CN107267599B - Method for accurately identifying nucleic acid - Google Patents

Abstract

The present disclosure relates to a method for identifying nucleic acids. The identification method comprises the following steps: a. contacting the sample with a hybridization probe, b. optionally, eluting the hybridization probe that is not bound to the target nucleic acid, and c. identifying whether the hybridization probe is bound to the target nucleic acid; wherein the hybridization probe comprises a reporter group and the hybridization probe differs in state in two cases, thereby generating an identifiable signal by the reporter group: (i) the hybridization probe is in a free state, (ii) the hybridization probe is bound to the target nucleic acid. The disclosure also relates to the use of hybridization probes in fluorescence microscopy imaging of target nucleic acids. The identification method disclosed by the disclosure can simplify the operation steps of nucleic acid identification, efficiently label target nucleic acid molecules, reduce the nonspecific binding of probes and nucleic acids except the target nucleic acids, improve the identification efficiency and accuracy, is particularly beneficial to the identification of nucleic acids with shorter length and/or non-repetitive sequences, and is suitable for obtaining super-resolution images of the target nucleic acids.

Description

Method for accurately identifying nucleic acid

Technical Field

The disclosure relates to the field of biological identification, in particular to an accurate identification method of nucleic acid.

Background

In order to study the structure and interaction of nucleic acid molecules in cells, it is necessary to identify the presence, position, form, and the like of nucleic acid molecules. One common method is Fluorescence In Situ Hybridization (FISH). In this method, a probe is hybridized with a target nucleic acid based on the high complementarity of the sequences, and then the target nucleic acid of a specific sequence is identified and localized by imaging with the aid of a fluorophore directly or indirectly labeled with the probe (non-patent document 1).

However, due to the limitation of diffraction limit, the conventional optical microscopy can only obtain the imaging resolution of about 200-300 nm in the lateral direction and about 600nm in the axial direction, so that the smaller-scale organelles or molecular structures cannot be distinguished. To overcome this problem, various super-resolution imaging methods have been proposed in recent years. For example, non-patent document 2 proposes labeling a chromosomal region containing a repetitive sequence with a DNA probe containing a 2.1kb HaeIII fragment, and obtaining an image with a resolution of about 50nm by photo-activated localization microscopy (PALM). However, the labeling and identification of non-repetitive nucleic acid regions, particularly nucleic acid regions of short length, is very difficult.

Patent document 1 discloses a super-resolution imaging method. In this method, the docking strand is hybridized to the target molecule to be identified, and then the fluorescently labeled imaging strand is bound to the docking strand, followed by imaging by activating the fluorophore on the imaging strand. This method requires the use of at least two functional molecules (docking strand and imaging strand), and thus is cumbersome to handle and is detrimental to the efficiency of labeling the target molecule.

For example, in an example provided in patent document 1, "DNA paint" (DNA paint) is used as a docking chain. The DNA coating is a marker for labeling a chromosomal region to be identified along its entire length (patent document 2). The user needs to establish a complicated system to prepare the DNA paint, and in the process of preparing the DNA paint by PCR, deviation may occur in the final concentration of the prepared DNA paint due to sequence preference, resulting in instability of FISH labeling.

In particular, the DNA coating may bind non-specifically to the sample to be tested, resulting in reduced imaging quality. To reduce non-specific binding, the temperature at which the DNA coating hybridizes to the sample needs to be increased. However, the increase in temperature leads to a decrease in the strength of the specific hybridization required, so that a greater number of imaging strands has to be required to label the target molecules to be detected sufficiently effectively. This prevents further improvement in resolution, and thus it is difficult to achieve high-resolution imaging of a shorter region of the target molecule.

List of citation documents

Patent document

Patent document 1: WO 2015/017586

Patent document 2: US 2010304994(A1)

Non-patent document

Non-patent document 1: Langer-Safer PR, Levine M, Ward DC. immunological method for mapping genes on Drosophila polytene chromosomes. Proc Natl Acad Sci USA.1982Jul; 79(14):4381-5.

Non-patent document 2: weiland, Y., Lemmer, P., Cremer, C.2011.combining FISH with localization microscopy, Super-resolution imaging of nuclear genome nanostructures Chromosome Res,19,5-23.

Non-patent document 3: rouilard, J.M., Zuker, M. & Gulari, E.2003.OligoArray2.0 design of oligonucleotide probes for DNA microarrays using a thermomechanical approach. nucleic Acids Res,31,3057-62.

Non-patent document 4: markham, N.R. & Zuker, M.2008.UNAFold: software for nucleic acid folding and hybridization. methods Mol Biol,453,3-31.

Non-patent document 5: huang, B, Wang, W., Bates, M. & Zhuang, X.2008.three-dimensional super-resolution imaging by means of magnetic recording microscopy. science,319,810-3.

