 'end, and in the formed stem-loop structure, the first unpaired base close to the 5' end is uracil (U).
(2) Sample treatment:
if the processed sample is gDNA, fragmenting 2 mu g of gDNA of the sample by utilizing a fragmenting enzyme of NEB company, selecting a DNA fragment of 100-250bp by a magnetic bead double-screen or gel cutting method, and treating the conditions of the fragmenting enzyme: dissolving the DNA after fragment selection with 30 mu L deionized water at 37 ℃ for 43 min;
if the processed sample is cfDNA, the sample can be directly used in the subsequent experimental process.
(3) Fragmented DNA end repair, 3' end addition a:
DNA end repair was performed using NEBNext Ultra II DNA Library Prep Kit for Illumina Kit, and A was added to the 3' end.
(4) And (3) label Barcode connection: the above product was reacted without purification according to the system shown in table 7:
TABLE 7
(5) To the above product, 3. mu.L of USER enzyme and 3. mu.L of recombinant shrimp alkaline phosphatase (rSAP) were added and reacted at 37 ℃ for 30min, see Table 8;
TABLE 8
| Components | Volume of μ L |
| DNA after the last step | 39 |
| rSAP | 2 |
| USER enzyme | 4 |
| |
5 |
| In total | 50 |
(6) Extending the label: the above product was purified and reacted according to the system shown in table 9:
TABLE 9
PCR cycling conditions are shown in table 10:
watch 10
| Temperature/. degree.C | Time |
| 95 | 45s |
| 61 | 30s |
| 68 | 5min |
| 4 | Holding |
(8) And purifying the product, repairing the DNA tail end by using a NEBNext Ultra II DNA Library Prep Kit for Illumina, adding A to the 3' end, and connecting by using a methylation connector.
(9) The product was purified, and then bisulfite-treated and column-purified according to ZYMO EZ DNA Methylation Gold Kit.
(10) Final library amplification increased INDEX: the purified product was subjected to the reaction shown in table 11:
TABLE 11
| Components | System μ L |
| Modified DNA | 30 |
| NEBNext universal PCR primer | 2.5 |
| NEBNext index (X) primer | 2.5 |
| dNTP(10mM) | 1 |
| 5 × EpiMark hot start Taq reaction buffer | 10 |
| EpiMark Hot Start Taq (2 units/. mu.L) | 0.25 |
| ddH2O | To 50 of |
PCR cycling conditions are shown in table 12:
TABLE 12
(10) The product was purified and stored at-20 ℃.
In conclusion, the invention provides a tag and application thereof, which realizes the marking of DNA double-end single-molecule coding by selecting a specific structure and a modified tag, more effectively locates the initial source of sequencing read length, and solves the problem that the read length cannot be determined from which template the initial read length is amplified due to the randomness of PCR amplification and fragmentation in the prior art; the provided label introduction method is simple and efficient, is matched with a specific label, is convenient to operate, plays a role in assistance, realizes the label marking function, saves manpower and material resources, and has popularization and application prospects and values.
The applicant states that the present invention is illustrated in detail by the above examples, but the present invention is not limited to the above detailed methods, i.e. it is not meant that the present invention must rely on the above detailed methods for its implementation. It should be understood by those skilled in the art that any modification of the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific modes, etc., are within the scope and disclosure of the present invention.
SEQUENCE LISTING
<110> Shenzhen haipraos medical examination laboratory
<120> a single-molecule tag for labeling DNA fragments
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 21
<212> DNA
<213> Artificial sequence
<220>
<221> misc_feature
<222> (7)..(14)
<223> n is a, c, g, or t
<400> 1
catagdunnn nnnnnctatg t 21
Claims (10)
1. A single molecular label for marking DNA fragments is characterized in that the single molecular label is any one of a stem-loop structure formed by annealing one oligo, a structure formed by annealing two oligos and complementary pairing at two ends, or a structure formed by annealing two oligos and complementary pairing at one end; the oligo is sequentially provided with a first nucleotide sequence, uracil, poly dNTP, a second nucleotide sequence and base protruding from the 3 'end from the 5' end to the 3 'end, the first nucleotide sequence and the second nucleotide sequence are complementarily paired, wherein the first unpaired base close to the 5' end has uracil modification on the annular structure without complementary pairing.
2. The single molecule tag of claim 1, wherein the first nucleotide sequence is a CATAG.
3. The single molecule tag of claim 1, wherein the second nucleotide sequence is CTATG.
4. The single molecule tag of claim 1, wherein the single molecule tag has an overhanging base at the 3 'end, wherein the overhanging base at the 3' end is thymine.
