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WO2019100842A1 - Procédé de détection d'une adsorption spécifique et/ou non spécifique de nucléotide - Google Patents

Procédé de détection d'une adsorption spécifique et/ou non spécifique de nucléotide Download PDF

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Publication number
WO2019100842A1
WO2019100842A1 PCT/CN2018/107249 CN2018107249W WO2019100842A1 WO 2019100842 A1 WO2019100842 A1 WO 2019100842A1 CN 2018107249 W CN2018107249 W CN 2018107249W WO 2019100842 A1 WO2019100842 A1 WO 2019100842A1
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image
chip
detecting
nucleotide
preprocessed
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Chinese (zh)
Inventor
赵陆洋
黄天逊
吴增丁
颜钦
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Direct Genomics Co Ltd
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Direct Genomics Co Ltd
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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
    • G06—COMPUTING OR CALCULATING; COUNTING
    • G06T—IMAGE DATA PROCESSING OR GENERATION, IN GENERAL
    • G06T7/00—Image analysis
    • G06T7/0002—Inspection of images, e.g. flaw detection
    • G06T7/0012—Biomedical image inspection
    • G—PHYSICS
    • G06—COMPUTING OR CALCULATING; COUNTING
    • G06T—IMAGE DATA PROCESSING OR GENERATION, IN GENERAL
    • G06T2207/00—Indexing scheme for image analysis or image enhancement
    • G06T2207/10—Image acquisition modality
    • G06T2207/10004—Still image; Photographic image
    • G—PHYSICS
    • G06—COMPUTING OR CALCULATING; COUNTING
    • G06T—IMAGE DATA PROCESSING OR GENERATION, IN GENERAL
    • G06T2207/00—Indexing scheme for image analysis or image enhancement
    • G06T2207/30—Subject of image; Context of image processing
    • G06T2207/30004—Biomedical image processing
    • G06T2207/30072—Microarray; Biochip, DNA array; Well plate

Definitions

  • the present invention relates to the field of nucleic acid detection, and in particular to a method for detecting nucleotide specific and/or non-specific adsorption.
  • nucleic acid detection on a chip for example, using a chip on which a probe is immobilized on a surface, and further detecting and detecting the captured nucleic acid, including sequence determination, information analysis, etc., probe/nucleic acid complex on the surface of the chip and/or thereon
  • the non-specific adsorption of the substrate to the reaction substrate affects the amount of valid data obtained and the accuracy of the test results.
  • Qualitative or quantitative detection of non-specific adsorption conditions can determine the quality of the chip and the prediction of the test results.
  • a substrate nucleotide (Terminator) on the chip, including non-specific adsorption of substrate nucleotides on the surface of the chip and chip surface.
  • Non-specific adsorption of nucleotides by the probe/probe complex can result in increased sequencing error rates, sequencing throughput, and/or reduced sequencing quality.
  • nucleotides include nucleotide analogs, and different nucleotide analogs may have different non-specific adsorption capacities on the chip surface, probe/nucleic acid complex. For example, performing nucleic acid sequence determination on a chip, the substrate, that is, the molecular size, structure of the nucleotide analog added in the biochemical reaction, and the charge property in the reaction system, etc., affecting the substrate and the chip and/or nucleic acid complex. Binding/adsorption.
  • nucleotide design including nucleotide design, preparation and production, chip production, and nucleic acid sequence detection using a chip.
  • Embodiments of the present invention are directed to at least one of the technical problems existing in the prior art or at least provide a useful commercial choice. To this end, the present invention provides a method of detecting nucleotide-specific and/or non-specific adsorption.
  • a method for detecting nucleotide-specific and/or non-specific adsorption comprising: reacting a nucleotide to be tested with a first template strand, the first template strand and the first chip a probe connection, the first chip comprising a surface of the substrate and a probe fixed at one end to the surface of the substrate, the first template strand carrying a first label, the nucleotide to be tested being capable of pairing with a corresponding base of the first template strand,
  • the other end of the probe extends one base
  • the nucleotide to be detected carries a second mark
  • the first mark can generate a first signal
  • the second mark can generate a second signal
  • the signal on the first chip is detected a first detection result
  • a method of detecting non-specific adsorption of a nucleotide comprising: reacting a nucleotide to be tested with a third template strand, the third template strand being linked to a probe on a third chip,
  • the third chip comprises a surface of the substrate and a probe fixed at one end to the surface of the substrate, and the third template chain carries a third mark, and the nucleotide to be tested cannot be paired with the corresponding base of the third template chain, and the probe cannot be The other end extends one base, the nucleotide to be tested carries a fourth mark, the third mark can generate a third signal, and the fourth mark can generate a fourth signal; the signal on the third chip is detected to obtain a third detection result;
  • the nucleotide to be tested is reacted with a fourth template strand, the fourth template strand is linked to the fourth chip, the fourth chip comprises a surface of the substrate without a probe, and the fourth template
  • a method for detecting nucleotide-specific and/or non-specific adsorption comprising: reacting a nucleotide to be tested with a fifth template strand, the fifth template strand and the fifth chip a probe connection, the fifth chip comprising a surface of the substrate and a probe fixed at one end to the surface of the substrate, the fifth template chain carrying a fifth label, the nucleotide to be tested being capable of pairing with the corresponding base of the fifth template strand,
  • the other end of the probe extends one base
  • the nucleotide to be tested carries a sixth mark
  • the fifth mark can generate a fifth signal
  • the sixth mark can generate a sixth signal
  • the signal on the fifth chip is detected.
