WO2019237405A1 - Dye encoding method based on fluorescence-labeled nucleotide - Google Patents

Dye encoding method based on fluorescence-labeled nucleotide Download PDF

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WO2019237405A1
WO2019237405A1 PCT/CN2018/091982 CN2018091982W WO2019237405A1 WO 2019237405 A1 WO2019237405 A1 WO 2019237405A1 CN 2018091982 W CN2018091982 W CN 2018091982W WO 2019237405 A1 WO2019237405 A1 WO 2019237405A1
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fluorescent
cds
cdse
probe molecule
sequence
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Chinese (zh)
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车团结
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苏州百源基因技术有限公司
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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
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
    • C12Q1/6837—Enzymatic or biochemical coupling of nucleic acids to a solid phase using probe arrays or probe chips
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54326—Magnetic particles
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids

Definitions

  • the invention belongs to the field of gene detection, and particularly relates to a dye coding method based on fluorescently labeled nucleotides, a tag sequence, a liquid chip, and uses.
  • Liquid chips also known as suspension arrays
  • the core of this technology is to encode polystyrene microspheres with fluorescent staining, and then covalently cross-link microspheres of each color (or fluorescently coded microspheres) to target Probes for specific nucleic acids.
  • the detection sample is subjected to a hybridization reaction with a plurality of microspheres with specific nucleic acid probes at the same time. After the reaction is completed, the instrument recognizes the encoded microspheres by two laser beams and detects the fluorescence intensity of the reporter molecule on the microspheres, so that Target nucleic acid is quantitatively detected.
  • the technical problem to be solved by the present invention is to provide a dye coding method, a tag sequence, and a liquid, which respectively label probes with different colors, have high luminous intensity, are not easily quenched, and have little overlap between spectra. Chips and uses.
  • the present invention provides a method for dye coding based on fluorescently labeled nucleotides, which uses a nucleotide double-stranded sequence as a tag sequence and divides the tag sequence along the extension direction of the nucleotide double-stranded sequence. For at least two fluorescent regions, the nucleotides in each fluorescent region are connected to at least one fluorescent dye, and adjacent two fluorescent regions are separated by at least 5 nucleotides.
  • the tag sequence is divided into at least four fluorescent regions, and the length of the nucleotides in the fluorescent region is 15-20bp.
  • the fluorescent dyes in any two of the fluorescent regions are different.
  • nucleotide linked to a fluorescent dye there is only one type of nucleotide linked to a fluorescent dye in each of the fluorescent regions, and the fluorescent dye in the same fluorescent region is the same.
  • the fluorescent dye includes BODIPY, FITC, rhodamine, coumarin, xanthene, anthocyanin, osmium or phthalocyanine;
  • the quantum dot is selected from MgS, MgSe, MgTe, CaS, CaSe, CaTe, ZnO , ZnS, ZnSe, ZnTe, SrS, SrSe, SeTe, CdS, CdSe, CdTe, BaS, BaSe, BaTe, HgS, HgSe, HgTe, PbSe, CaAs, InP, InAs, InCaAs, ZnS / CdS, ZnS / CdS / ZnS , ZnS / HgS / ZnS / CdS, CdS / ZnS, CdS / Ag2S, CdS / HgS, CdS / HgS,
  • the tag sequence is a DNA double-stranded sequence or an RNA double-stranded sequence.
  • the present invention provides a tag sequence encoded by the dye-coding method based on a fluorescently labeled nucleotide.
  • the invention provides a liquid phase chip, and the invention provides:
  • a probe molecule P2 connected to the magnetic microsphere, the probe molecule P1 and the probe molecule P2 are not bound to each other.
  • the probe molecule P1 comprises a nucleotide sequence, an antigen or an antibody
  • the probe molecule P2 comprises a nucleotide sequence, an antigen or an antibody
  • biotin or a fluorescent dye is connected to the probe molecule P2, and the fluorescent dye connected to the probe molecule P2 is different from the fluorescent dye in the tag sequence.
  • the invention provides a method for preparing the liquid phase chip, which includes the following steps:
  • the invention provides a use of the liquid-phase chip in the field of nucleic acid or protein detection.
  • the present invention has the following advantages:
  • a method for dye coding based on fluorescently labeled nucleotides which uses a nucleotide double-stranded sequence as a tag sequence and divides the tag sequence into at least two along the extension direction of the nucleotide double-stranded sequence Two fluorescent regions, and at least 5 nucleotides between two adjacent fluorescent regions, so that the fluorescence emitted between the two regions does not interfere with each other.
  • each fluorescent region is connected with a fluorescent dye or quantum dot, and the label can be achieved by arranging and combining the fluorescent dye or quantum dot connected with each fluorescent region.
  • the sequence is numbered for the purpose.
  • the tag sequence is divided into four fluorescent regions. By combining 4 different fluorescent dyes or quantum dots, 24 different combinations can be obtained. If different fluorescent regions can use the same fluorescent dye or quantum dot, 4 Fluorescent areas can be obtained in 256 different combinations. By analogy, the more fluorescent regions are divided into the tag sequence, the more combinations can be obtained, and more numbered tag sequences can be realized, which greatly expands the number range of the tag sequence. In addition, by using different colors of fluorescent dyes or quantum dots, it is possible to effectively avoid overlap between the spectra.
  • the number of fluorescent dyes or quantum dots connected in each fluorescent region of the tag sequence is different, so that the light intensity of each fluorescent region is different.
  • Permutation and combination of the luminous intensity of each fluorescent region can achieve the purpose of numbering the tag sequence; for example, the tag sequence is divided into 4 fluorescent regions, each fluorescent region can be connected to 10 quantum dots, and a fluorescent intensity is set for each two quantum dots.
  • Level, divided into 5 fluorescence intensity levels, 4 fluorescent regions can be obtained in 5 4 different arrangements, and so on, the label sequence is set to N fluorescent regions, each fluorescent region is set to M fluorescent intensity levels, you can get M N different arrangement methods greatly expand the number range of the tag sequence.
  • the liquid-phase chip provided by the present invention comprises a tag sequence, a probe molecule P1 connected to the tag sequence, and a magnetic microsphere-labeled probe molecule P2, and detects the fluorescence emitted by the fluorescent dye of each fluorescent region of the tag sequence to obtain a tag.
  • the sequence number is used to know whether the target nucleic acid sequence exists in the test sequence, and the target nucleic acid sequence can be qualitatively detected.
  • the liquid-phase chip provided by the present invention can utilize the fluorescence intensity of the fluorescent dye or biotin on the probe molecule P2 to be proportional to the concentration of the target nucleic acid sequence, so that the target nucleic acid sequence can be quantitatively detected.
  • the liquid-phase chip provided by the present invention can detect a plurality of different target sequences at one time, and realize high-throughput detection.
  • the probe molecule P1 includes a nucleotide sequence, an antigen, or an antibody
  • the probe molecule P2 includes a nucleotide sequence, an antigen, or an antibody, so as to realize the detection of the nucleotide sequence, antigen, or antibody .
  • FIG. 1 is a coding schematic diagram of a dye coding method for fluorescently labeled nucleotides in an embodiment of the present invention
  • FIG. 2 is a schematic diagram of detection of a liquid phase chip in an embodiment of the present invention.
  • FIG. 3 is a liquid crystal chip detection result of ALK, APC, BRAF and EGFR genes in an experimental example of the present invention.
  • This embodiment provides a method for dye coding based on fluorescently labeled nucleotides.
  • a double-stranded DNA sequence is used as a tag sequence with a length of 95 bp.
  • the tag sequence is divided into four fluorescent regions, respectively. It is fluorescent region I, fluorescent region II, fluorescent region III, and fluorescent region IV, all of which are 20 bp in length, with at least 5 nucleotides spaced between two adjacent fluorescent regions. In this embodiment, it is two adjacent fluorescent regions.
  • base A in fluorescent region I is connected to rhodamine
  • base C in fluorescent region II is connected to anthocyanin
  • base T in fluorescent region III is connected to coumarin
  • fluorescent region The base G in IV is attached to the phthalocyanine.
  • the fluorescent dyes connected to each fluorescent region are arranged and combined.
  • Each fluorescent region uses different fluorescent dyes to obtain 24 different arrangements, and for each arrangement Numbering is used as the number of the label sequence to realize numbering of the label sequence.
  • the coding method divides the tag sequence into a plurality of fluorescent regions, and the interval between two adjacent fluorescent regions is at least 5 bp, so that the emitted fluorescence between the two regions does not interfere with each other.
  • a fluorescent dye is connected to each fluorescent region, and the number of tag sequences can be achieved by arranging and combining the fluorescent dyes connected to each fluorescent region. For example, as shown in Table 1, an example of the combination method of arrangement is given. By combining 4 different fluorescent dyes, 24 different combinations can be obtained. If different fluorescent regions can use the same fluorescent dye, 4 There are 256 different combinations of fluorescence regions.
  • Dividing the tag sequence into 5 fluorescent regions can get 625 different combinations, and so on.
  • the more the tag sequence is divided into more fluorescent regions the more combinations can be obtained, and more numbered tag sequences can be achieved, greatly expanding.
  • the number range of the tag sequence In addition, by using fluorescent dyes of different colors, it is possible to effectively avoid overlap between the spectra.
  • This embodiment provides a method for dye coding based on fluorescently labeled nucleotides.
  • a double-stranded DNA sequence is used as a tag sequence with a length of 66 bp.
  • the tag sequence is divided into four fluorescent regions, which are respectively fluorescent region I and fluorescent.
  • Region II, fluorescence region III, and fluorescence region IV are all 15 bp in length, with at least 5 nucleotides spaced between two adjacent fluorescent regions, and in this embodiment, 7 spaces between two adjacent fluorescent regions Nucleotide; base T in fluorescent region I is linked to ⁇ , base C in fluorescent region II is connected to BODIPY, base T in fluorescent region III is connected to FITC, and base G in fluorescent region IV is connected to rhodamine.
  • the fluorescent dyes connected to each fluorescent region are arranged and combined.
  • Each fluorescent region uses different fluorescent dyes to obtain 24 different arrangements, and for each arrangement Numbering is used as the number of the label sequence to realize numbering of the label sequence.
  • This embodiment provides a method for dye coding based on fluorescently labeled nucleotides.
  • a double-stranded DNA sequence is used as a tag sequence with a length of 100 bp.
  • the tag sequence is divided into five fluorescent regions, which are respectively fluorescent region I and fluorescent.
  • Region II, fluorescent region III, fluorescent region IV, and fluorescent region V are all 15 bp in length, with at least 5 nucleotides spaced between two adjacent fluorescent regions, in this embodiment, between two adjacent fluorescent regions 5 nucleotides apart; base C in fluorescent region I is linked to xanthene, base G in fluorescent region II is linked to anthocyanins, base A in fluorescent region III is linked to pyrene, and The base T is connected to BODIPY, and the base G in the fluorescent region V is connected to rhodamine.
  • the above five fluorescent dyes (xanthene, anthocyanin, hydrazone, BODIPY, and rhodamine) are selected and combined to label the above five fluorescent regions, and realize 625 tag sequences Numbering.
  • This embodiment provides a method for dye coding based on fluorescently labeled nucleotides.
  • a double-stranded ribonucleic acid sequence is used as a tag sequence with a length of 50 bp.
  • the tag sequence is divided into two fluorescent regions, namely a fluorescent region I and a fluorescent region. II, both are 20 bp in length, with at least 5 nucleotides spaced between two adjacent fluorescent regions, in this embodiment, 10 nucleotides spaced between two adjacent fluorescent regions;
  • the base A is linked to the phthalocyanine, and the base C in the fluorescent region II is linked to anthocyanins.
  • This embodiment provides a method for dye coding based on fluorescently labeled nucleotides.
  • a double-stranded DNA sequence is used as a tag sequence with a length of 105 bp.
  • the tag sequence is divided into six fluorescent regions, which are respectively fluorescent region I and fluorescent.
  • Region II, fluorescent region III, fluorescent region IV, fluorescent region V, and fluorescent region VI are all 15 bp in length, with at least 5 nucleotides spaced between two adjacent fluorescent regions, which in this embodiment are two adjacent
  • the fluorescent regions are separated by 5 nucleotides; the base T in the fluorescent region I is connected to coumarin, the base G in the fluorescent region II is connected to anthocyanins, and the base A in the fluorescent region III is connected to coumarin.