5. The single molecule tag of claim 1, wherein the 5' end of the single molecule tag is phosphorylated.
6. The single molecule tag according to claim 1, wherein the number of uracil modifications of the single molecule tag is not less than one.
7. The single-molecule tag of claim 1, wherein the single-molecule tag labels the DNA fragments by ligation thereto, and wherein the DNA fragments are end-repaired before the single-molecule tag is ligated thereto.
8. The single-molecule tag of claim 7, wherein the single-molecule tag labels the DNA fragment by linking to the DNA fragment, and further adenine is added to the 3' end of the DNA fragment before the single-molecule tag is linked to the DNA fragment.
9. The single molecule tag of claim 7 or 8, wherein after the single molecule tag is ligated to the DNA fragment, the DNA fragment is ligated to a universal primer for Illumina second generation library amplification.
10. The single molecule tag of claim 9, wherein the universal primers for amplification of the Illumina second generation library are P5 and P7.
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Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20030050453A1 (en) * | 1997-10-06 | 2003-03-13 | Joseph A. Sorge | Collections of uniquely tagged molecules |
| US20060234230A1 (en) * | 2002-05-30 | 2006-10-19 | Yusuke Nakamura | Method of detecting gene polymorphism |
| WO2010048605A1 (en) * | 2008-10-24 | 2010-04-29 | Epicentre Technologies Corporation | Transposon end compositions and methods for modifying nucleic acids |
| CN106086162A (en) * | 2015-11-09 | 2016-11-09 | 厦门艾德生物医药科技股份有限公司 | A kind of double label joint sequences for detecting Tumor mutations and detection method |
| CN106148503A (en) * | 2015-04-22 | 2016-11-23 | 王金 | A kind of method detecting DNA sequence dna |
| CN106755451A (en) * | 2017-01-05 | 2017-05-31 | 苏州艾达康医疗科技有限公司 | Nucleic acid is prepared and analyzed |
| CN107124888A (en) * | 2014-11-21 | 2017-09-01 | 深圳华大基因科技有限公司 | Bubbling straight coupling element and the method that sequencing library is built using it |
| CN108774640A (en) * | 2018-06-20 | 2018-11-09 | 深圳海普洛斯医学检验实验室 | A kind of unimolecule label and its application |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110643680B (en) * | 2016-05-27 | 2023-05-26 | 深圳市海普洛斯生物科技有限公司 | Joint suitable for ultra-trace DNA sequencing and application thereof |
| GB201615486D0 (en) * | 2016-09-13 | 2016-10-26 | Inivata Ltd | Methods for labelling nucleic acids |
| CN106906211B (en) * | 2017-04-13 | 2020-11-20 | 苏州普瑞迈德医学检验所有限公司 | Molecular joint and application thereof |
| CN109797197A (en) * | 2019-02-11 | 2019-05-24 | 杭州纽安津生物科技有限公司 | It a kind of single chain molecule label connector and single stranded DNA banking process and its is applied in detection Circulating tumor DNA |
-
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- 2018-06-20 CN CN201810638123.6A patent/CN108774640B/en active Active
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Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20030050453A1 (en) * | 1997-10-06 | 2003-03-13 | Joseph A. Sorge | Collections of uniquely tagged molecules |
| US20060234230A1 (en) * | 2002-05-30 | 2006-10-19 | Yusuke Nakamura | Method of detecting gene polymorphism |
| WO2010048605A1 (en) * | 2008-10-24 | 2010-04-29 | Epicentre Technologies Corporation | Transposon end compositions and methods for modifying nucleic acids |
| CN107124888A (en) * | 2014-11-21 | 2017-09-01 | 深圳华大基因科技有限公司 | Bubbling straight coupling element and the method that sequencing library is built using it |
| CN106148503A (en) * | 2015-04-22 | 2016-11-23 | 王金 | A kind of method detecting DNA sequence dna |
| CN106086162A (en) * | 2015-11-09 | 2016-11-09 | 厦门艾德生物医药科技股份有限公司 | A kind of double label joint sequences for detecting Tumor mutations and detection method |
| CN106755451A (en) * | 2017-01-05 | 2017-05-31 | 苏州艾达康医疗科技有限公司 | Nucleic acid is prepared and analyzed |
| CN108774640A (en) * | 2018-06-20 | 2018-11-09 | 深圳海普洛斯医学检验实验室 | A kind of unimolecule label and its application |
Non-Patent Citations (1)
| Title |
|---|
| 文思远, 张敏丽, 陈苏红, 王升启: "基因芯片结合可环化探针对单碱基突变的检测", 军事医学科学院院刊, no. 06, pages 530 - 533 * |
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