Disclosure of Invention

As used herein, the following terms/words have the following meanings, unless otherwise indicated:

"DNA" means deoxyribonucleic acid.

"RNA" means ribonucleic acid.

"molecular beacon" means an oligonucleotide fragment with a stem-loop structure, conjugated at the 5 'and 3' ends with a fluorophore and a quencher, respectively, that is morphologically distinct in the two following states, thereby producing a recognizable signal: (i) the molecular beacon is not bound to the target nucleic acid but exists freely, (ii) the molecular beacon is bound to the target nucleic acid.

“T_mThe value "represents the temperature at which the double helix structure of the double-stranded nucleic acid is half dissociated.

Problems to be solved by the invention

The present disclosure provides a method for identifying a nucleic acid, which can simplify the steps of nucleic acid identification, and can efficiently label a target nucleic acid molecule to improve the efficiency and accuracy of identification.

Means for solving the problems

The present disclosure provides a method for identifying a nucleic acid, the method comprising the steps of:

a. the sample is brought into contact with the hybridization probe,

b. optionally, eluting hybridization probes that are not bound to the target nucleic acid, and

c. identifying whether the hybridization probe binds to the target nucleic acid;

wherein the hybridization probe comprises a reporter group and the hybridization probe differs in state in two cases, thereby generating an identifiable signal by the reporter group: (i) the hybridization probe is in a free state, (ii) the hybridization probe is bound to the target nucleic acid.

According to an aspect of the present disclosure, step c of the identification method is performed by fluorescence spectrophotometer reading or fluorescence microscopy imaging, preferably said fluorescence microscopy imaging is super-resolution imaging.

According to one aspect of the disclosure, a hybridization probe comprises a binding region and a dissociation region, the dissociation region self-hybridizes when the hybridization probe is in a free state, and the reporter group does not emit an identifiable signal; when the hybridization probe is specifically bound to the target sequence segment of the target nucleic acid through the binding region, the dissociation region dissociates and the reporter group emits an identifiable signal.

According to one aspect of the disclosure, the reporter comprises a fluorophore and a quencher, and the identifiable signal is fluorescence; preferably, the fluorophore is Alexa-647 and/or the quencher is BHQ 3.

According to an aspect of the present disclosure, the hybridization probe comprises a loop structure serving as the binding region, and at least two arm structures serving as the dissociation region, wherein the at least two arm structures are located on both sides of the loop structure, respectively.

According to an aspect of the disclosure, the length of the loop structure is 20nt to 60nt, preferably 25nt to 55nt, more preferably 30nt to 50nt, more preferably 35nt to 45nt, still further preferably 40nt to 44nt, and/or the length of the arm structure is 4nt to 10nt, preferably 5nt to 9nt, preferably 6nt to 8nt, more preferably 7 nt.

According to an aspect of the disclosure, the sequence identity between the loop structure and the target sequence fragment of the target nucleic acid is between 90% and 100%, preferably between 95% and 100%, more preferably between 98% and 100%.

According to one aspect of the disclosure, the target nucleic acid is less than 4.9kb in length; preferably, the target nucleic acid has a length of 3.3kb or less, more preferably 2.5kb or less.

According to one aspect of the disclosure, the target nucleic acid is free of repetitive sequences.

According to an aspect of the disclosure, step a of the identification method is performed according to the following method: the sample and the hybridization probe are incubated together at a temperature of 70 ℃ to 80 ℃, preferably 73 ℃ to 77 ℃, more preferably 75 ℃, and then hybridized at a temperature of 10 ℃ to 42 ℃, more preferably 12 ℃ to 38 ℃, more preferably 14 ℃ to 34 ℃, more preferably 16 ℃ to 30 ℃, more preferably 18 ℃ to 26 ℃, more preferably 22 ℃.

According to an aspect of the disclosure, the standard deviation of the accuracy of single molecule repeat localization in x-y direction of the super resolution imaging is 50nm or less, preferably 20nm or less, further preferably 9nm or less; alternatively, the full width at half maximum of the accuracy of the single molecule repeat localization in the x-y direction of the super-resolution imaging is 120nm or less, preferably 50nm or less, and more preferably 20nm or less.

According to an aspect of the disclosure, the standard deviation of the accuracy of single molecule repeat localization in the z-direction of the super-resolution imaging is 100nm or less, preferably 40nm or less, further preferably 20nm or less; alternatively, the full width at half maximum of the positioning accuracy of the single molecule repeat in the z direction in the super-resolution imaging is 250nm or less, preferably 100nm or less, and more preferably 50nm or less.

According to one aspect of the disclosure, the laser power used to excite the fluorophore is 0.80kW/cm²～2.00kW/cm²Preferably 0.90kW/cm²～1.45kW/cm²Further preferably 1.00kW/cm²。

According to an aspect of the present disclosure, the sampling frame frequency when the fluorescence is recognized is 10Hz or more, preferably 50Hz or more, more preferably 60Hz or more, and further preferably 85Hz or more.