  • the base T in the fluorescent region IV is connected to rhodamine, the base C in the fluorescent region V is connected to coumarin, and the base G in the fluorescent region VI is connected to BODIPY.
  • the fluorescent dyes connected to each fluorescent region are arranged and combined, and each arrangement is numbered as the number of the tag sequence to realize numbering of the tag sequence.
  • the tag sequence can be divided into two, three, four, five or more fluorescent regions, as long as the interval between adjacent two fluorescent regions is at least 5bp, and there is only one base in each fluorescent region Attach a fluorescent dye.
  • the fluorescent dye may be BODIPY, FITC, rhodamine, coumarin, xanthene, anthocyanins, osmium, and phthalocyanine.
  • This embodiment provides a method for dye coding based on fluorescently labeled nucleotides.
  • a double-stranded DNA sequence is used as a tag sequence with a length of 95 bp.
  • the tag sequence is divided into four fluorescent regions, which are respectively fluorescent region I and fluorescent.
  • Region II, fluorescence region III, and fluorescence region IV are all 20 bp in length, with at least 5 nucleotides spaced between two adjacent fluorescent regions, and in this embodiment, 5 nuclei spaced between two adjacent fluorescent regions.
  • Glycine base A in fluorescent region I is connected to CdTe
  • base C in fluorescent region II is connected to CaS
  • base T in fluorescent region III is connected to CdS
  • base G in fluorescent region IV is connected to CdSe.
  • the coding method divides the tag sequence into a plurality of fluorescent regions, and the interval between two adjacent fluorescent regions is at least 5 bp, so that the emitted fluorescence between the two regions does not interfere with each other.
  • a quantum dot is connected to each fluorescent region, and the number of tag sequences can be achieved by arranging and combining the quantum dots connected to each fluorescent region. For example, as shown in Table 2, an example of the arrangement of the combination is given. By combining 4 different fluorescent dyes, 24 different combinations can be obtained. If different fluorescent regions can use the same quantum dot, 4 There are 256 different combinations of fluorescence regions.
  • Dividing the tag sequence into 5 fluorescent regions can get 625 different combinations, and so on.
  • the more the tag sequence is divided into more fluorescent regions the more combinations can be obtained, and more numbered tag sequences can be achieved, greatly expanding.
  • the number range of the tag sequence In addition, by using fluorescent dyes of different colors, it is possible to effectively avoid overlap between the spectra.
  • This embodiment provides a method for dye coding based on fluorescently labeled nucleotides.
  • a double-stranded DNA sequence is used as a tag sequence with a length of 95 bp.
  • the tag sequence is divided into four fluorescent regions, which are respectively fluorescent region I and fluorescent.
  • Region II, fluorescence region III, and fluorescence region IV are all 20 bp in length, with at least 5 nucleotides spaced between two adjacent fluorescent regions, and in this embodiment, 5 nuclei spaced between two adjacent fluorescent regions.
  • nucleotide; nucleotides in fluorescent region I are connected to CdTe
  • nucleotides in fluorescent region II are connected to CaS
  • nucleotides in fluorescent region III are connected to CdS
  • nucleotides in fluorescent region IV are connected to CdSe.
  • Each fluorescence region is connected to two quantum dots in one fluorescence intensity level. For example, two nucleotides in fluorescent region I are connected to CdTe, then the fluorescence intensity level in fluorescent region I is CdTe intensity I, and four in fluorescence region II.
  • the fluorescence intensity level of fluorescent region II is CaS intensity II
  • the fluorescence intensity level of fluorescent region III is CdS intensity III
  • the fluorescence intensity level of the fluorescent region IV is CdSe intensity IV.
  • each fluorescent region is divided into five fluorescent intensity levels: intensity I, intensity II, intensity III, intensity IV, and intensity V.
  • Fluorescent region I Fluorescent Region II Fluorescent Region III Fluorescent Region IV 1 CdTe intensity I CaS intensity II CdS intensity III CdS intensity IV 2 CdT intensity III CaS intensity I CdS intensity IV CdS intensity V 3 CdT intensity III CdS intensity IV CdSe intensity I CaS intensity V 4 CdT intensity IV CdS intensity III CaS intensity II CdSe intensity I
  • This embodiment provides a method for dye coding based on fluorescently labeled nucleotides.
  • a double-stranded DNA sequence is used as a tag sequence with a length of 125 bp.
  • the tag sequence is divided into five fluorescent regions, which are respectively fluorescent region I and fluorescent.
  • Region II, fluorescent region III, fluorescent region IV, and fluorescent region V are all 20 bp in length, with at least 5 nucleotides spaced between two adjacent fluorescent regions, in this embodiment between two adjacent fluorescent regions 5 nucleotides apart; nucleotides in fluorescent region I are linked to CdTe, nucleotides in fluorescent region II are linked to CaS, nucleotides in fluorescent region III are linked to CdS, and nucleotides in fluorescent region IV are linked CdSe, nucleotides in fluorescent region V are linked to HgS.
  • Each fluorescence region is connected to two quantum dots in one fluorescence intensity level.
  • the fluorescence intensity level in fluorescent region I is CdTe intensity I, and four in fluorescence region II.
  • the fluorescence intensity level of fluorescent region II is CaS intensity II
  • six nucleotides in fluorescent region III are connected to CdS
  • the fluorescence intensity level of fluorescent region III is CdS intensity III
  • the fluorescence intensity level of fluorescent region IV is CdSe intensity IV
  • ten nucleotides in the fluorescent region V are connected to HgS
  • the fluorescence intensity level of the fluorescent region V is HgS intensity V.
  • each fluorescent region is divided into six fluorescence intensity levels: intensity I, intensity II, intensity III, intensity IV, intensity V, and intensity VI.
  • the combination and arrangement of the fluorescence intensity levels of the quantum dots connected to each fluorescent region can achieve Objective tag sequence numbering, by five different quantum dots, 6 fluorescence intensity levels are combined can be obtained 65 different combinations, that is numbered 65.
  • the tag sequence can be divided into two, three, four, five or more fluorescent regions, as long as the interval between adjacent two fluorescent regions is at least 5bp, and one type of nucleotide is connected in each fluorescent region Quantum dots.
  • the quantum dots may be MgS, MgSe, MgTe, CaS, CaSe, CaTe, ZnO, ZnS, ZnSe, ZnTe, SrS, SrSe, SeTe, CdS, CdSe, CdTe, BaS, BaSe, BaTe, HgS , HgSe, HgTe, PbSe, CaAs, InP, InAs, InCaAs, ZnS / CdS, ZnS / CdS / ZnS, ZnS / HgS / ZnS / CdS, CdS / ZnS, CdS / Ag2S, CdS / HgS, CdS / HgS / CdS , CdS / PbS, CdS / Cd (OH) 2, CdSe / CuSe, CdSe / ZnS, CdS
  • the tag sequence is divided into N fluorescent regions with an interval of at least 5bp between two adjacent fluorescent regions.
  • a nucleotide is connected to a quantum dot in each fluorescent region.
  • Each fluorescent region is divided into M fluorescence intensity levels. Different kinds of quantum dots and M fluorescence intensity levels are combined to obtain M N different combinations, that is, M N number.
  • a liquid phase chip provided in this embodiment includes a tag sequence encoded by the dye coding method based on a fluorescently labeled nucleotide in Embodiment 1;
  • a probe molecule P1 and a probe molecule P2 are designed.
  • the probe molecule P1 can hybridize and bind to the 5 'end of the target nucleic acid sequence, and the probe molecule P2 can hybridize and bind to the 3' end of the target nucleic acid sequence.
  • the probe molecule P1 and probe molecule P2 are not complementary, and a fluorescent dye FITC is connected to the probe molecule P2.
  • the probe molecule P1 is connected to a tag sequence, and the probe molecule P2 is connected to a magnetic microsphere; the tag sequence to which the probe molecule P1 is connected and the magnetism to which the probe molecule P2 is connected. Microsphere mix.
  • the PCR amplification product simultaneously performs hybridization reaction with the tag sequence and magnetic microspheres. If the sample to be tested contains the target nucleic acid Sequence, the probe molecule P1 on the tag sequence and the probe molecule P2 on the magnetic microsphere can specifically bind to the target nucleic acid sequence through base complementation. The resulting complex can be magnetically separated from the reaction system using magnetic microspheres.
  • the target nucleic acid sequence is quantitatively detected based on the fluorescence intensity of the fluorescent dye on the tag sequence and the concentration of the target nucleic acid sequence.
  • the probe molecule P1 for the target nucleic acid cannot be bound to the magnetic microsphere-labeled probe molecule P2 through the target nucleic acid sequence, and is not magnetic, so it can be taken out during magnetic separation.
  • This embodiment provides a liquid phase chip, which includes a tag sequence encoded by the dye-encoding method based on a fluorescently labeled nucleotide of Example 2, that is, the tag sequence is a double-stranded DNA sequence with a length of 95 bp, as shown in FIG.
  • the tag sequence is divided into four fluorescent regions, namely, fluorescent region I, fluorescent region II, fluorescent region III, and fluorescent region IV, each having a length of 20 bp, and the interval between adjacent two fluorescent regions is at least 5 bp;
  • Anthocyanin, coumarin and phthalocyanine are each connected to a nucleotide in four fluorescent regions, where base A is connected to rhodamine, base C is connected to anthocyanin, and base T is connected to coumarin
  • the base G is connected to the phthalocyanine, and the fluorescent dyes connected to each fluorescent region are arranged and combined.
  • Each fluorescent region uses different fluorescent dyes to obtain 24 different arrangements, and each arrangement is numbered as a label sequence. Number.
  • Probe molecule P1 and probe molecule P2 are designed according to 24 different target nucleic acid sequences.
  • Probe molecule P1 can hybridize and bind to the 5 'end of the target nucleic acid sequence, and probe molecule P2 can hybridize to the 3' end of the target nucleic acid sequence.
  • the probe molecule P1 and the probe molecule P2 are not complementary, and a fluorescent dye anthocyanin is connected to the probe molecule P2.
  • the probe molecule P1 is connected to a tag sequence, and the probe molecule P2 is connected to a magnetic microsphere; the tag sequence to which the probe molecule P1 is connected and the magnetism to which the probe molecule P2 is connected. Microsphere mix.
  • the PCR amplification product performs hybridization reaction with the tag sequence and magnetic microspheres at the same time.
  • Target nucleic acid sequence, the probe molecule P1 on the tag sequence and the probe molecule P2 on the magnetic microsphere can specifically bind to the target nucleic acid sequence through base complementation, and the resulting complex can be magnetically separated from the magnetic microsphere It is separated from the reaction system, and the fluorescence emitted by the fluorescent dye in each fluorescent region of the tag sequence is detected to obtain the number of the tag sequence.
  • the target nucleic acid sequence exists in the test sequence, and the target nucleic acid sequence can be qualitatively detected.
  • the fluorescence intensity of the fluorescent dye anthocyanin on the needle molecule P2 is directly proportional to the concentration of the target nucleic acid sequence, and the target nucleic acid sequence is quantitatively detected.
  • This embodiment provides a liquid phase chip, which includes a tag sequence encoded by the dye-coding method based on a fluorescently labeled nucleotide in Example 7, that is, the tag sequence is a double-stranded DNA sequence with a length of 95 bp.
  • fluorescent region I Divided into four fluorescent regions, namely fluorescent region I, fluorescent region II, fluorescent region III and fluorescent region IV, both of which are 20 bp in length, and the interval between two adjacent fluorescent regions is at least 5 bp; CdTe, CaS, CdS, and CdSe, respectively Linked to a nucleotide in four fluorescent regions, where base A in fluorescent region I is connected to CdTe, base C in fluorescent region II is connected to CaS, base T in fluorescent region III is connected to CdS, and fluorescence The base G in region IV is connected to CdSe, and each fluorescent region is connected to two quantum dots as a fluorescence intensity level.
  • each fluorescent region is divided into five fluorescent intensity levels: intensity I, intensity II, intensity III, intensity IV, and intensity V.