  • a fifth detection result reacting the nucleotide to be tested with a sixth template chain, the sixth template chain being connected to a probe on the sixth chip, the sixth chip comprising a substrate surface and a probe having one end fixed to the surface of the substrate
  • the sixth template chain carries a fifth label, and the nucleotide to be tested cannot be paired with the corresponding base of the sixth template strand, and the other end of the probe cannot be extended by one base; the signal on the sixth chip is detected to obtain the first Six test results;
  • reacting the nucleotide to be tested with a seventh template chain the seventh template chain is connected to the seventh chip, the seventh chip comprises a surface of the substrate without the probe; detecting the signal on the seventh chip, obtaining the seventh detection result And detecting the specificity and/or non-specific adsorption of the nucleotide to be detected based on at least two of the fifth detection result, the sixth detection result, and the seventh detection result.
  • the above method for detecting nucleic acid-specific and/or non-specific adsorption can qualitatively or quantitatively detect non-specific adsorption conditions and/or specific adsorption conditions of a probe/template strand on a substrate surface and/or a substrate surface; By detecting the signal on the surface of the substrate by the detection system, it is possible to qualitatively or quantitatively distinguish between specific and non-specific adsorption, and obtain information such as quantification and distribution of non-specific or specific adsorption, which can be used for structural design and preparation of nucleoside analogs, and chip production.
  • FIG. 1 is a schematic diagram showing a pairing/unpairing relationship between a nucleotide to be tested and a corresponding base on a template strand in an embodiment of the present invention
  • FIG. 2 is a schematic flowchart diagram of an image processing method according to an embodiment of the present invention.
  • FIG. 3 is another schematic flowchart of an image processing method according to an embodiment of the present invention.
  • FIG. 4 is a schematic diagram showing the principle of probe immobilization, template strand hybridization, and nucleotide polymerization/adsorption on a substrate surface according to an embodiment of the present invention
  • FIG. 5 is a schematic diagram showing the principle of probe immobilization, template strand hybridization, and nucleotide polymerization/adsorption on the surface of another substrate in an embodiment of the present invention
  • FIG. 6 is a schematic diagram showing the principle of template strand hybridization and nucleotide adsorption on the surface of still another substrate in the embodiment of the present invention.
  • FIG. 7 is an image of a Cy3 fluorescent dot (left image) and an ATTO647N fluorescent dot (right image) in a field of view obtained by photographing an experimental group in an embodiment of the present invention
  • Fig. 9 is a view showing Cy3 fluorescence dots (left image) and ATTO647N fluorescent dots (right image) in one field of view obtained by the control group 2 in the embodiment of the present invention.
  • nucleotides include ribonucleotides, deoxyribonucleotides, and the like, including A, T, C, G, and U, and the like.
  • C is used for cytosine or cytosine analogs
  • G for guanine or guanine analogs
  • A for adenine or adenine analogs
  • T for thymine or thymine analogs
  • U is used to represent uracil or uracil analogs.
  • the "template strand” referred to in the embodiments of the present invention may be DNA and/or RNA, etc., and may also be referred to as a "hybrid strand” in some embodiments, for example, a target DNA strand in the process of detecting nucleic acid using a chip.
  • the "probe” referred to in the embodiments of the present invention may be DNA and/or RNA, etc., and may also be referred to as "primer”, “capture strand” or “fixed strand” in some embodiments.
  • the so-called “probes” can be randomly distributed or regularly distributed on the surface of the substrate, such as arrays, such as capture chips currently on the market, where the probes are generally distributed in an array on the surface of the chip.
  • the so-called “substrate” can be any solid support that can be used to immobilize a nucleic acid sequence, such as a nylon membrane, a glass slide, a plastic, a silicon wafer, a magnetic bead, etc., and the surface of the chip can be used interchangeably with the surface of the substrate unless otherwise specified.
  • the probes are typically attached/immobilized on the surface of the substrate by chemical bonds.
  • the surface of the substrate is a chemically modified surface with reactive groups that can be attached to the probe. Surface modification, fixation, etc. can be carried out using known methods or directly customized or purchased.
  • non-specific adsorption generally refers to the adsorption caused by the force of non-covalent bonds, the force of non-covalent bonds Including hydrophobic interaction, van der Waals force, electrostatic force, etc.; on the chip with probe on the surface, in the process of nucleic acid detection based on the principle of base complementation, generally, the non-specific adsorption of nucleic acid refers to the non-covalent attachment of nucleic acid to the chip. On the surface and / or probe.
  • the so-called “marker” may be any physical, chemical or biologically detectable label.
  • the “signal” referred to in the embodiments of the present invention is a signal generated by the “marker” in a specific situation, typically but not limited.
  • a sexual "marker” is an optically detectable label, such as a fluorescent dye, and correspondingly, the “signal” is a fluorescent signal.
  • the fluorescent dyes such as Cy3 and/or ATTO-647N, are capable of emitting a green fluorescent signal and a red fluorescent signal, respectively, under the excitation of 532 nm and 640 nm wavelengths.
  • test result can be any suitable qualitative or quantitative result, such as visual results, instrumental test results, and the like.
  • the non-specific adsorption of the nucleotide to be tested can be judged by visually measuring the signal on the surface of the substrate, such as the distribution, intensity, etc. of the fluorescent signal.
  • the signal on the surface of the substrate can be detected by means of an instrument/signal detection device to determine the non-specific adsorption of the nucleotide to be tested, for example, the instrument can be an optical detection device with an imaging system, etc. Including the light source, the objective lens, and the camera, correspondingly, the so-called "detection result” includes obtaining an image.
  • the so-called “bright spot” refers to the spot/spot on the image, and one spot/spot occupies at least one pixel.
  • the so-called “pixel” is the same as “pixel.”