The present disclosure also provides for the use of a hybridization probe in the fluorospectrophotometric reading or fluorescence microscopy imaging of a target nucleic acid, wherein the hybridization probe comprises a reporter group and the hybridization probe differs in state between: (i) the hybridization probe is in a free state, (ii) the hybridization probe is bound to the target nucleic acid.

In the use according to the present disclosure, preferably the fluorescence microscopy imaging is super-resolution imaging; preferably the target nucleic acid is less than 4.9kb in length, e.g.less than 3.3kb in length, e.g.less than 2.5kb in length; preferably the target nucleic acid does not contain repetitive sequences; preferably, the standard deviation of the accuracy of the single molecule repeat locations in the x-y direction for super-resolution imaging is 50nm or less, e.g., 20nm or less, e.g., 9nm or less; alternatively, the full width at half maximum of the accuracy of single molecule repeat localization in the x-y direction of the super-resolution imaging is 120nm or less, such as 50nm or less, such as 20nm or less; alternatively, the standard deviation of the accuracy of the single molecule repeat localization in the z-direction of the super-resolution imaging is preferably 100nm or less, such as 40nm or less, for example 20nm or less; alternatively, the full width at half maximum of the resolution of the single molecule repeat localization in the z-direction of the super-resolution imaging is preferably 250nm or less, such as 100nm or less, for example 50nm or less.

ADVANTAGEOUS EFFECTS OF INVENTION

The nucleic acid identification method disclosed by the invention can simplify the operation steps of nucleic acid identification, efficiently label target nucleic acid molecules, reduce the nonspecific binding of probes and nucleic acids except the target nucleic acids, improve the identification efficiency and accuracy, is particularly beneficial to the identification of nucleic acids with shorter lengths and/or non-repetitive sequences, and is particularly suitable for obtaining super-resolution images of the target nucleic acids.

In particular, by using the nucleic acid identification method of the present disclosure, the dilemma of selecting the hybridization temperature is eliminated, and non-specific binding can be attenuated while increasing the specific hybridization strength, thereby enabling high-resolution imaging of shorter (e.g., less than 4.9kb) target nucleic acid fragments.

On the other hand, the nucleic acid identification method simplifies the operation steps and the requirements on reagents, does not need a user to establish an expensive and complicated nucleic acid coating generation system, saves the cost, has stable and controllable quality of the nucleic acid coating and the probe, and is more convenient and feasible to operate.

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate exemplary embodiments, features, and aspects of the disclosure and, together with the description, serve to explain the principles of the disclosure.

FIG. 1a shows the structure of a target nucleic acid fragment inserted into the genome of a cell prepared in example 1.

FIG. 1b shows the results of PCR detection of the cell sample (I) prepared in example 1, showing that the positive sample contains the target nucleic acid fragment and the negative sample does not contain the target nucleic acid fragment.

FIG. 2 shows the sequence of the antisense strand of the target nucleic acid fragment of 3.3kb in example 1.

FIG. 3a shows fluorescence emission readings of hybridization probes MB1 through MB29 after co-reaction with corresponding complementary or non-complementary oligonucleotides at room temperature (22 ℃).

FIG. 3b shows fluorescence emission readings of hybridization probes MB1 through MB29 after co-incubation with the corresponding complementary or non-complementary oligonucleotides at different temperatures.

Fig. 3c shows the change over time in the number of recorded events when imaging is performed with different sampling frame rates and laser powers.

Fig. 4a shows 1378 pieces of positional information obtained when the positive samples prepared in example 4 were subjected to the STORM imaging.

Fig. 4b shows the distribution of the position information in fig. 4a in the x, y, z direction.

FIG. 5 shows images of 3 exemplary target nucleic acids obtained after reconstruction using conventional optical microscopy and STORM super-resolution imaging for positive samples prepared in example 4.

Fig. 6 shows intensity normalized distributions of the regions where red lines are present in the three exemplary sight field images obtained by the STORM method in fig. 5.

FIG. 7 shows the results of PCR detection of the cell sample (II) prepared in example 7, showing that the positive sample contains the target nucleic acid fragment and the negative sample does not contain the target nucleic acid fragment.

FIG. 8 shows the sequence of the sense strand of the 2.5kb target nucleic acid fragment in example 7.

FIG. 9a shows fluorescence emission readings of hybridization probes MB30 through MB63 after co-reaction with corresponding complementary or non-complementary oligonucleotides at room temperature (22 ℃).

FIG. 9b shows fluorescence emission readings of hybridization probes MB30 through MB63 after co-incubation with the corresponding complementary or non-complementary oligonucleotides at different temperatures.