  • the combination and arrangement of the fluorescence intensity levels of the quantum dots connected to each fluorescent region can achieve The label sequence is numbered for the purpose. By combining four different quantum dot fluorescence intensity levels, 625 different combinations can be obtained, and each arrangement is numbered as the number of the tag sequence.
  • probe molecules P1 and P2 are designed.
  • the probe molecule P1 can hybridize and bind to the 5 'end of the target nucleic acid sequence, and the probe molecule P2 can hybridize and bind to the 3' end of the target nucleic acid sequence.
  • the probe molecule P1 and the probe molecule P2 are not complementary, and a fluorescent dye FITC is connected to the probe molecule P2.
  • the probe molecule P1 is connected to a tag sequence, and the probe molecule P2 is connected to a magnetic microsphere; the tag sequence to which the probe molecule P1 is connected and the magnetic microsphere to which the probe molecule P2 is connected are mixed.
  • the PCR amplification product performs hybridization reaction with the tag sequence and magnetic microspheres at the same time.
  • Target nucleic acid sequence, the probe molecule P1 on the tag sequence and the probe molecule P2 on the magnetic microsphere can specifically bind to the target nucleic acid sequence through base complementation, and the resulting complex can be magnetically separated from the magnetic microsphere It is separated from the reaction system, and the fluorescence emitted by the fluorescent dye in each fluorescent region of the tag sequence is detected to obtain the number of the tag sequence.
  • the fluorescence intensity of the fluorescent dye FITC on the needle molecule P2 is directly proportional to the concentration of the target nucleic acid sequence, and the target nucleic acid sequence is quantitatively detected.
  • This experimental example uses the liquid-phase chip in Example 11 to detect four tumor-targeting drug-related genes, including ALK, APC, BRAF, and EGFR.
  • the tag sequence is shown in SEQ NO.1, the probe molecule P1 of the ALK gene is shown in SEQ NO.2, the probe molecule P2 is shown in SEQ NO.3, and the probe molecule P1 of the APC gene is shown in SEQ NO.4
  • the probe molecule P2 is shown in SEQ NO.5; the probe molecule P1 of the BRAF gene is shown in SEQ NO.6, the probe molecule P2 is shown in SEQ NO.7; the probe molecule P1 of the EGFR gene is shown in SEQ.
  • the probe molecule P2 is shown in SEQ. NO.9.
  • FIG. 3 is a standard curve for detecting a gene-mixed sample related to tumor-targeting drug administration using a flow cytometer using the liquid-phase chip of the present invention, and a regression equation of each standard curve is obtained by a least square method.
  • R2 is higher than 0.99, indicating fluorescence
  • the fluorescence intensity of the dye FITC is directly proportional to the concentration of the target nucleic acid sequence, and the target nucleic acid sequence can be quantitatively detected based on this.

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Abstract

Provided are a dye encoding method based on fluorescence-labeled nucleotide and an obtained label sequence. That is, a nucleotide double-chain label sequence is divided into at least two fluorescence areas, at least five pieces of nucleotide are spaced between the two fluorescence areas, and the nucleotide is at least connected with one type of fluorescence dye or quantum dot. Also provided are a probe P1 connected with the label sequence, a liquid phase chip of a probe P2 connected with a magnetic microsphere, a manufacturing method for the liquid phase chip, and the use of the liquid phase chip in the field of nucleotide or protein detection.

Description

基于荧光标记核苷酸的染料编码方法Dye coding method based on fluorescently labeled nucleotides 技术领域Technical field
本发明属于基因检测领域，具体涉及基于荧光标记核苷酸的染料编码方法、标签序列、液体芯片以及用途。The invention belongs to the field of gene detection, and particularly relates to a dye coding method based on fluorescently labeled nucleotides, a tag sequence, a liquid chip, and uses.
背景技术Background technique
液体芯片，又称悬浮阵列，该技术的核心是将聚苯乙烯微球用荧光染色的方法进行编码，然后将每种颜色的微球(或称为荧光编码微球)共价交联上针对特定核酸的探针。应用时，将检测样本与多种连有特定核酸探针的微球同时进行杂交反应，反应完成后，仪器通过两束激光分别识别编码微球和检测微球上报告分子的荧光强度，从而对靶核酸进行定量检测。由于整个反应在液相中完成，反应速度快，杂交效率高，并可以在一个微量反应体系中同时检测多达上百条靶序列。然而，近年研究结果显示，液体芯片技术尚存在灵敏度较低、重复性较差等不足，其原因主要是不同荧光标记物的发射光谱在液相中广泛重叠，导致大量假阳性结果产生，限制了液体芯片技术的进一步推广应用。因此，寻找一种能以不同颜色分别标记探针，且发光强度高，不易淬灭，光谱之间重叠少的荧光标记物是提高液体芯片检测灵敏度和重复性的关键。Liquid chips, also known as suspension arrays, the core of this technology is to encode polystyrene microspheres with fluorescent staining, and then covalently cross-link microspheres of each color (or fluorescently coded microspheres) to target Probes for specific nucleic acids. In application, the detection sample is subjected to a hybridization reaction with a plurality of microspheres with specific nucleic acid probes at the same time. After the reaction is completed, the instrument recognizes the encoded microspheres by two laser beams and detects the fluorescence intensity of the reporter molecule on the microspheres, so that Target nucleic acid is quantitatively detected. Because the entire reaction is completed in the liquid phase, the reaction speed is fast, the hybridization efficiency is high, and up to hundreds of target sequences can be detected simultaneously in one micro reaction system. However, research results in recent years show that the liquid chip technology still has shortcomings such as low sensitivity and poor reproducibility. The main reason is that the emission spectra of different fluorescent labels overlap widely in the liquid phase, leading to a large number of false positive results, which limits the Further promotion and application of liquid chip technology. Therefore, finding a fluorescent label that can separately label probes with different colors, has high luminous intensity, is not easy to quench, and has little overlap between spectra is the key to improving the sensitivity and repeatability of liquid chip detection.
发明内容Summary of the Invention
为此，本发明要解决的技术问题是提供一种以不同颜色分别标记探针且发光强度高，不易淬灭，光谱之间重叠少的荧光标记核苷酸的染料编码方法、标签序列、液体芯片以及用途。Therefore, the technical problem to be solved by the present invention is to provide a dye coding method, a tag sequence, and a liquid, which respectively label probes with different colors, have high luminous intensity, are not easily quenched, and have little overlap between spectra. Chips and uses.
为此，本发明提供了一种基于荧光标记核苷酸的染料编码方法，以核苷酸双链序列为标签序列，沿着所述核苷酸双链序列延伸方向，将所述标签序列分为至少两个荧光区域，每个荧光区域中的核苷酸至少连接一种荧光染料，相邻两个荧光区域间隔至少5个核苷酸。To this end, the present invention provides a method for dye coding based on fluorescently labeled nucleotides, which uses a nucleotide double-stranded sequence as a tag sequence and divides the tag sequence along the extension direction of the nucleotide double-stranded sequence. For at least two fluorescent regions, the nucleotides in each fluorescent region are connected to at least one fluorescent dye, and adjacent two fluorescent regions are separated by at least 5 nucleotides.
优选的，所述标签序列分为至少四个荧光区域，所述荧光区域中核苷酸的长度为15-20bp。Preferably, the tag sequence is divided into at least four fluorescent regions, and the length of the nucleotides in the fluorescent region is 15-20bp.
优选的，任意两个所述荧光区域内的荧光染料不相同。Preferably, the fluorescent dyes in any two of the fluorescent regions are different.
优选的，每个所述荧光区域内只有一种核苷酸连接荧光染料，且同一个荧光区域内的荧光染料相同。Preferably, there is only one type of nucleotide linked to a fluorescent dye in each of the fluorescent regions, and the fluorescent dye in the same fluorescent region is the same.
优选的，所述荧光染料包括BODIPY、FITC、罗丹明、香豆素、呫吨、花青素、芘或酞菁；所述量子点选自MgS、MgSe、MgTe、CaS、CaSe、CaTe、ZnO、ZnS、ZnSe、ZnTe、SrS、SrSe、SeTe、CdS、CdSe、CdTe、BaS、BaSe、BaTe、HgS、HgSe、HgTe、PbSe、CaAs、InP、InAs、InCaAs、ZnS/CdS、ZnS/CdS/ZnS、ZnS/HgS/ZnS/CdS、CdS/ZnS、CdS/Ag2S、CdS/HgS、CdS/HgS/CdS、CdS/PbS、CdS/Cd(OH)2、CdSe/CuSe、CdSe/ZnS、CdSe/ZnSe、CdSe/CdS、CdSe/HgSe、CdSe/HgSe/CdSe、CdSe/HgTe、CdTe/HgS、CdTe/HgTe、InAs/ZnSe、InAs/CdSe、InAs/InP、ZnS:Mn、ZnS:Cu、CdS:Mn和CdS:Cu中的任一种，以及以上述任一种为核、二氧化硅为壳的核壳型量子点。Preferably, the fluorescent dye includes BODIPY, FITC, rhodamine, coumarin, xanthene, anthocyanin, osmium or phthalocyanine; the quantum dot is selected from MgS, MgSe, MgTe, CaS, CaSe, CaTe, ZnO , ZnS, ZnSe, ZnTe, SrS, SrSe, SeTe, CdS, CdSe, CdTe, BaS, BaSe, BaTe, HgS, HgSe, HgTe, PbSe, CaAs, InP, InAs, InCaAs, ZnS / CdS, ZnS / CdS / ZnS , ZnS / HgS / ZnS / CdS, CdS / ZnS, CdS / Ag2S, CdS / HgS, CdS / HgS / CdS, CdS / PbS, CdS / Cd (OH) 2, CdSe / CuSe, CdSe / ZnS, CdSe / ZnSe , CdSe / CdS, CdSe / HgSe, CdSe / HgSe / CdSe, CdSe / HgTe, CdTe / HgS, CdTe / HgTe, InAs / ZnSe, InAs / CdSe, InAs / InP, ZnS: Mn, ZnS: Cu, CdS: Mn And CdS: Cu, and a core-shell type quantum dot using any of the above as a core and silica as a shell.
优选的，所述标签序列是DNA双链序列或RNA双链序列。Preferably, the tag sequence is a DNA double-stranded sequence or an RNA double-stranded sequence.
本发明提供了一种由所述基于荧光标记核苷酸的染料编码方法编码得到的标签序列。The present invention provides a tag sequence encoded by the dye-coding method based on a fluorescently labeled nucleotide.
本发明提供了一种液相芯片，本发明提供了包括：The invention provides a liquid phase chip, and the invention provides:
所述的标签序列；The tag sequence;
与所述标签序列连接的探针分子P1；A probe molecule P1 linked to the tag sequence;
磁性微球；Magnetic microsphere
与磁性微球连接的探针分子P2，所述探针分子P1与所述探针分子P2之间不互相结合。A probe molecule P2 connected to the magnetic microsphere, the probe molecule P1 and the probe molecule P2 are not bound to each other.
优选的，所述探针分子P1包括核苷酸序列、抗原或抗体，所述探针分子P2包括核苷酸序列、抗原或抗体。Preferably, the probe molecule P1 comprises a nucleotide sequence, an antigen or an antibody, and the probe molecule P2 comprises a nucleotide sequence, an antigen or an antibody.
优选的，所述探针分子P2上连接有生物素或荧光染料，且探针分子P2上连接的荧光染料与所述标签序列中的荧光染料不相同。Preferably, biotin or a fluorescent dye is connected to the probe molecule P2, and the fluorescent dye connected to the probe molecule P2 is different from the fluorescent dye in the tag sequence.
本发明提供了一种制备上述液相芯片的方法，包括以下步骤：The invention provides a method for preparing the liquid phase chip, which includes the following steps:
S1.将所述的标签序列连接探针分子P1；S1. Connecting the tag sequence to a probe molecule P1;
S2.将生物素或荧光染料连接探针分子P2S2. Link biotin or fluorescent dye to probe molecule P2
S3.将磁性微球连接上探针分子P2。。S3. Attach the magnetic microsphere to the probe molecule P2. .
本发明提供了一种利用所述的液相芯片在核酸或蛋白检测领域的用途。The invention provides a use of the liquid-phase chip in the field of nucleic acid or protein detection.