  • the so-called “bright spot detection” corresponds to the detection of an optical signal of the nucleic acid or base or base cluster.
  • a method for detecting non-specific adsorption of a nucleotide comprises: S100: reacting a nucleotide to be tested with a first template strand, the first template strand being linked to a probe on the first chip
  • the first chip comprises a surface of the substrate and a probe fixed at one end to the surface of the substrate, the first template chain carries a first label, and the nucleotide to be tested can be paired with the corresponding base of the first template strand, thereby enabling the probe
  • the other end extends one base, the nucleotide to be tested carries a second mark, the first mark can generate a first signal, the second mark can generate a second signal;
  • S300 reacting the nucleotide to be tested with the second template chain, the second template chain is connected to the probe on the second chip, and the second chip comprises a surface of the substrate and a probe fixed to the surface of the substrate at
  • the non-specific adsorption and/or specific adsorption of the probe/template strand on the substrate surface and/or the substrate surface can be qualitatively or quantitatively detected; the detection system can detect the signal on the surface of the substrate, Qualitative or quantitative distinction between specific and non-specific adsorption, obtaining information on the quantification and distribution of non-specific or specific adsorption, can be used for nucleoside analog structure design and preparation, chip production, and all methods involving chip detection of nucleic acid processes or In applications, for example, evaluation of performance of an idle chip (without a probe) or a capture chip (with a probe), quality control of chip production, prediction of effects of chip capture nucleic acid, analysis and comparison, and the like.
  • the first signal and the second signal are detection-distinguishable signals, that is, the first signal and the second signal can be detected as two different signals, such as fluorescent signals of different colors.
  • S100 and S300 are performed in no order, and can be performed sequentially or simultaneously. Similarly, the same is true for S200 and S400. In some embodiments, S100 and S300 are parallel tests performed simultaneously.
  • the materials and surface characteristics of the substrates of the first chip and the second chip in S100 and S300 are substantially the same or identical, such as hydrophilicity, surface size, and the like.
  • the substrate surfaces of the first chip and the second chip are substrates of the same surface properties that are subjected to the same surface treatment.
  • the substrate surfaces of the first chip and the second chip are different surface areas of the same substrate that are subjected to the same surface treatment.
  • the substrate surfaces of the first chip and the second chip in S100 and S300 are in various aspects such as surface size, probe type and nucleotide sequence, probe immobilization density, probe distribution, amount of nucleotide to be detected,
  • the reaction conditions and the like are basically the same.
  • the terms "substantially identical”, “substantially identical” and “consistent” or “identical” mean that the differences produced by different batch preparation, processing, and/or parallel tests are within the allowable tolerances.
  • the first template strand differs from the second template strand in that the nucleotide to be tested is capable of pairing with the corresponding base of the first template strand and not with the corresponding base of the second template strand, as shown in FIG. 1 shows a paired/unpaired relationship of an exemplary nucleotide to be tested and a corresponding base on a template strand in an embodiment of the present invention, in this example, the nucleotide C to be tested and the first template strand Corresponding base G pairing, but not paired with the corresponding base A, C, T or U on the second template strand, thus, reacting the nucleotide C to be tested with the first template strand can keep the probe away One end of the surface of the substrate extends one base C to form a CG pairing relationship; and the nucleotide C to be tested reacts with the second template strand to extend one end of the probe away from the surface of the substrate by one base.
  • the nucleotide to be tested is another nucleotide
  • the distribution or arrangement of the probes on the surface of the substrate is not limited.
  • the probes on the first chip and the second chip are randomly distributed and/or regularly distributed (for example, in an array) on the surface of the substrate. on.
  • the probes were identical on different chip surfaces or in different surface areas of the same chip.
  • the first marker and the second marker can be different fluorescent dyes.
  • detecting a signal on the first chip, obtaining the first detection result includes: taking a photo of the first chip surface by the imaging system to obtain a first image; and detecting the first image to obtain the first detection result.
  • Detecting a signal on the second chip, obtaining a second detection result includes: taking a photo of the second chip surface by the imaging system to obtain a second image; and detecting the second image to obtain a second detection result.
  • the imaging system may be an optical detection device or the like with an imaging system, including a light source, an objective lens, and a camera.
  • detecting the first image to obtain the first detection result further comprises: detecting the first image to determine the number Na1 of the positions of the first signal and the second signal simultaneously on the surface of the first chip and only There is a number Na3 of positions of the second signal.
  • Detecting the second image to obtain the second detection result further comprises: detecting the second image to determine the number Nb1 of locations where the first signal and the second signal are simultaneously present on the surface of the second chip, and the number of locations where only the second signal is present Nb3.
  • the image comprises a plurality of pixel points.
  • the detection of the first image is taken as an example, and it should be understood that the following image detection method is also applicable to the processing of the second image, and the parallel test of the detection example of the same nucleotide-specific/non-specific adsorption.
  • the same treatment can be used to obtain reliable and comparable detection results.
  • an image detecting method includes: an optional image pre-processing step S11, wherein the image pre-processing step S11 includes pre-processing the first image to obtain a first pre-processed image.
  • Image includes pre-processing the first image to obtain a first pre-processed image.
  • Bright spot detecting step S12 the bright spot detecting step S12 includes the steps of: S21, analyzing the first image to calculate a bright spot determination threshold, S22, analyzing the first image to obtain a candidate bright spot, and S23, determining whether the candidate bright spot is a bright spot according to the bright spot determination threshold.
  • the first image is processed by the image pre-processing step, and the calculation amount of the bright spot detecting step can be reduced.
  • whether the candidate bright spot is a bright spot is determined by the bright spot determining threshold, and the accuracy of determining the bright spot of the image can be improved.