本发明相对现有技术具有如下优点：Compared with the prior art, the present invention has the following advantages:
1.本发明提供的基于荧光标记核苷酸的染料编码方法，以核苷酸双链序列为标签序列，沿着所述核苷酸双链序列延伸方向，将所述标签序列分为至少两个荧光区域，并且相邻两个荧光区域之间间隔至少5个核苷酸，使得两个区域之间的发出的荧光不互相干扰。1. A method for dye coding based on fluorescently labeled nucleotides provided by the present invention, which uses a nucleotide double-stranded sequence as a tag sequence and divides the tag sequence into at least two along the extension direction of the nucleotide double-stranded sequence Two fluorescent regions, and at least 5 nucleotides between two adjacent fluorescent regions, so that the fluorescence emitted between the two regions does not interfere with each other.
2.本发明提供的基于荧光标记核苷酸的染料编码方法，每个荧光区域连接一种荧光染料或量子点，通过对每个荧光区域连接的荧光染料或量子点进行排列组合可以达到对标签序列进行编号的目的。将标签序列分为四个 荧光区域，通过对4种不同的荧光染料或量子点进行组合，可以得到24种不同的组合方式，如果不同的荧光区域可以使用相同的荧光染料或量子点，4个荧光区域可以得到256种不同的组合方式。依次类推，将标签序列分得荧光区域越多，可以获得更多的组合方式，实现更多编号的标签序列，极大的扩展了标签序列的编号范围。另外，通过使用不同颜色的荧光染料或量子点，可以有效的避免光谱之间的重叠。2. The dye-coding method based on fluorescently labeled nucleotides provided by the present invention, each fluorescent region is connected with a fluorescent dye or quantum dot, and the label can be achieved by arranging and combining the fluorescent dye or quantum dot connected with each fluorescent region. The sequence is numbered for the purpose. The tag sequence is divided into four fluorescent regions. By combining 4 different fluorescent dyes or quantum dots, 24 different combinations can be obtained. If different fluorescent regions can use the same fluorescent dye or quantum dot, 4 Fluorescent areas can be obtained in 256 different combinations. By analogy, the more fluorescent regions are divided into the tag sequence, the more combinations can be obtained, and more numbered tag sequences can be realized, which greatly expands the number range of the tag sequence. In addition, by using different colors of fluorescent dyes or quantum dots, it is possible to effectively avoid overlap between the spectra.
3.本发明提供的基于荧光标记核苷酸的染料编码方法，标签序列每个荧光区域内连接的荧光染料或量子点的个数不同，使得每个荧光区域的放光强度不同，通过对每个荧光区域的发光强度进行排列组合可以达到对标签序列进行编号的目的；例如将标签序列分为4个荧光区域，每个荧光区域可以连接10个量子点，每两个量子点设置一个荧光强度等级，分为5个荧光强度等级，4个荧光区域就可以得到5 4中不同的排列方式，依次类推，标签序列设置N个荧光区域，每个荧光区域内设置M个荧光强度等级，可以得到M N种不同的排列方式，极大的扩展了标签序列的编号范围。 3. The dye-coding method based on fluorescently labeled nucleotides provided by the present invention, the number of fluorescent dyes or quantum dots connected in each fluorescent region of the tag sequence is different, so that the light intensity of each fluorescent region is different. Permutation and combination of the luminous intensity of each fluorescent region can achieve the purpose of numbering the tag sequence; for example, the tag sequence is divided into 4 fluorescent regions, each fluorescent region can be connected to 10 quantum dots, and a fluorescent intensity is set for each two quantum dots. Level, divided into 5 fluorescence intensity levels, 4 fluorescent regions can be obtained in 5 4 different arrangements, and so on, the label sequence is set to N fluorescent regions, each fluorescent region is set to M fluorescent intensity levels, you can get M N different arrangement methods greatly expand the number range of the tag sequence.
4.本发明提供的液相芯片，包括标签序列，与标签序列连接的探针分子P1，磁性微球标记的探针分子P2，检测标签序列每个荧光区域的荧光染料发出的荧光，得到标签序列的编号，得知待测序列中是否存在靶核酸序列，可以对靶核酸序列进行定性检测。4. The liquid-phase chip provided by the present invention comprises a tag sequence, a probe molecule P1 connected to the tag sequence, and a magnetic microsphere-labeled probe molecule P2, and detects the fluorescence emitted by the fluorescent dye of each fluorescent region of the tag sequence to obtain a tag. The sequence number is used to know whether the target nucleic acid sequence exists in the test sequence, and the target nucleic acid sequence can be qualitatively detected.
5.本发明提供的液相芯片，利用探针分子P2上的荧光染料或生物素的荧光强度与靶核酸序列浓度成正比，能够对靶核酸序列进行定量检测。5. The liquid-phase chip provided by the present invention can utilize the fluorescence intensity of the fluorescent dye or biotin on the probe molecule P2 to be proportional to the concentration of the target nucleic acid sequence, so that the target nucleic acid sequence can be quantitatively detected.
6.本发明提供的液相芯片，可以一次检测多种不同的靶序列，实现高通量的检测。6. The liquid-phase chip provided by the present invention can detect a plurality of different target sequences at one time, and realize high-throughput detection.
7.本发明提供的液相芯片，探针分子P1包括核苷酸序列、抗原或抗体，探针分子P2包括核苷酸序列、抗原或抗体，实现对核苷酸序列、抗原或抗体进行检测。7. The liquid-phase chip provided by the present invention, the probe molecule P1 includes a nucleotide sequence, an antigen, or an antibody, and the probe molecule P2 includes a nucleotide sequence, an antigen, or an antibody, so as to realize the detection of the nucleotide sequence, antigen, or antibody .
附图说明BRIEF DESCRIPTION OF THE DRAWINGS
图1为本发明实施例中荧光标记核苷酸的染料编码方法的编码示意图；FIG. 1 is a coding schematic diagram of a dye coding method for fluorescently labeled nucleotides in an embodiment of the present invention; FIG.
图2为本发明实施例中液相芯片的检测示意图；2 is a schematic diagram of detection of a liquid phase chip in an embodiment of the present invention;
图3为本发明实验例中液相芯片检测ALK、APC、BRAF和EGFR基因的检测结果。FIG. 3 is a liquid crystal chip detection result of ALK, APC, BRAF and EGFR genes in an experimental example of the present invention.
具体实施方式detailed description
以下通过具体实施例来说明本发明的实施方式，除非另外说明，本发明中所公开的实验方法均采用本技术领域常规技术。The following describes the embodiments of the present invention through specific examples. Unless otherwise stated, the experimental methods disclosed in the present invention adopt conventional techniques in the technical field.
实施例1Example 1
本实施例提供一种基于荧光标记核苷酸的染料编码方法，以双链脱氧核糖核酸序列为标签序列，长度为95bp，如图1所示，将所述标签序列分成四个荧光区域，分别为荧光区域I，荧光区域II，荧光区域III和荧光区域IV，长度均为20bp，相邻两个荧光区域之间间隔至少5个核苷酸，在本实施例中为相邻两个荧光区域之间间隔5个核苷酸；其中荧光区域I中的碱基A连接罗丹明，荧光区域II中的碱基C连接花青素，荧光区域III中的碱基T连接香豆素，荧光区域IV中的碱基G连接酞菁。This embodiment provides a method for dye coding based on fluorescently labeled nucleotides. A double-stranded DNA sequence is used as a tag sequence with a length of 95 bp. As shown in FIG. 1, the tag sequence is divided into four fluorescent regions, respectively. It is fluorescent region I, fluorescent region II, fluorescent region III, and fluorescent region IV, all of which are 20 bp in length, with at least 5 nucleotides spaced between two adjacent fluorescent regions. In this embodiment, it is two adjacent fluorescent regions. 5 nucleotides apart; base A in fluorescent region I is connected to rhodamine, base C in fluorescent region II is connected to anthocyanin, base T in fluorescent region III is connected to coumarin, and fluorescent region The base G in IV is attached to the phthalocyanine.
按照上述基于荧光标记核苷酸的染料编码方法，对每个荧光区域连接的荧光染料进行排列组合，每个荧光区域使用不同的荧光染料，得到24种不同的排列方式，并对每种排列方式进行编号作为标签序列的编号，实现 对标签序列编号。According to the above-mentioned method for encoding dyes based on fluorescently labeled nucleotides, the fluorescent dyes connected to each fluorescent region are arranged and combined. Each fluorescent region uses different fluorescent dyes to obtain 24 different arrangements, and for each arrangement Numbering is used as the number of the label sequence to realize numbering of the label sequence.
所述编码方法将标签序列分成了多个荧光区域，并且相邻两个荧光区域之间间隔至少5bp，使得两个区域之间的发出的荧光不互相干扰。每个荧光区域连接一种荧光染料，通过对每个荧光区域连接的荧光染料进行排列组合可以达到对标签序列进行编号的目的。例如，如表1所示，对排列的组合方式进行了举例，通过对4种不同的荧光染料进行组合，可以得到24种不同的组合方式，如果不同的荧光区域可以使用相同的荧光染料，4个荧光区域可以得到256种不同的组合方式。The coding method divides the tag sequence into a plurality of fluorescent regions, and the interval between two adjacent fluorescent regions is at least 5 bp, so that the emitted fluorescence between the two regions does not interfere with each other. A fluorescent dye is connected to each fluorescent region, and the number of tag sequences can be achieved by arranging and combining the fluorescent dyes connected to each fluorescent region. For example, as shown in Table 1, an example of the combination method of arrangement is given. By combining 4 different fluorescent dyes, 24 different combinations can be obtained. If different fluorescent regions can use the same fluorescent dye, 4 There are 256 different combinations of fluorescence regions.
表1Table 1
IDID 荧光区域IFluorescent region I 荧光区域IIFluorescent Region II 荧光区域IIIFluorescent Region III 荧光区域IVFluorescent Region IV
11 罗丹明Rodin 花青素anthocyanin 香豆素Coumarin 酞菁Phthalocyanine
22 罗丹明Rodin 花青素anthocyanin 酞菁Phthalocyanine 香豆素Coumarin
33 罗丹明Rodin 香豆素Coumarin 酞菁Phthalocyanine 花青素anthocyanin
44 罗丹明Rodin 香豆素Coumarin 花青素anthocyanin 酞菁Phthalocyanine
55 罗丹明Rodin 酞菁Phthalocyanine 花青素anthocyanin 香豆素Coumarin
66 罗丹明Rodin 酞菁Phthalocyanine 香豆素Coumarin 花青素anthocyanin
77 花青素anthocyanin 罗丹明Rodin 香豆素Coumarin 酞菁Phthalocyanine
88 花青素anthocyanin 罗丹明Rodin 酞菁Phthalocyanine 香豆素Coumarin
99 花青素anthocyanin 香豆素 Coumarin 罗丹明Rodin 酞菁Phthalocyanine
1010 花青素anthocyanin 香豆素Coumarin 酞菁Phthalocyanine 罗丹明Rodin
1111 花青素anthocyanin 酞菁Phthalocyanine 罗丹明Rodin 香豆素Coumarin
1212 花青素anthocyanin 酞菁Phthalocyanine 香豆素Coumarin 罗丹明Rodin
1313 香豆素Coumarin 罗丹明Rodin 花青素anthocyanin 酞菁Phthalocyanine
1414 香豆素Coumarin 罗丹明Rodin 酞菁Phthalocyanine 花青素anthocyanin
1515 香豆素Coumarin 花青素anthocyanin 罗丹明Rodin 酞菁Phthalocyanine
1616 香豆素Coumarin 花青素anthocyanin 酞菁Phthalocyanine 罗丹明Rodin
1717 香豆素Coumarin 酞菁Phthalocyanine 罗丹明Rodin 花青素anthocyanin
1818 香豆素Coumarin 酞菁Phthalocyanine 花青素anthocyanin 罗丹明Rodin
1919 酞菁Phthalocyanine 罗丹明Rodin 花青素anthocyanin 香豆素Coumarin
2020 酞菁Phthalocyanine 罗丹明Rodin 香豆素Coumarin 花青素anthocyanin
21twenty one 酞菁Phthalocyanine 花青素anthocyanin 罗丹明Rodin 香豆素Coumarin
22twenty two 酞菁Phthalocyanine 花青素anthocyanin 香豆素Coumarin 罗丹明Rodin
23twenty three 酞菁Phthalocyanine 香豆素Coumarin 花青素anthocyanin 罗丹明Rodin
24twenty four 酞菁Phthalocyanine 香豆素Coumarin 罗丹明Rodin 花青素anthocyanin
将标签序列分成5个荧光区域可以得到625种不同的组合方式，依次类推，将标签序列分得荧光区域越多，可以获得更多的组合方式，实现更多编号的标签序列，极大的扩展了标签序列的编号范围。另外，通过使用不同颜色的荧光染料，可以有效的避免光谱之间的重叠。Dividing the tag sequence into 5 fluorescent regions can get 625 different combinations, and so on. The more the tag sequence is divided into more fluorescent regions, the more combinations can be obtained, and more numbered tag sequences can be achieved, greatly expanding. The number range of the tag sequence. In addition, by using fluorescent dyes of different colors, it is possible to effectively avoid overlap between the spectra.
实施例2Example 2
本实施例提供一种基于荧光标记核苷酸的染料编码方法，以双链脱氧 核糖核酸序列为标签序列，长度为66bp，将所述标签序列分成四个荧光区域，分别为荧光区域I，荧光区域II，荧光区域III和荧光区域IV，长度均为15bp，相邻两个荧光区域之间间隔至少5个核苷酸，在本实施例中为相邻两个荧光区域之间间隔间隔7个核苷酸；其中荧光区域I中的碱基T连接芘，荧光区域II中的碱基C连接BODIPY，荧光区域III中的碱基T连接FITC，荧光区域IV中的碱基G连接罗丹明。This embodiment provides a method for dye coding based on fluorescently labeled nucleotides. A double-stranded DNA sequence is used as a tag sequence with a length of 66 bp. The tag sequence is divided into four fluorescent regions, which are respectively fluorescent region I and fluorescent. Region II, fluorescence region III, and fluorescence region IV are all 15 bp in length, with at least 5 nucleotides spaced between two adjacent fluorescent regions, and in this embodiment, 7 spaces between two adjacent fluorescent regions Nucleotide; base T in fluorescent region I is linked to 芘, base C in fluorescent region II is connected to BODIPY, base T in fluorescent region III is connected to FITC, and base G in fluorescent region IV is connected to rhodamine.
按照上述基于荧光标记核苷酸的染料编码方法，对每个荧光区域连接的荧光染料进行排列组合，每个荧光区域使用不同的荧光染料，得到24种不同的排列方式，并对每种排列方式进行编号作为标签序列的编号，实现对标签序列编号。According to the above-mentioned method for encoding dyes based on fluorescently labeled nucleotides, the fluorescent dyes connected to each fluorescent region are arranged and combined. Each fluorescent region uses different fluorescent dyes to obtain 24 different arrangements, and for each arrangement Numbering is used as the number of the label sequence to realize numbering of the label sequence.
实施例3Example 3
本实施例提供一种基于荧光标记核苷酸的染料编码方法，以双链脱氧核糖核酸序列为标签序列，长度为100bp，将所述标签序列分成五个荧光区域，分别为荧光区域I，荧光区域II，荧光区域III、荧光区域IV和荧光区域V，长度均为15bp，相邻两个荧光区域之间间隔至少5个核苷酸，在本实施例中为相邻两个荧光区域之间间隔间隔5个核苷酸；其中荧光区域I中的碱基C连接呫吨，荧光区域II中的碱基G连接花青素，荧光区域III中的碱基A连接芘，荧光区域IV中的碱基T连接BODIPY，荧光区域V中的碱基G连接罗丹明。This embodiment provides a method for dye coding based on fluorescently labeled nucleotides. A double-stranded DNA sequence is used as a tag sequence with a length of 100 bp. The tag sequence is divided into five fluorescent regions, which are respectively fluorescent region I and fluorescent. Region II, fluorescent region III, fluorescent region IV, and fluorescent region V are all 15 bp in length, with at least 5 nucleotides spaced between two adjacent fluorescent regions, in this embodiment, between two adjacent fluorescent regions 5 nucleotides apart; base C in fluorescent region I is linked to xanthene, base G in fluorescent region II is linked to anthocyanins, base A in fluorescent region III is linked to pyrene, and The base T is connected to BODIPY, and the base G in the fluorescent region V is connected to rhodamine.
按照上述基于荧光标记核苷酸的染料编码方法，选择上述5种荧光染料(呫吨、花青素、芘、BODIPY和罗丹明)排列组合对上述5个荧光区域进行标记，实现625个标签序列编号。According to the above-mentioned method for dye coding based on fluorescently labeled nucleotides, the above five fluorescent dyes (xanthene, anthocyanin, hydrazone, BODIPY, and rhodamine) are selected and combined to label the above five fluorescent regions, and realize 625 tag sequences Numbering.
实施例4Example 4
本实施例提供一种基于荧光标记核苷酸的染料编码方法，以双链核糖核酸序列为标签序列，长度为50bp，将所述标签序列分成2个荧光区域，分别为荧光区域I，荧光区域II，长度均为20bp，相邻两个荧光区域之间间隔至少5个核苷酸，在本实施例中为相邻两个荧光区域之间间隔间隔10个核苷酸；其中荧光区域I中的碱基A连接酞菁，荧光区域II中的碱基C连接花青素。This embodiment provides a method for dye coding based on fluorescently labeled nucleotides. A double-stranded ribonucleic acid sequence is used as a tag sequence with a length of 50 bp. The tag sequence is divided into two fluorescent regions, namely a fluorescent region I and a fluorescent region. II, both are 20 bp in length, with at least 5 nucleotides spaced between two adjacent fluorescent regions, in this embodiment, 10 nucleotides spaced between two adjacent fluorescent regions; The base A is linked to the phthalocyanine, and the base C in the fluorescent region II is linked to anthocyanins.
按照上述基于荧光标记核苷酸的染料编码方法，对每个荧光区域连接的荧光染料进行排列组合，每个荧光区域使用不同的荧光染料，得到2种不同的排列方式，并对每种排列方式进行编号作为标签序列的编号，实现对标签序列编号。According to the above-mentioned method for encoding dyes based on fluorescently labeled nucleotides, arrange and combine the fluorescent dyes connected to each fluorescent region, and use different fluorescent dyes for each fluorescent region to obtain 2 different arrangements, and for each arrangement Numbering is used as the number of the label sequence to realize numbering of the label sequence.
实施例5Example 5
本实施例提供一种基于荧光标记核苷酸的染料编码方法，以双链脱氧核糖核酸序列为标签序列，长度为105bp，将所述标签序列分成六个荧光区域，分别为荧光区域I，荧光区域II，荧光区域III、荧光区域IV、荧光区域V和荧光区域VI，长度均为15bp，相邻两个荧光区域之间间隔至少5个核苷酸，在本实施例中为相邻两个荧光区域之间间隔间隔5个核苷酸；其中荧光区域I中的碱基T连接香豆素，荧光区域II中的碱基G连接花青素，荧光区域III中的碱基A连接香豆素，荧光区域IV中的碱基T连接罗丹明，荧光区域V中的碱基C连接香豆素，荧光区域VI中的碱基G连接BODIPY。This embodiment provides a method for dye coding based on fluorescently labeled nucleotides. A double-stranded DNA sequence is used as a tag sequence with a length of 105 bp. The tag sequence is divided into six fluorescent regions, which are respectively fluorescent region I and fluorescent. Region II, fluorescent region III, fluorescent region IV, fluorescent region V, and fluorescent region VI are all 15 bp in length, with at least 5 nucleotides spaced between two adjacent fluorescent regions, which in this embodiment are two adjacent The fluorescent regions are separated by 5 nucleotides; the base T in the fluorescent region I is connected to coumarin, the base G in the fluorescent region II is connected to anthocyanins, and the base A in the fluorescent region III is connected to coumarin. The base T in the fluorescent region IV is connected to rhodamine, the base C in the fluorescent region V is connected to coumarin, and the base G in the fluorescent region VI is connected to BODIPY.
按照上述基于荧光标记核苷酸的染料编码方法，对每个荧光区域连接 的荧光染料进行排列组合，并对每种排列方式进行编号作为标签序列的编号，实现对标签序列编号。According to the above-mentioned fluorescently-labeled nucleotide-based dye coding method, the fluorescent dyes connected to each fluorescent region are arranged and combined, and each arrangement is numbered as the number of the tag sequence to realize numbering of the tag sequence.
作为可替换的实施方式，标签序列可以分为两个、三个、四个、五个以上荧光区域，只要相邻两个荧光区域之间间隔至少5bp，每个荧光区域中只有一种碱基连接一种荧光染料。As an alternative embodiment, the tag sequence can be divided into two, three, four, five or more fluorescent regions, as long as the interval between adjacent two fluorescent regions is at least 5bp, and there is only one base in each fluorescent region Attach a fluorescent dye.
作为可替换的实施方式，荧光染料可以是BODIPY、FITC、罗丹明、香豆素、呫吨、花青素、芘和酞菁。As an alternative embodiment, the fluorescent dye may be BODIPY, FITC, rhodamine, coumarin, xanthene, anthocyanins, osmium, and phthalocyanine.
实施例6Example 6
本实施例提供一种基于荧光标记核苷酸的染料编码方法，以双链脱氧核糖核酸序列为标签序列，长度为95bp，将所述标签序列分成四个荧光区域，分别为荧光区域I，荧光区域II，荧光区域III和荧光区域IV，长度均为20bp，相邻两个荧光区域之间间隔至少5个核苷酸，在本实施例中为相邻两个荧光区域之间间隔5个核苷酸；其中荧光区域I中的碱基A连接CdTe，荧光区域II中的碱基C连接CaS，荧光区域III中的碱基T连接CdS，荧光区域IV中的碱基G连接CdSe。This embodiment provides a method for dye coding based on fluorescently labeled nucleotides. A double-stranded DNA sequence is used as a tag sequence with a length of 95 bp. The tag sequence is divided into four fluorescent regions, which are respectively fluorescent region I and fluorescent. Region II, fluorescence region III, and fluorescence region IV are all 20 bp in length, with at least 5 nucleotides spaced between two adjacent fluorescent regions, and in this embodiment, 5 nuclei spaced between two adjacent fluorescent regions. Glycine; base A in fluorescent region I is connected to CdTe, base C in fluorescent region II is connected to CaS, base T in fluorescent region III is connected to CdS, and base G in fluorescent region IV is connected to CdSe.
所述编码方法将标签序列分成了多个荧光区域，并且相邻两个荧光区域之间间隔至少5bp，使得两个区域之间的发出的荧光不互相干扰。每个荧光区域连接一种量子点，通过对每个荧光区域连接的量子点进行排列组合可以达到对标签序列进行编号的目的。例如，如表2所示，对排列的组合方式进行了举例，通过对4种不同的荧光染料进行组合，可以得到24种不同的组合方式，如果不同的荧光区域可以使用相同的量子点，4个荧光区域可以得到256种不同的组合方式。The coding method divides the tag sequence into a plurality of fluorescent regions, and the interval between two adjacent fluorescent regions is at least 5 bp, so that the emitted fluorescence between the two regions does not interfere with each other. A quantum dot is connected to each fluorescent region, and the number of tag sequences can be achieved by arranging and combining the quantum dots connected to each fluorescent region. For example, as shown in Table 2, an example of the arrangement of the combination is given. By combining 4 different fluorescent dyes, 24 different combinations can be obtained. If different fluorescent regions can use the same quantum dot, 4 There are 256 different combinations of fluorescence regions.