  • the image pre-processing step S11 is a step taken to obtain a better detection effect.
  • the image may be directly subjected to a bright spot detection step.
  • the input first image to be detected may be a 16-bit tiff format image of 512*512 or 2048*2048, and the image of the tiff format may be a grayscale image. In this way, the processing of the image detecting method can be simplified.
  • the image pre-processing step S11 includes performing a background subtraction process on the first image to obtain a pre-processed first image. In this way, the noise of the first image can be further reduced, and the accuracy of the image detecting method is higher.
  • the image pre-processing step S11 includes: performing a simplification process on the first image subjected to the subtractive background processing to obtain a pre-processed first image. In this way, the amount of calculation of the subsequent image detecting method can be reduced.
  • the image pre-processing step S11 includes filtering the first image to obtain a pre-processed first image. In this way, filtering the first image can obtain the pre-processed first image under the condition that the image detail features are retained as much as possible, thereby improving the accuracy of the image detection method.
  • the image pre-processing step S11 includes performing a subtractive background process on the first image and then performing a filtering process to obtain the pre-processed first image. In this way, after the background is subtracted from the first image and then filtered, the noise of the first image can be further reduced, and the accuracy of the image detection method is higher.
  • the image pre-processing step S11 includes: performing a simplification process on the first image after performing the subtractive background processing and then performing the filtering process to obtain the pre-processed first image. In this way, the amount of calculation of the subsequent image detecting method can be reduced.
  • the image pre-processing step S11 includes performing a simplification process on the first image to obtain a pre-processed first image. In this way, the amount of calculation of the subsequent image detecting method can be reduced.
  • the step of determining whether the candidate bright spot is a bright spot according to the bright spot determination threshold includes: step S31, searching for the greater than (p*p-1) connectivity in the preprocessed first image. Pixel points and the found pixel points as the center of the candidate bright points, p*p and the bright points are in one-to-one correspondence, each value in p*p corresponds to one pixel point, p is a natural number and is an odd number greater than 1; S32.
  • I max can be understood as the center strongest intensity of the candidate bright spot.
  • I max is the strongest intensity of the center of the 3*3 window.
  • a BI is the ratio of the set value in the first image preprocessed in the 3*3 window, and ceof guass is the pixel and 2D of the 3*3 window. Correlation coefficient of Gaussian distribution.
  • the preprocessed first image is a simplified processed image.
  • the preprocessed first image may be a binarized image, that is, the set value in the binarized image may be that the pixel meets the set condition. The value corresponding to the time.
  • the binarized image may contain two values of 0 and 1 characterizing different attributes of the pixel, the set value is 1, and A BI is the ratio of 1 in the binarized image in the p*p window. .
  • the image includes a plurality of pixel points.
  • detecting the first image includes: highlighting the first image by using a k*k matrix.
  • the detecting comprises determining that the matrix whose central pixel value of the matrix is not less than any pixel value of the matrix non-center corresponds to a bright point, k is an odd number greater than 1, and the k*k matrix comprises k*k pixels.
  • the method is based on the difference between the brightness/intensity of the signal generated by the mark and the background brightness/intensity, enabling simple and rapid detection of the image, detection and acquisition of signal information.
  • the pixel value of the matrix center is greater than a first preset value, and any one of the matrix non-center pixel values is greater than the second pixel value.
  • the first preset value and the second preset value may be set according to experience or a certain amount of pixel/intensity data of a normal bright spot of a normal image, and the so-called "normal image” and "normal bright spot” may be normal to the naked eye. If the image looks clear, the background is clean, the size and intensity of the highlights are relatively uniform.
  • the first preset value and the second preset value are related to an average pixel value of the image. For example, the first preset value is set to be 1.4 times the average pixel value of the image, and the second preset value is 1.1 times the average pixel value of the image, which can eliminate interference and obtain a better bright spot detection result.
  • the first image is a color image
  • one pixel of the color image has three pixel values
  • the color image can be converted into a gray image, and then image detection is performed to reduce the calculation of the image detection process.
  • Quantity and complexity may be selected to be converted into a grayscale image using, but not limited to, a floating point algorithm, an integer method, a shift method, or an average value method.
  • the color image can also be directly detected.
  • the size comparison of the pixel values mentioned above can be regarded as a three-dimensional value or a comparison of the size of an array of three elements, and the relative sizes of the plurality of multi-dimensional values can be customized according to experience and needs, for example. When any two-dimensional value in the three-dimensional value a is larger than the value of the corresponding dimension of the three-dimensional value b, the three-dimensional value a is considered to be larger than the three-dimensional value b.
  • the first image is a grayscale image
  • the pixel value of the grayscale image is a one-dimensional value
  • the pixel value of the grayscale image is the same as the grayscale value
  • the surface of the substrate also referred to as the surface of the chip, is a surface-modified surface with an epoxy group: a probe (DNA capture strand, Capture DNA)
  • the probe is an amino-modified probe having an amino group at the end, and the probe is reacted with an epoxy group on the surface of the chip by -NH 3 , fixed on the surface of the substrate, and then the chip is passivated using a passivation solution. To block unreacted epoxy groups. Further, a hybrid template strand is added, which is a DNA strand of a specific sequence which is end-labeled with a Cy3 fluorescent dye and which is complementary to the base of the probe.
  • the nucleotide to be tested which is complementary to the first base to be tested of the hybrid template strand and labeled with ATTO-647N fluorescent dye is added, and then the surface is irradiated by a fluorescence microscope such as total internal reflection fluorescence microscopy (TIRF). Take a picture and get an image with signal points/spots.