表2Table 2
IDID 荧光区域IFluorescent region I 荧光区域IIFluorescent Region II 荧光区域IIIFluorescent Region III 荧光区域IVFluorescent Region IV
11 CdTeCdTe CaSCaS CdSCdS CdSeCdSe
22 CdTeCdTe CaSCaS CdSeCdSe CdSCdS
33 CdTeCdTe CdSCdS CdSeCdSe CaSCaS
44 CdTeCdTe CdSCdS CaSCaS CdSeCdSe
55 CdTeCdTe CdSeCdSe CaSCaS CdSCdS
66 CdTeCdTe CdSeCdSe CdSCdS CaSCaS
77 CaSCaS CdTeCdTe CdSCdS CdSeCdSe
88 CaSCaS CdTeCdTe CdSeCdSe CdSCdS
99 CaSCaS CdS CdS CdTeCdTe CdSeCdSe
1010 CaSCaS CdSCdS CdSeCdSe CdTeCdTe
1111 CaSCaS CdSeCdSe CdTeCdTe CdSCdS
1212 CaSCaS CdSeCdSe CdSCdS CdTeCdTe
1313 CdSCdS CdTeCdTe CaSCaS CdSeCdSe
1414 CdSCdS CdTeCdTe CdSeCdSe CaSCaS
1515 CdSCdS CaSCaS CdTeCdTe CdSeCdSe
1616 CdSCdS CaSCaS CdSeCdSe CdTeCdTe
1717 CdSCdS CdSeCdSe CdTeCdTe CaSCaS
1818 CdSCdS CdSeCdSe CaSCaS CdTeCdTe
1919 CdSeCdSe CdTeCdTe CaSCaS CdSCdS
2020 CdSeCdSe CdTeCdTe CdSCdS CaSCaS
21twenty one CdSeCdSe CaSCaS CdTeCdTe CdSCdS
22twenty two CdSeCdSe CaSCaS CdSCdS CdTeCdTe
23twenty three CdSeCdSe CdSCdS CaSCaS CdTeCdTe
24twenty four CdSeCdSe CdSCdS CdTeCdTe CaSCaS
将标签序列分成5个荧光区域可以得到625种不同的组合方式，依次类推，将标签序列分得荧光区域越多，可以获得更多的组合方式，实现更多编号的标签序列，极大的扩展了标签序列的编号范围。另外，通过使用不同颜色的荧光染料，可以有效的避免光谱之间的重叠。Dividing the tag sequence into 5 fluorescent regions can get 625 different combinations, and so on. The more the tag sequence is divided into more fluorescent regions, the more combinations can be obtained, and more numbered tag sequences can be achieved, greatly expanding. The number range of the tag sequence. In addition, by using fluorescent dyes of different colors, it is possible to effectively avoid overlap between the spectra.
实施例7Example 7
本实施例提供一种基于荧光标记核苷酸的染料编码方法，以双链脱氧核糖核酸序列为标签序列，长度为95bp，将所述标签序列分成四个荧光区域，分别为荧光区域I，荧光区域II，荧光区域III和荧光区域IV，长度均为20bp，相邻两个荧光区域之间间隔至少5个核苷酸，在本实施例中为相邻两个荧光区域之间间隔5个核苷酸；其中荧光区域I中的核苷酸连接CdTe，荧光区域II中的核苷酸连接CaS，荧光区域III中的核苷酸连接CdS，荧光区域IV中的核苷酸连接CdSe。每个荧光区域中每连接两个量子点为一个荧光强度等级，例如荧光区域I中有两个核苷酸连接CdTe，则荧光区域I的荧光强度等级为CdTe强度I，荧光区域II中有四个核苷酸连接CaS，则 荧光区域II的荧光强度等级为CaS强度II，荧光区域III中有六个核苷酸连接CdS，则荧光区域III的荧光强度等级为CdS强度III，荧光区域IV中有八个核苷酸连接CdSe，则荧光区域IV的荧光强度等级为CdSe强度IV。本实施例中每个荧光区域分为5个荧光强度等级：强度I，强度II，强度III，强度IV和强度V，通过对每个荧光区域连接的量子点的荧光强度等级进行排列组合可以达到对标签序列进行编号的目的。例如，如表3所示，对排列的组合方式进行了举例，通过对4种不同的量子点荧光强度等级进行组合，可以得到5 4种不同的组合方式，即有5 4个编号。 This embodiment provides a method for dye coding based on fluorescently labeled nucleotides. A double-stranded DNA sequence is used as a tag sequence with a length of 95 bp. The tag sequence is divided into four fluorescent regions, which are respectively fluorescent region I and fluorescent. Region II, fluorescence region III, and fluorescence region IV are all 20 bp in length, with at least 5 nucleotides spaced between two adjacent fluorescent regions, and in this embodiment, 5 nuclei spaced between two adjacent fluorescent regions. Nucleotide; nucleotides in fluorescent region I are connected to CdTe, nucleotides in fluorescent region II are connected to CaS, nucleotides in fluorescent region III are connected to CdS, and nucleotides in fluorescent region IV are connected to CdSe. Each fluorescence region is connected to two quantum dots in one fluorescence intensity level. For example, two nucleotides in fluorescent region I are connected to CdTe, then the fluorescence intensity level in fluorescent region I is CdTe intensity I, and four in fluorescence region II. If nucleotides are connected to CaS, the fluorescence intensity level of fluorescent region II is CaS intensity II, and six nucleotides in fluorescent region III are connected to CdS, then the fluorescence intensity level of fluorescent region III is CdS intensity III, and in fluorescent region IV With eight nucleotides linked to CdSe, the fluorescence intensity level of the fluorescent region IV is CdSe intensity IV. In this embodiment, each fluorescent region is divided into five fluorescent intensity levels: intensity I, intensity II, intensity III, intensity IV, and intensity V. By arranging and combining the fluorescence intensity levels of the quantum dots connected to each fluorescent region, it can be achieved The purpose of numbering the tag sequence. For example, as shown in Table 3, are arranged in combination is exemplified by 4 different intensity levels of fluorescence quantum dots composition can be obtained 54 different combinations, that is numbered 54.
表3table 3
IDID 荧光区域IFluorescent region I 荧光区域IIFluorescent Region II 荧光区域IIIFluorescent Region III 荧光区域IVFluorescent Region IV
11 CdTe强度ICdTe intensity I CaS强度IICaS intensity II CdS强度IIICdS intensity III CdS强度IVCdS intensity IV
22 CdT强度IIICdT intensity III CaS强度ICaS intensity I CdS强度IVCdS intensity IV CdS强度VCdS intensity V
33 CdT强度IIICdT intensity III CdS强度IVCdS intensity IV CdSe强度ICdSe intensity I CaS强度VCaS intensity V
44 CdT强度IVCdT intensity IV CdS强度IIICdS intensity III CaS强度IICaS intensity II CdSe强度ICdSe intensity I
实施例8Example 8
本实施例提供一种基于荧光标记核苷酸的染料编码方法，以双链脱氧核糖核酸序列为标签序列，长度为125bp，将所述标签序列分成五个荧光区域，分别为荧光区域I，荧光区域II，荧光区域III，荧光区域IV和荧光区域V，长度均为20bp，相邻两个荧光区域之间间隔至少5个核苷酸，在本实施例中为相邻两个荧光区域之间间隔5个核苷酸；其中荧光区域I中的核苷酸连接CdTe，荧光区域II中的核苷酸连接CaS，荧光区域III中的核苷 酸连接CdS，荧光区域IV中的核苷酸连接CdSe，荧光区域V中的核苷酸连接HgS。每个荧光区域中每连接两个量子点为一个荧光强度等级，例如荧光区域I中有两个核苷酸连接CdTe，则荧光区域I的荧光强度等级为CdTe强度I，荧光区域II中有四个核苷酸连接CaS，则荧光区域II的荧光强度等级为CaS强度II，荧光区域III中有六个核苷酸连接CdS，则荧光区域III的荧光强度等级为CdS强度III，荧光区域IV中有八个核苷酸连接CdSe，则荧光区域IV的荧光强度等级为CdSe强度IV；荧光区域V中有十个核苷酸连接HgS，则荧光区域V的荧光强度等级为HgS强度V，本实施例中每个荧光区域分为6个荧光强度等级：强度I，强度II，强度III，强度IV，强度V和强度VI，对每个荧光区域连接的量子点的荧光强度等级进行排列组合可以达到对标签序列进行编号的目的，通过对5种不同的量子点，6个荧光强度等级进行组合，可以得到6 5种不同的组合方式，即有6 5个编号。 This embodiment provides a method for dye coding based on fluorescently labeled nucleotides. A double-stranded DNA sequence is used as a tag sequence with a length of 125 bp. The tag sequence is divided into five fluorescent regions, which are respectively fluorescent region I and fluorescent. Region II, fluorescent region III, fluorescent region IV, and fluorescent region V are all 20 bp in length, with at least 5 nucleotides spaced between two adjacent fluorescent regions, in this embodiment between two adjacent fluorescent regions 5 nucleotides apart; nucleotides in fluorescent region I are linked to CdTe, nucleotides in fluorescent region II are linked to CaS, nucleotides in fluorescent region III are linked to CdS, and nucleotides in fluorescent region IV are linked CdSe, nucleotides in fluorescent region V are linked to HgS. Each fluorescence region is connected to two quantum dots in one fluorescence intensity level. For example, two nucleotides in fluorescent region I are connected to CdTe, then the fluorescence intensity level in fluorescent region I is CdTe intensity I, and four in fluorescence region II. If nucleotides are connected to CaS, the fluorescence intensity level of fluorescent region II is CaS intensity II, and six nucleotides in fluorescent region III are connected to CdS, then the fluorescence intensity level of fluorescent region III is CdS intensity III, and in fluorescent region IV With eight nucleotides connected to CdSe, the fluorescence intensity level of the fluorescent region IV is CdSe intensity IV; ten nucleotides in the fluorescent region V are connected to HgS, and the fluorescence intensity level of the fluorescent region V is HgS intensity V. This implementation In the example, each fluorescent region is divided into six fluorescence intensity levels: intensity I, intensity II, intensity III, intensity IV, intensity V, and intensity VI. The combination and arrangement of the fluorescence intensity levels of the quantum dots connected to each fluorescent region can achieve Objective tag sequence numbering, by five different quantum dots, 6 fluorescence intensity levels are combined can be obtained 65 different combinations, that is numbered 65.
作为可替换的实施方式，标签序列可以分为两个、三个、四个、五个以上荧光区域，只要相邻两个荧光区域之间间隔至少5bp，每个荧光区域中核苷酸连接一种量子点。As an alternative embodiment, the tag sequence can be divided into two, three, four, five or more fluorescent regions, as long as the interval between adjacent two fluorescent regions is at least 5bp, and one type of nucleotide is connected in each fluorescent region Quantum dots.
作为可替换的实施方式，量子点可以是MgS、MgSe、MgTe、CaS、CaSe、CaTe、ZnO、ZnS、ZnSe、ZnTe、SrS、SrSe、SeTe、CdS、CdSe、CdTe、BaS、BaSe、BaTe、HgS、HgSe、HgTe、PbSe、CaAs、InP、InAs、InCaAs、ZnS/CdS、ZnS/CdS/ZnS、ZnS/HgS/ZnS/CdS、CdS/ZnS、CdS/Ag2S、CdS/HgS、CdS/HgS/CdS、CdS/PbS、CdS/Cd(OH)2、CdSe/CuSe、CdSe/ZnS、CdSe/ZnSe、CdSe/CdS、CdSe/HgSe、CdSe/HgSe/CdSe、CdSe/HgTe、CdTe/HgS、CdTe/HgTe、InAs/ZnSe、InAs/CdSe、InAs/InP、ZnS:Mn、ZnS:Cu、CdS:Mn和CdS:Cu 中的任一种，以及以上述任一种为核、二氧化硅为壳的核壳型量子点。As an alternative embodiment, the quantum dots may be MgS, MgSe, MgTe, CaS, CaSe, CaTe, ZnO, ZnS, ZnSe, ZnTe, SrS, SrSe, SeTe, CdS, CdSe, CdTe, BaS, BaSe, BaTe, HgS , HgSe, HgTe, PbSe, CaAs, InP, InAs, InCaAs, ZnS / CdS, ZnS / CdS / ZnS, ZnS / HgS / ZnS / CdS, CdS / ZnS, CdS / Ag2S, CdS / HgS, CdS / HgS / CdS , CdS / PbS, CdS / Cd (OH) 2, CdSe / CuSe, CdSe / ZnS, CdSe / ZnSe, CdSe / CdS, CdSe / HgSe, CdSe / HgSe / CdSe, CdSe / HgTe, CdTe / HgS, CdTe / HgTe , InAs / ZnSe, InAs / CdSe, InAs / InP, ZnS: Mn, ZnS: Cu, CdS: Mn, and CdS: Cu, and a core with any of the above as the core and silica as the shell Shell-type quantum dots.
将标签序列分为N个荧光区域，相邻两个荧光区域之间间隔至少5bp，每个荧光区域中核苷酸连接一种量子点，每个荧光区域分为M个荧光强度等级，通过对N种不同的量子点，M个荧光强度等级进行组合，可以得到M N种不同的组合方式，即有M N个编号。 The tag sequence is divided into N fluorescent regions with an interval of at least 5bp between two adjacent fluorescent regions. A nucleotide is connected to a quantum dot in each fluorescent region. Each fluorescent region is divided into M fluorescence intensity levels. Different kinds of quantum dots and M fluorescence intensity levels are combined to obtain M N different combinations, that is, M N number.
实施例9Example 9
本实施例提供的一种液相芯片，包括通过实施例1的基于荧光标记核苷酸的染料编码方法编码得到的标签序列；A liquid phase chip provided in this embodiment includes a tag sequence encoded by the dye coding method based on a fluorescently labeled nucleotide in Embodiment 1;
根据靶核酸序列设计一段探针分子P1和一段探针分子P2，探针分子P1能与靶核酸序列5’端杂交结合，探针分子P2能与靶核酸序列3’端杂交结合，探针分子P1和探针分子P2不互补，探针分子P2上连接有荧光染料FITC。如图2所示，将探针分子P1连接到标签序列上，将探针分子P2连接到磁性微球上；将连接探针分子P1的所述标签序列与连接探针分子P2的所述磁性微球混合。According to the target nucleic acid sequence, a probe molecule P1 and a probe molecule P2 are designed. The probe molecule P1 can hybridize and bind to the 5 'end of the target nucleic acid sequence, and the probe molecule P2 can hybridize and bind to the 3' end of the target nucleic acid sequence. The probe molecule P1 and probe molecule P2 are not complementary, and a fluorescent dye FITC is connected to the probe molecule P2. As shown in FIG. 2, the probe molecule P1 is connected to a tag sequence, and the probe molecule P2 is connected to a magnetic microsphere; the tag sequence to which the probe molecule P1 is connected and the magnetism to which the probe molecule P2 is connected. Microsphere mix.
使用通用引物将待测样本进行PCR扩增，再将PCR扩增产物加入至所述液相芯片中，PCR扩增产物同时与标签序列和磁性微球进行杂交反应，如果待测样本含有靶核酸序列，则标签序列上的探针分子P1和磁性微球上的探针分子P2可以与靶核酸序列通过碱基互补发生特异性结合，所得的复合物利用磁性微球可以通过磁性分离从反应体系中分离出来，再根据标签序列上的荧光染料的荧光强度与靶核酸序列浓度成正比，对靶核酸序列进行定量检测。如果待测样本中不含有靶核酸序列，则针对该靶核酸的探针分子P1无法通过靶核酸序列与磁性微球标记的探针分子P2结合，不具有 磁性，从而在磁性分离中取出。Use universal primers to perform PCR amplification on the sample to be tested, and then add the PCR amplification product to the liquid-phase chip. The PCR amplification product simultaneously performs hybridization reaction with the tag sequence and magnetic microspheres. If the sample to be tested contains the target nucleic acid Sequence, the probe molecule P1 on the tag sequence and the probe molecule P2 on the magnetic microsphere can specifically bind to the target nucleic acid sequence through base complementation. The resulting complex can be magnetically separated from the reaction system using magnetic microspheres. The target nucleic acid sequence is quantitatively detected based on the fluorescence intensity of the fluorescent dye on the tag sequence and the concentration of the target nucleic acid sequence. If the target nucleic acid sequence is not contained in the test sample, the probe molecule P1 for the target nucleic acid cannot be bound to the magnetic microsphere-labeled probe molecule P2 through the target nucleic acid sequence, and is not magnetic, so it can be taken out during magnetic separation.
实施例10Example 10
本实施例提供一种液相芯片，包括通过实施例2的基于荧光标记核苷酸的染料编码方法编码得到的标签序列，即标签序列为双链脱氧核糖核酸序列，长度为95bp，如图1所示，将标签序列分成四个荧光区域，分别为荧光区域I，荧光区域II，荧光区域III和荧光区域IV，长度均为20bp，相邻两个荧光区域之间间隔至少5bp；将罗丹明，花青素，香豆素和酞菁分别连接到四个荧光区域中的一种核苷酸上，其中碱基A连接罗丹明，碱基C连接花青素，碱基T连接香豆素，碱基G连接酞菁，对每个荧光区域连接的荧光染料进行排列组合，每个荧光区域使用不同的荧光染料，得到24种不同的排列方式，并对每种排列方式进行编号作为标签序列的编号。This embodiment provides a liquid phase chip, which includes a tag sequence encoded by the dye-encoding method based on a fluorescently labeled nucleotide of Example 2, that is, the tag sequence is a double-stranded DNA sequence with a length of 95 bp, as shown in FIG. 1 As shown, the tag sequence is divided into four fluorescent regions, namely, fluorescent region I, fluorescent region II, fluorescent region III, and fluorescent region IV, each having a length of 20 bp, and the interval between adjacent two fluorescent regions is at least 5 bp; Anthocyanin, coumarin and phthalocyanine are each connected to a nucleotide in four fluorescent regions, where base A is connected to rhodamine, base C is connected to anthocyanin, and base T is connected to coumarin The base G is connected to the phthalocyanine, and the fluorescent dyes connected to each fluorescent region are arranged and combined. Each fluorescent region uses different fluorescent dyes to obtain 24 different arrangements, and each arrangement is numbered as a label sequence. Number.
根据24种不同的靶核酸序列设计不同的探针分子P1和探针分子P2，探针分子P1能与靶核酸序列5’端杂交结合，探针分子P2能与靶核酸序列3’端杂交结合，探针分子P1和探针分子P2不互补，探针分子P2上连接有荧光染料花青素。如图2所示，将探针分子P1连接到标签序列上，将探针分子P2连接到磁性微球上；将连接探针分子P1的所述标签序列与连接探针分子P2的所述磁性微球混合。Different probe molecule P1 and probe molecule P2 are designed according to 24 different target nucleic acid sequences. Probe molecule P1 can hybridize and bind to the 5 'end of the target nucleic acid sequence, and probe molecule P2 can hybridize to the 3' end of the target nucleic acid sequence. The probe molecule P1 and the probe molecule P2 are not complementary, and a fluorescent dye anthocyanin is connected to the probe molecule P2. As shown in FIG. 2, the probe molecule P1 is connected to a tag sequence, and the probe molecule P2 is connected to a magnetic microsphere; the tag sequence to which the probe molecule P1 is connected and the magnetism to which the probe molecule P2 is connected. Microsphere mix.
使用通用引物将待测样本进行PCR扩增，再将PCR扩增产物加入至所述液相芯片中，所述PCR扩增产物同时与标签序列和磁性微球进行杂交反应，如果待测样本含有靶核酸序列，则标签序列上的探针分子P1和磁性微球上的探针分子P2可以与靶核酸序列通过碱基互补发生特异性结合，所得的复合物利用磁性微球可以通过磁性分离从反应体系中分离出来，检测标 签序列每个荧光区域的荧光染料发出的荧光，得到标签序列的编号，得知待测序列中是否存在靶核酸序列，可以对靶核酸序列进行定性检测；再利用探针分子P2上的荧光染料花青素的荧光强度与靶核酸序列浓度成正比，对靶核酸序列进行定量检测。Use universal primers to perform PCR amplification on the sample to be tested, and then add the PCR amplification product to the liquid-phase chip. The PCR amplification product performs hybridization reaction with the tag sequence and magnetic microspheres at the same time. Target nucleic acid sequence, the probe molecule P1 on the tag sequence and the probe molecule P2 on the magnetic microsphere can specifically bind to the target nucleic acid sequence through base complementation, and the resulting complex can be magnetically separated from the magnetic microsphere It is separated from the reaction system, and the fluorescence emitted by the fluorescent dye in each fluorescent region of the tag sequence is detected to obtain the number of the tag sequence. It can be known whether the target nucleic acid sequence exists in the test sequence, and the target nucleic acid sequence can be qualitatively detected. The fluorescence intensity of the fluorescent dye anthocyanin on the needle molecule P2 is directly proportional to the concentration of the target nucleic acid sequence, and the target nucleic acid sequence is quantitatively detected.
实施例11Example 11
本实施例提供一种液相芯片，包括通过实施例7的基于荧光标记核苷酸的染料编码方法编码得到的标签序列，即标签序列为双链脱氧核糖核酸序列，长度为95bp，将标签序列分成四个荧光区域，分别为荧光区域I，荧光区域II，荧光区域III和荧光区域IV，长度均为20bp，相邻两个荧光区域之间间隔至少5bp；将CdTe，CaS，CdS和CdSe分别连接到四个荧光区域中的一种核苷酸上，其中荧光区域I中的碱基A连接CdTe，荧光区域II中的碱基C连接CaS，荧光区域III中的碱基T连接CdS，荧光区域IV中的碱基G连接CdSe，每个荧光区域中每连接两个量子点为一个荧光强度等级，例如荧光区域I中有两个核苷酸连接CdTe，则荧光区域I的荧光强度等级为CdTe强度I，荧光区域II中有四个核苷酸连接CaS，则荧光区域II的荧光强度等级为CaS强度II，荧光区域III中有六个核苷酸连接CdS，则荧光区域III的荧光强度等级为CdS强度III，荧光区域IV中有八个核苷酸连接CdSe，则荧光区域IV的荧光强度等级为CdSe强度IV。本实施例中每个荧光区域分为5个荧光强度等级：强度I，强度II，强度III，强度IV和强度V，对每个荧光区域连接的量子点的荧光强度等级进行排列组合可以达到对标签序列进行编号的目的。通过对4种不同的量子点荧光强度等级进行组合，可以得到625种不同的组合方式，并对每种排列方式进行编 号作为标签序列的编号。This embodiment provides a liquid phase chip, which includes a tag sequence encoded by the dye-coding method based on a fluorescently labeled nucleotide in Example 7, that is, the tag sequence is a double-stranded DNA sequence with a length of 95 bp. Divided into four fluorescent regions, namely fluorescent region I, fluorescent region II, fluorescent region III and fluorescent region IV, both of which are 20 bp in length, and the interval between two adjacent fluorescent regions is at least 5 bp; CdTe, CaS, CdS, and CdSe, respectively Linked to a nucleotide in four fluorescent regions, where base A in fluorescent region I is connected to CdTe, base C in fluorescent region II is connected to CaS, base T in fluorescent region III is connected to CdS, and fluorescence The base G in region IV is connected to CdSe, and each fluorescent region is connected to two quantum dots as a fluorescence intensity level. For example, if two nucleotides in fluorescent region I are connected to CdTe, the fluorescence intensity level of fluorescent region I is CdTe intensity I, four nucleotides in the fluorescent region II are connected to CaS, then the fluorescence intensity level of the fluorescent region II is CaS intensity II, and six nucleotides in the fluorescent region III are connected to CdS, then the fluorescent region III's CdS light intensity level of intensity III, IV fluorescent area in eight nucleotides connecting CdSe, the fluorescence intensity level of fluorescence intensity of CdSe region IV is IV. In this embodiment, each fluorescent region is divided into five fluorescent intensity levels: intensity I, intensity II, intensity III, intensity IV, and intensity V. The combination and arrangement of the fluorescence intensity levels of the quantum dots connected to each fluorescent region can achieve The label sequence is numbered for the purpose. By combining four different quantum dot fluorescence intensity levels, 625 different combinations can be obtained, and each arrangement is numbered as the number of the tag sequence.
根据625种不同的靶核酸序列设计不同的探针分子P1和探针分子P2，探针分子P1能与靶核酸序列5’端杂交结合，探针分子P2能与靶核酸序列3’端杂交结合，探针分子P1和探针分子P2不互补，探针分子P2上连接有荧光染料FITC。将探针分子P1连接到标签序列上，将探针分子P2连接到磁性微球上；将连接探针分子P1的所述标签序列与连接探针分子P2的所述磁性微球混合。According to 625 different target nucleic acid sequences, different probe molecules P1 and P2 are designed. The probe molecule P1 can hybridize and bind to the 5 'end of the target nucleic acid sequence, and the probe molecule P2 can hybridize and bind to the 3' end of the target nucleic acid sequence. The probe molecule P1 and the probe molecule P2 are not complementary, and a fluorescent dye FITC is connected to the probe molecule P2. The probe molecule P1 is connected to a tag sequence, and the probe molecule P2 is connected to a magnetic microsphere; the tag sequence to which the probe molecule P1 is connected and the magnetic microsphere to which the probe molecule P2 is connected are mixed.