  • TIRF total internal reflection fluorescence microscopy
  • the surface of the substrate is also referred to as a chip surface, and is a surface-modified epoxy group-bearing surface: the same reaction system and time, the same concentration, the same concentration
  • the probe by reacting -NH 3 with an epoxy group on the surface of the chip, bonds the probe to the surface of the chip by a chemical bond, and then passivates the chip with a passivation solution to block the unreacted epoxy group. Further, a hybrid template strand is added, which is a DNA strand with a Cy3 fluorescent dye label at the end and a complementary pair with the probe sequence, but the first base to be tested is not paired with the nucleotide to be added to be added.
  • the nucleotide to be tested (which is the same as the nucleotide to be tested in the example of Fig. 4) is added, labeled with ATTO-647N fluorescent dye, and then the surface is subjected to total internal reflection fluorescence microscopy (TIRF). The image is taken to obtain an image with fluorescent signal spots/spots.
  • TIRF total internal reflection fluorescence microscopy
  • a method for detecting non-specific adsorption of a nucleotide comprises: S1000: reacting a nucleotide to be tested with a third template strand, the third template strand being linked to a probe on a third chip
  • the third chip includes a surface of the substrate and a probe fixed to the surface of the substrate at one end, and the third template chain carries a third mark, and the nucleotide to be tested cannot be paired with the corresponding base of the third template chain, and the probe cannot be made.
  • the other end extends one base, the nucleotide to be tested carries a fourth mark, the third mark can generate a third signal, and the fourth mark can generate a fourth signal; S2000: detects the signal on the third chip, obtains a third The detection result; S3000: reacting the nucleotide to be tested with the fourth template chain, the fourth template chain is connected to the fourth chip, the fourth chip includes the surface of the substrate without the probe, and the fourth template chain has the third mark
  • the nucleotide to be tested can be paired with the corresponding base of the fourth template chain; S4000: detecting the signal on the fourth chip to obtain the fourth detection result; S5000: detecting the test based on the third detection result and the fourth detection result Nucleotide Specific adsorption.
  • the non-specific adsorption of the probe/template strand on the substrate surface and/or the surface of the substrate can be qualitatively or quantitatively detected; and the non-specific adsorption can be qualitatively or quantitatively distinguished by detecting the signal on the surface of the substrate by the detection system.
  • Type, information such as non-specific quantification, distribution, etc. can be used in chip production and in all methods or applications involving chip detection of nucleic acid processes, such as for no-load chips (without probes) or capture chips (with probes) Evaluation of performance, quality control of chip production, prediction, analysis and comparison of effects of chip capture nucleic acid.
  • the S1000 and S3000 are carried out in no order, and can be carried out sequentially or simultaneously. Similarly, the S2000 and S4000 are also carried out without order. In some embodiments, S1000 and S3000 are parallel tests for simultaneous ⁇ ⁇
  • the materials and surface characteristics of the substrates of the third chip and the fourth chip in S1000 and S3000 are substantially the same or identical, such as hydrophilicity, surface size, and the like.
  • the substrate surfaces of the third and fourth chips are substrates of the same surface properties that are subjected to the same surface treatment.
  • the substrate surfaces of the third and fourth chips are different surface areas of the same substrate that have been subjected to the same surface treatment.
  • the substrate surfaces of the third chip and the fourth chip in S1000 and S3000 are substantially identical except for whether or not the probe is immobilized, such as the surface size, the amount of the nucleotide to be detected, the reaction conditions, and the like.
  • the terms “substantially identical”, “substantially identical” and “consistent” or “identical” mean that the differences produced by different batch preparation, processing, and/or parallel tests are within the allowable tolerances.
  • the distribution or arrangement of the probes on the surface of the third chip is not limited. In the embodiment of the invention, the probes are randomly distributed or regularly distributed on the surface of the third chip (for example, in an array).
  • the third and fourth labels are different fluorescent dyes.
  • detecting a signal on the third chip, obtaining the third detection result includes: taking a photo of the third chip surface by the imaging system to obtain a third image; and detecting the third image to obtain the third detection result.
  • Detecting a signal on the fourth chip, obtaining a fourth detection result includes: taking a photo of the fourth chip surface by the imaging system to obtain a fourth image; and detecting the fourth image to obtain a fourth detection result.
  • the imaging system may be an optical detection device or the like with an imaging system, including a light source, an objective lens, and a camera.
  • detecting the third image to obtain the third detection result further comprises: detecting the third image to determine the number Nc1 of positions at which the third signal and the fourth signal appear simultaneously on the surface of the third chip, and only The number of positions Nc3 at which the fourth signal appears.
  • Detecting the fourth image to obtain the fourth detection result further includes: detecting the fourth image to determine the number Nd1 of positions at which the third signal and the fourth signal appear simultaneously on the surface of the fourth chip, and the number of positions where only the fourth signal appears Nd3.
  • the detection of the image can be referred to the above-described detection example of the first image.
  • the formula (Nc1+Nc3-Nd1-Nd3)/(Nc1+Nc3) can be used to determine the non-specific adsorption change ratio of the nucleotide to be measured after the substrate surface is fixed by the probe, thereby realizing the third detection result and the fourth detection result. Non-specific adsorption of the nucleotide to be tested is detected.
  • the surface of the substrate also referred to as the surface of the chip, is a surface-modified surface with an epoxy group: a probe (DNA capture strand, Capture DNA)
  • the probe is an amino-modified probe having an amino group at the end, and the probe is reacted with an epoxy group on the surface of the chip by -NH 3 , fixed on the surface of the substrate, and then the chip is passivated using a passivation solution. To block unreacted epoxy groups.