使用通用引物将待测样本进行PCR扩增，再将PCR扩增产物加入至所述液相芯片中，所述PCR扩增产物同时与标签序列和磁性微球进行杂交反应，如果待测样本含有靶核酸序列，则标签序列上的探针分子P1和磁性微球上的探针分子P2可以与靶核酸序列通过碱基互补发生特异性结合，所得的复合物利用磁性微球可以通过磁性分离从反应体系中分离出来，检测标签序列每个荧光区域的荧光染料发出的荧光，得到标签序列的编号，得知待测序列中是否存在靶核酸序列，可以对靶核酸序列进行定性检测；再利用探针分子P2上的荧光染料FITC的荧光强度与靶核酸序列浓度成正比，对靶核酸序列进行定量检测。Use universal primers to perform PCR amplification on the sample to be tested, and then add the PCR amplification product to the liquid-phase chip. The PCR amplification product performs hybridization reaction with the tag sequence and magnetic microspheres at the same time. Target nucleic acid sequence, the probe molecule P1 on the tag sequence and the probe molecule P2 on the magnetic microsphere can specifically bind to the target nucleic acid sequence through base complementation, and the resulting complex can be magnetically separated from the magnetic microsphere It is separated from the reaction system, and the fluorescence emitted by the fluorescent dye in each fluorescent region of the tag sequence is detected to obtain the number of the tag sequence. It can be known whether the target nucleic acid sequence exists in the test sequence, and the target nucleic acid sequence can be qualitatively detected. The fluorescence intensity of the fluorescent dye FITC on the needle molecule P2 is directly proportional to the concentration of the target nucleic acid sequence, and the target nucleic acid sequence is quantitatively detected.
实验例1Experimental example 1
本实验例使用实施例11中的液相芯片检测4个肿瘤靶向药物用药相关的基因，包括ALK、APC、BRAF和EGFR。标签序列如SEQ NO.1所示，ALK基因的探针分子P1如SEQ NO.2所示，探针分子P2如SEQ NO.3所示，APC基因的探针分子P1如SEQ NO.4所示，探针分子P2如SEQ NO.5所示；BRAF基因的探针分子P1如SEQ NO.6所示，探针分子P2如SEQ NO.7 所示；EGFR基因的探针分子P1如SEQ NO.8所示，探针分子P2如SEQ NO.9所示。This experimental example uses the liquid-phase chip in Example 11 to detect four tumor-targeting drug-related genes, including ALK, APC, BRAF, and EGFR. The tag sequence is shown in SEQ NO.1, the probe molecule P1 of the ALK gene is shown in SEQ NO.2, the probe molecule P2 is shown in SEQ NO.3, and the probe molecule P1 of the APC gene is shown in SEQ NO.4 The probe molecule P2 is shown in SEQ NO.5; the probe molecule P1 of the BRAF gene is shown in SEQ NO.6, the probe molecule P2 is shown in SEQ NO.7; the probe molecule P1 of the EGFR gene is shown in SEQ. The probe molecule P2 is shown in SEQ. NO.9.
图3为采用本发明液相芯片使用流式细胞仪检测肿瘤靶向药物用药相关的基因混合样本的标准曲线，用最小平方法求得各标准曲线的回归方程，R2均高于0.99，表明荧光染料FITC荧光强度与靶核酸序列浓度成正比，可以据此对靶核酸序列进行定量检测。FIG. 3 is a standard curve for detecting a gene-mixed sample related to tumor-targeting drug administration using a flow cytometer using the liquid-phase chip of the present invention, and a regression equation of each standard curve is obtained by a least square method. R2 is higher than 0.99, indicating fluorescence The fluorescence intensity of the dye FITC is directly proportional to the concentration of the target nucleic acid sequence, and the target nucleic acid sequence can be quantitatively detected based on this.
上述实施例仅仅是为清楚地说明所作的举例，而并非对实施方式的限定。对于所属领域的普通技术人员来说，在上述说明的基础上还可以做出其它不同形式的变化或变动，由此所引伸出的显而易见的变化或变动仍处于本发明创造的保护范围之中。The above-mentioned embodiment is merely an example for clear description, and is not a limitation on the implementation. For those of ordinary skill in the art, on the basis of the above description, other different forms of changes or changes can also be made, and the obvious changes or changes derived therefrom are still within the protection scope of the present invention.
Figure PCTCN2018091982-appb-000001
Figure PCTCN2018091982-appb-000001
Figure PCTCN2018091982-appb-000002
Figure PCTCN2018091982-appb-000002
Figure PCTCN2018091982-appb-000003
Figure PCTCN2018091982-appb-000003
Figure PCTCN2018091982-appb-000004
Figure PCTCN2018091982-appb-000004
Figure PCTCN2018091982-appb-000005
Figure PCTCN2018091982-appb-000005

Claims (12)

  1. 一种基于荧光标记核苷酸的染料编码方法，其特征在于，A dye coding method based on fluorescently labeled nucleotides, characterized in that:
    以核苷酸双链序列为标签序列，沿着所述核苷酸双链序列延伸方向，将所述标签序列分为至少两个荧光区域，每个荧光区域中的核苷酸至少连接一种荧光染料或量子点，相邻两个荧光区域间隔至少5个核苷酸。A nucleotide double-stranded sequence is used as a tag sequence, and the tag sequence is divided into at least two fluorescent regions along the extension direction of the nucleotide double-stranded sequence, and at least one kind of nucleotide is connected in each fluorescent region A fluorescent dye or quantum dot with two adjacent fluorescent regions separated by at least 5 nucleotides.
  2. 根据权利要求1所述基于荧光标记核苷酸的染料编码方法，其特征在于，所述标签序列分为至少四个荧光区域，所述荧光区域中核苷酸的长度为15-20bp。The method of claim 1, wherein the tag sequence is divided into at least four fluorescent regions, and the length of the nucleotides in the fluorescent region is 15-20 bp.
  3. 根据权利要求1或2所述基于荧光标记核苷酸的染料编码方法，其特征在于，任意两个所述荧光区域内的荧光染料或量子点不相同。The method according to claim 1 or 2, wherein the fluorescent dyes or quantum dots in any two of the fluorescent regions are different.
  4. 根据权利要求1-3任一项所述基于荧光标记核苷酸的染料编码方法，其特征在于，每个所述荧光区域内只有一种核苷酸连接荧光染料或量子点，且同一个荧光区域内的荧光染料或量子点相同。The method for encoding a dye based on a fluorescently labeled nucleotide according to any one of claims 1 to 3, wherein only one nucleotide in each fluorescent region is connected to a fluorescent dye or a quantum dot, and the same fluorescence The fluorescent dyes or quantum dots in the region are the same.
  5. 根据权利要求1-4任一所述基于荧光标记核苷酸的染料编码方法，其特征在于，所述荧光染料包括BODIPY、FITC、罗丹明、香豆素、呫吨、花青素、芘或酞菁；所述量子点选自MgS、MgSe、MgTe、CaS、CaSe、CaTe、ZnO、ZnS、ZnSe、ZnTe、SrS、SrSe、SeTe、CdS、CdSe、CdTe、BaS、BaSe、BaTe、HgS、HgSe、HgTe、PbSe、CaAs、InP、InAs、InCaAs、ZnS/CdS、ZnS/CdS/ZnS、ZnS/HgS/ZnS/CdS、CdS/ZnS、CdS/Ag2S、CdS/HgS、CdS/HgS/CdS、CdS/PbS、CdS/Cd(OH)2、CdSe/CuSe、CdSe/ZnS、CdSe/ZnSe、CdSe/CdS、CdSe/HgSe、CdSe/HgSe/CdSe、CdSe/HgTe、CdTe/HgS、CdTe/HgTe、InAs/ZnSe、InAs/CdSe、InAs/InP、ZnS:Mn、ZnS:Cu、CdS:Mn和CdS:Cu 中的任一种，以及以上述任一种为核、二氧化硅为壳的核壳型量子点。The method for encoding a dye based on a fluorescently labeled nucleotide according to any one of claims 1 to 4, wherein the fluorescent dye comprises BODIPY, FITC, rhodamine, coumarin, xanthene, anthocyanin, osmium or Phthalocyanine; the quantum dot is selected from MgS, MgSe, MgTe, CaS, CaSe, CaTe, ZnO, ZnS, ZnSe, ZnTe, SrS, SrSe, SeTe, CdS, CdSe, CdTe, BaS, BaSe, BaTe, HgS, HgSe , HgTe, PbSe, CaAs, InP, InAs, InCaAs, ZnS / CdS, ZnS / CdS / ZnS, ZnS / HgS / ZnS / CdS, CdS / ZnS, CdS / Ag2S, CdS / HgS, CdS / HgS / CdS, CdS / PbS, CdS / Cd (OH) 2, CdSe / CuSe, CdSe / ZnS, CdSe / ZnSe, CdSe / CdS, CdSe / HgSe, CdSe / HgSe / CdSe, CdSe / HgTe, CdTe / HgS, CdTe / HgTe, InAs / ZnSe, InAs / CdSe, InAs / InP, ZnS: Mn, ZnS: Cu, CdS: Mn, and CdS: Cu, and a core-shell type with any of the above as the core and silica as the shell Quantum dots.
  6. 根据权利要求1-5任一项所述基于荧光标记核苷酸的染料编码方法，其特征在于，所述标签序列是DNA双链序列或RNA双链序列。The method according to any one of claims 1 to 5, wherein the tag sequence is a DNA double-stranded sequence or an RNA double-stranded sequence.
  7. 由权利要求1-6任一项所述基于荧光标记核苷酸的染料编码方法编码得到的标签序列。The tag sequence encoded by the dye-coding method based on a fluorescently labeled nucleotide according to any one of claims 1-6.
  8. 一种液相芯片，其特征在于，包括：A liquid-phase chip, comprising:
    权利要求7所述的标签序列；The tag sequence of claim 7;
    与所述标签序列连接的探针分子P1；A probe molecule P1 linked to the tag sequence;
    磁性微球；Magnetic microsphere
    与磁性微球连接的探针分子P2，所述探针分子P1与所述探针分子P2之间不互相结合。A probe molecule P2 connected to the magnetic microsphere, the probe molecule P1 and the probe molecule P2 are not bound to each other.
  9. 根据权利要求8所述的液相芯片，其特征在于，所述探针分子P1包括核苷酸序列、抗原或抗体，所述探针分子P2包括核苷酸序列、抗原或抗体。The liquid-phase chip according to claim 8, wherein the probe molecule P1 comprises a nucleotide sequence, an antigen or an antibody, and the probe molecule P2 comprises a nucleotide sequence, an antigen or an antibody.
  10. 根据权利要求9所述的液相芯片，其特征在于，所述探针分子P2上连接有生物素或荧光染料，且探针分子P2上连接的荧光染料与所述标签序列中的荧光染料不相同。The liquid-phase chip according to claim 9, wherein biotin or a fluorescent dye is connected to the probe molecule P2, and the fluorescent dye connected to the probe molecule P2 is not the same as the fluorescent dye in the tag sequence. the same.
  11. 一种制备权利要求8-10所述的液相芯片的方法，其特征在于，包括以下步骤：A method for preparing a liquid-phase chip according to claims 8-10, comprising the following steps:
    S1.将权利要求7所述的标签序列连接探针分子P1；S1. Linking the tag sequence of claim 7 to a probe molecule P1;
    S2.将生物素或荧光染料连接探针分子P2S2. Link biotin or fluorescent dye to probe molecule P2
    S3.将磁性微球连接上探针分子P2。S3. Attach the magnetic microsphere to the probe molecule P2.
  12. 一种利用权利要求8-10所述的液相芯片在核酸或蛋白检测领域的用途。A use of the liquid-phase chip according to claims 8-10 in the field of nucleic acid or protein detection.
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