  • a hybrid template strand is added, which is a DNA strand with a Cy3 fluorescent dye label at the end and a complementary pair with the probe sequence, but the first base to be tested is not paired with the nucleotide to be added to be added.
  • the nucleotide to be tested labeled with ATTO-647N fluorescent dye is added to carry out hybridization.
  • the surface is photographed by a fluorescence microscope including a total internal reflection fluorescence microscope (TIRF) to obtain an image with a fluorescent signal.
  • TIRF total internal reflection fluorescence microscope
  • the tendency is to indicate that there is a non-specific adsorption of the template strand and the nucleotide at the same position, the number is Nc1; (2) if only the green fluorescent signal is observed, the position tends to indicate the position For the non-specific adsorption of the template strand, the number is Nc2; (3) If only the red fluorescent signal is observed, the tendency is to indicate that the position is a non-specific adsorption of nucleotides, the number is Nc3.
  • the surface of the substrate is also referred to as a chip surface, and is a surface-modified epoxy group-bearing surface.
  • a fixing solution containing no probe is added.
  • the reaction system and reaction time were the same as in the above-described example of Fig. 5, and then the chip was passivated using a passivation liquid.
  • a hybrid template strand is added, which is a DNA strand of a specific sequence tagged with a Cy3 fluorescent dye at the end.
  • the nucleotide to be tested labeled with ATTO-647N fluorescent dye is added, and then the surface is imaged by a fluorescence microscope including a total internal reflection fluorescence microscope (TIRF) to obtain an image with a fluorescent signal.
  • TIRF total internal reflection fluorescence microscope
  • the formula (Nc1+Nc3-Nd1-Nd3)/(Nc1+Nc3) can be further used to determine the proportion of non-specific adsorption changes of the nucleotide to be measured after the substrate surface passes through the immobilized probe.
  • a method for detecting nucleotide-specific and/or non-specific adsorption comprises: S10000: reacting a nucleotide to be tested with a fifth template strand, the fifth template strand and a fifth chip a probe connection, the fifth chip comprising a surface of the substrate and a probe fixed to the surface of the substrate at one end, the fifth template chain carrying a fifth label, the nucleotide to be tested being capable of pairing with the corresponding base of the fifth template strand So that the other end of the probe extends one base, the nucleotide to be tested carries a sixth mark, the fifth mark can generate a fifth signal, and the sixth mark can generate a sixth signal; S20000: detecting the fifth chip Signal, obtaining a fifth detection result; S30000: reacting the nucleotide to be tested with a sixth template chain, the sixth template chain being connected to the probe on the sixth chip, the sixth chip including the surface of the substrate and one end fixed thereto The probe
  • the specificity and/or non-specific adsorption of the probe/template strand on the substrate surface and/or the substrate surface can be qualitatively or quantitatively detected; the detection system can detect the signal on the surface of the substrate, and can be qualitative or Quantitatively distinguish between specific and/or non-specific types of adsorption, obtain specific and/or non-specific quantification, distribution, etc., can be used for nucleotide analog performance evaluation, chip production, and all methods involving chip detection of nucleic acid processes or In applications, for example, evaluation of performance of an idle chip (without a probe) or a capture chip (with a probe), quality control of chip production, prediction of effects of chip capture nucleic acid, analysis and comparison, and the like.
  • S10000, S30000, and S50000 are performed in no order, and may be performed sequentially or simultaneously. Similarly, the execution of S20000, S40000, and S60000 is also not limited in order. In some embodiments, S10000, S30000, and S50000 are parallel tests, performed simultaneously.
  • the materials, surface characteristics such as hydrophilicity, surface size, and the like of the substrates of the fifth chip, the sixth chip, and the seventh chip in S10000, S30000, and S50000 are substantially the same or identical.
  • the substrate surfaces of the fifth, sixth, and seventh chips are substrates of the same surface properties that are subjected to the same surface treatment.
  • the substrate surfaces of the fifth, sixth, and seventh chips are different surface areas of the same substrate that are subjected to the same surface treatment.
  • the substrate surfaces of the fifth chip and the sixth chip in S10000 and S30000 are in various aspects such as surface size, probe type and nucleotide sequence, probe immobilization density, probe distribution, amount of nucleotide to be detected,
  • the reaction conditions and the like are basically the same.
  • the seventh chip in S50000 is basically the same as the fifth chip and the sixth chip in S10000 and S30000, except for the absence of the fixed probe, such as the surface size, the amount of the nucleotide to be tested, and the reaction conditions. .
  • the terms “substantially identical”, “substantially identical” and “consistent” or “identical” mean that the differences produced by different batch preparation, processing, and/or parallel tests are within the allowable tolerances.
  • the distribution or arrangement of the probes on the surface of the fifth chip and the sixth chip is not limited.
  • the probes are randomly distributed or regularly distributed on the surfaces of the fifth chip and the sixth chip (for example, in an array). distributed). In parallel experiments, the probes were distributed in the same state on different chip surfaces.
  • the fifth and sixth indicia are different fluorescent dyes.
  • detecting a signal on the fifth chip, obtaining a fifth detection result includes: taking a photo of the fifth chip surface by the imaging system to obtain a fifth image; and detecting the fifth image to obtain a fifth detection result.
  • Detecting the signal on the sixth chip, obtaining the sixth detection result includes: taking a picture of the sixth chip surface by the imaging system to obtain a sixth image; and detecting the sixth image to obtain a sixth detection result.
  • Detecting the signal on the seventh chip, and obtaining the seventh detection result includes: taking a photo of the seventh chip surface by the imaging system to obtain a seventh image; and detecting the seventh image to obtain a seventh detection result.
  • the imaging system may be an optical detection device or the like with an imaging system, including a light source, an objective lens, and a camera.
  • detecting the fifth image to obtain the fifth detection result further comprises: detecting the fifth image to determine the number Ne1 of the positions of the fifth signal and the sixth signal simultaneously on the surface of the fifth chip and only There is a number Ne3 of positions of the sixth signal.
  • Detecting the sixth image to obtain the sixth detection result further includes: detecting the sixth image to determine the number Nf1 of positions at which the fifth signal and the sixth signal are simultaneously present on the surface of the sixth chip, and the number of positions where only the sixth signal exists Nf3.
  • Detecting the seventh image to obtain the seventh detection result further includes: detecting the seventh image to determine the number Ng1 of positions at which the fifth signal and the sixth signal appear simultaneously on the surface of the seventh chip, and the number of positions where only the sixth signal appears Ng3.
  • the detection of the image can be referred to the above-described detection example of the first image.
  • Detecting the specificity and/or non-specific adsorption of the nucleotide to be tested (a) determining the ratio of non-specific adsorption of the nucleotide to be detected on the surface of the substrate and the nucleic acid strand using the formula (Nf1+Nf3)/(Ne1+Ne3); (b) Using the formula Nf1/(Ne1+Ne3) to determine the ratio of non-specific adsorption of the nucleotide to be detected in the nucleic acid strand; (c) using the formula (Ne1+Ne3-Nf1-Nf3)/(Ne1+Ne3) to determine the test The effective ratio of nucleotide to template strand; (d) using the formula (Ng1+Ng3-Nf1-Nf3)
  • the surface of the substrate also referred to as the surface of the chip, is a surface-modified surface with an epoxy group: a probe (DNA capture strand, Capture DNA)
  • the probe is an amino-modified probe having an amino group at the end, and the probe is reacted with an epoxy group on the surface of the chip by -NH 3 , fixed on the surface of the substrate, and then the chip is passivated using a passivation solution. To block unreacted epoxy groups. Further, a hybrid template strand is added, which is a DNA strand of a specific sequence which is end-labeled with a Cy3 fluorescent dye and which is complementary to the base of the probe.
  • the nucleotide to be tested which is complementary to the first base to be tested of the hybrid template strand and labeled with ATTO-647N fluorescent dye is added, and then the surface is irradiated by a fluorescence microscope such as total internal reflection fluorescence microscopy (TIRF). Take a picture and get an image with signal points/spots.
  • TIRF total internal reflection fluorescence microscopy
  • the surface of the substrate is also referred to as a chip surface, and is a surface-modified epoxy group-bearing surface: the same reaction system and time, the same concentration, the same concentration
  • the probe by reacting -NH 3 with an epoxy group on the surface of the chip, bonds the probe to the surface of the chip by a chemical bond, and then passivates the chip with a passivation solution to block the unreacted epoxy group. Further, a hybrid template strand is added, which is a DNA strand with a Cy3 fluorescent dye label at the end and a complementary pair with the probe sequence, but the first base to be tested is not paired with the nucleotide to be added to be added.
  • the nucleotide to be tested (which is the same as the nucleotide to be tested in the example of Fig. 4) is added, labeled with ATTO-647N fluorescent dye, and then the surface is subjected to total internal reflection fluorescence microscopy (TIRF). The image is taken to obtain an image with fluorescent signal spots/spots.
  • TIRF total internal reflection fluorescence microscopy
  • the nucleotides at this position are non-specifically adsorbed with the template strand, the number is Nf1; (2) If only the green fluorescent signal is observed, the position is The non-specific adsorption of the template strand is in the number of Nf2; (3) If only the red fluorescent signal is observed, the position is the surface non-specific adsorption of the nucleotide, and the number is Nf3.
  • the surface of the substrate is also referred to as a chip surface, and is a surface-modified epoxy group-bearing surface.
  • a fixing solution containing no probe is added.
  • the reaction system and reaction time were the same as in the above-described example of Fig. 5, and then the chip was passivated using a passivation liquid.
  • a hybrid template strand is added, which is a DNA strand of a specific sequence tagged with a Cy3 fluorescent dye at the end.
  • the nucleotide to be tested labeled with ATTO-647N fluorescent dye is added, and then the surface is imaged by a fluorescence microscope including a total internal reflection fluorescence microscope (TIRF) to obtain an image with a fluorescent signal.
  • TIRF total internal reflection fluorescence microscope
  • Example 1 Investigating the effects of non-specific adsorption during a nucleotide reaction
  • Chip glass with epoxy group on the surface (purchased from SCHOTT);
  • EKB-6P Fixed strand
  • Hybridization chain 1 (EKB-6T-2-Cy3): a specific sequence synthesized by a complementary pair with a fixed strand, with a short sequence of 33 bp of the sequence to be tested and a fluorophore Cy3 at the end.
  • the specific sequence is:
  • Hybridization chain 2 (EKB-6T-6-Cy3): a synthetically specific sequence that is not complementary to a fixed strand, with a 33 bp length to be tested (inconsistent with the EKB-6T-2-Cy3 sequence to be tested), terminus A short sequence of DNA with the fluorophore Cy3, the specific sequence is:
  • immobilized chain 1 (experimental group, control group 1): After washing and drying the glass, placed in a solution of 150 mM K 2 HPO 4 and containing a concentration of 1.0 M amino-modified EKB-6P nucleic acid sequence, 37 ° C conditions The reaction was carried out for 0.5 hour, washed successively with 3X SSC solution (containing 0.1% Triton), 3XSSC, 0.15 MK 2 HPO 4 solution, and then passivated with 1 M K 2 HPO 4 at 37 ° C for 17 hours.
  • 3X SSC solution containing 0.1% Triton
  • 3XSSC 0.15 MK 2 HPO 4 solution
  • Fixation of the fixed chain 2 (control group 2): After the glass was washed and dried, it was placed in a 150 mM K 2 HPO 4 solution, and reacted at 37 ° C for 0.5 hour, followed by 3XSSC solution (containing 0.1% Triton), 3XSSC, After washing with 0.15 MK 2 HPO 4 solution, 1 M K 2 HPO 4 was added and passivated at 37 ° C for 17 hours.
  • Chip (Flow Cell) Assembly The passivated chips are assembled into a four-channel flow cell.
  • Hybridization of hybridization chain 1 (experimental group, control group 2): The assembled four-channel flow cell was added to Rinse buffer (1XSSC + 150 mM HEPES + 0.1% SDS), reconstituted at 55 ° C for 0.5 hour, and then hybridized. A 3X SSC solution having a chain (EKB-6T-2-Cy3) concentration of 1 nM was reacted at 55 ° C for 0.5 hour. The channels were then washed sequentially using Rinse buffer (1XSSC + 150 mM HEPES + 0.1% SDS) and Buffer H (150 mM HEPES + 150 mM NaCl).
  • Hybridization of Hybrid Chain 2 (Control 1): The assembled four-channel flow cell was added to Rinse buffer (1XSSC + 150 mM HEPES + 0.1% SDS), reconstituted at 55 ° C for 0.5 hour, and then added to the hybrid strand (EKB). -6T-6-Cy3) A 3XSSC solution having a concentration of 1 nM was reacted at 55 ° C for 0.5 hour. The channels were then washed sequentially using Rinse buffer (1XSSC + 150 mM HEPES + 0.1% SDS) and Buffer H (150 mM HEPES + 150 mM NaCl).
  • Nucleotide reaction Add a reaction solution containing a concentration of 200 nM of nucleotide A to the channel, and react at 37 ° C for 90 s, then use Rinse buffer (1XSSC + 150 mM HEPES + 0.1% SDS) and Buffer H (150 mM HEPES + 150 mM). NaCl) Wash the channels sequentially and then add imaging buffer.
  • Photo detection Photographing the chip channel using TIRF.
  • the results of the experimental group are shown in Fig. 7.
  • the left image shows the Cy3 fluorescence point in one field of view.
  • the number of Cy3 fluorescence points in all the fields of the multiple channels is 19852.
  • the right picture shows the ATTO647N fluorescence points in the same field of view.
  • the ATTO647N fluorescence point of the field of view is 16744, and the coincidence points are 13358.
  • the results of the control group 1 are shown in Fig. 8.
  • the left image shows the Cy3 fluorescence point in one field of view.
  • the number of Cy3 fluorescence points in all the fields of the multiple channels is counted as 19971.
  • the right image shows the ATTO647N fluorescence points in the same field of view.
  • the ATTO647N fluorescence point of all fields of view was 619, and the coincidence points were 358.
  • the results of the control group 2 are shown in Fig. 9.
  • the left image shows the Cy3 fluorescence point in one field of view.
  • the number of Cy3 fluorescence points in all the fields of the multiple channels is 603.
  • the right image shows the ATTO647N fluorescence points in the same field of view.
  • the ATTO647N fluorescence point of all fields of view is 524.
  • the Cy3 fluorescence point is the impurity on the surface of the chip, and the brightness is weak.
  • the ATTO647N fluorescence point is the non-specific adsorption of nucleotides on the surface of the chip.
  • the ratio of the non-specific adsorption of the nucleotide to be detected on the surface of the substrate and the nucleic acid strand is 3.25%; the ratio of the non-specific adsorption of the nucleotide to be detected in the nucleic acid strand is 1.19%; the nucleotide to be tested and the template The effective proportion of the chain was 96.75%; the ratio of non-specific adsorption of the nucleotide to be measured after the surface of the substrate was fixed by the probe was -86.5%.

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Abstract

L'invention concerne un procédé pour détecter une adsorption spécifique et/ou non spécifique de nucléotide, comprenant les étapes consistant : à amener un nucléotide à tester à réagir avec un premier brin matrice, et à lier le premier brin matrice à une sonde sur une première puce, le nucléotide à tester pouvant être apparié avec une base correspondante sur le premier brin matrice, de telle sorte que la sonde soit étendue par une base, le premier brin matrice et le nucléotide à tester présentant des marqueurs qui produisent des signaux différents; à détecter un signal sur la première puce en vue d'obtenir un premier résultat de détection; à amener le nucléotide à tester à réagir avec un second brin matrice, et à lier le second brin matrice à une sonde sur une seconde puce, le second brin matrice et le nucléotide à tester présentant différents marqueurs, et le nucléotide à tester étant incapable de s'apparier avec une base correspondante sur le second brin matrice; à détecter un signal sur la seconde puce en vue d'obtenir un second résultat de détection; sur la base des résultats de détection, à détecter l'adsorption spécifique et/ou non spécifique du nucléotide à détecter, et à détecter l'adsorption spécifique et/ou non spécifique du nucléotide avec une surface d'un substrat ou d'une sonde sur ce dernier, qui sont utilisés pour un contrôle de qualité dans la production de puces.
PCT/CN2018/107249 2017-11-22 2018-09-25 Procédé de détection d'une adsorption spécifique et/ou non spécifique de nucléotide Ceased WO2019100842A1 (fr)

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CN108034699A (zh) * 2017-11-22 2018-05-15 深圳市瀚海基因生物科技有限公司 一种检测核苷酸特异性和/或非特异性吸附的方法
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