FIG. 10 shows images of 3 exemplary target nucleic acids obtained after reconstruction using conventional optical microscopy and STORM super-resolution imaging for positive samples prepared in example 10.

Fig. 11 shows intensity normalized distribution of the region where the red line is located in the three exemplary sight field images obtained by the STORM method in fig. 10.

Fig. 12 shows a schematic diagram of an exemplary embodiment according to the present disclosure.

Detailed Description

Various exemplary embodiments, features and aspects of the present disclosure will be described in detail below with reference to the accompanying drawings. The word "exemplary" is used exclusively herein to mean "serving as an example, embodiment, or illustration. Any embodiment described herein as "exemplary" is not necessarily to be construed as preferred or advantageous over other embodiments.

Furthermore, in the following detailed description, numerous specific details are set forth in order to provide a better understanding of the present disclosure. It will be understood by those skilled in the art that the present disclosure may be practiced without some of these specific details. In some instances, methods, means, reagents and devices well known to those skilled in the art have not been described in detail in order to avoid obscuring the subject matter of the present disclosure.

The present disclosure provides a method for identifying a nucleic acid, the method comprising the steps of:

a. the sample is brought into contact with the hybridization probe,

b. optionally, eluting the hybridization probes not bound to the target nucleic acid; and

c. identifying whether the hybridization probe binds to the target nucleic acid;

wherein the hybridization probe comprises a reporter group and the hybridization probe differs in state in two cases, thereby generating an identifiable signal by the reporter group: (i) the hybridization probe is in a free state, (ii) the hybridization probe is bound to the target nucleic acid.

Sample (I)

The sample to be identified by the identification method of the present disclosure may be a natural or synthetic nucleic acid, or may be a tissue or cell obtained from an organism, a cultured cell, or the like, and for example, the sample may be a tissue frozen section, a paraffin section, a cell slide, a tissue/cell lysate, or the like. The pretreatment of the sample can be carried out by a usual pretreatment method for in situ hybridization.

Hybridization probes

The hybridization probe comprises a reporter group, and the hybridization probe differs in state in the following two cases, thereby generating a recognizable signal by the reporter group: (i) the hybridization probe is in a free state, (ii) the hybridization probe is bound to the target nucleic acid. When the above conditions are satisfied, the specific structure of the hybridization probe is not particularly limited. For example, a hybridization probe may comprise a binding region and a dissociation region, the dissociation region self-hybridizes when the hybridization probe is in a free state, and the reporter group does not emit an identifiable signal; when the hybridization probe is specifically bound to the target sequence segment of the target nucleic acid through the binding region, the dissociation region dissociates and the reporter group emits an identifiable signal.

The association between the binding region and the dissociation region of the hybridization probe may be, for example, such that the dissociation region is contained in the binding region, or the dissociation region partially overlaps with the binding region, or the dissociation region is located outside the binding region.

Preferably, the reporter group comprises a fluorophore and a quencher, and the identifiable signal is fluorescence. That is, when the hybridization probe is in a free state, the dissociation region self-hybridizes, the fluorophore is quenched by the quencher, and thus the reporter does not emit fluorescence; when the hybridization probe is specifically bound to the target sequence fragment of the target nucleic acid through the binding region, the dissociation region dissociates, the quenching of the fluorophore by the quencher is released, and the reporter fluoresces.

Hybridization probes can be DNA probes, RNA probes, chemically modified oligonucleotide nucleic acid probes, nucleic acid analogs, and the like. Preferably, the hybridization probe is a single-stranded DNA probe or a single-stranded RNA probe.

In a preferred embodiment of the present disclosure, the hybridization probe has a stem-loop structure, such as a hairpin structure. For example, a hybridization probe comprises a loop structure serving as a binding region, and at least two arm structures serving as a dissociation region, wherein the at least two arm structures are located on both sides of the loop structure, respectively, and when the hybridization probe is not contacted with a target nucleic acid, the arm structure located on the 5 'side of the loop structure (hereinafter referred to as 5' arm structure) and the arm structure located on the 3 'side of the loop structure (hereinafter referred to as 3' arm structure) are complementarily combined to form a stable stem structure; the fluorophore and the quencher are located at the 5 'end of the 5' arm structure and the 3 'end of the 3' arm structure, respectively, and the positions of the fluorophore and the quencher can be interchanged, i.e., either the fluorophore is located at the 5 'end of the 5' arm structure and the quencher is located at the 3 'end of the 3' arm structure, or the fluorophore is located at the 3 'end of the 3' arm structure and the quencher is located at the 5 'end of the 5' arm structure.

The length of the loop structure is not particularly limited as long as the loop structure has sufficient binding strength with the target nucleic acid at the hybridization temperature. From various considerations such as the difficulty of design and the cost of probe preparation, the length of the loop structure is preferably 20 to 60 nucleotides (i.e., 20nt to 60nt), more preferably 25nt to 55nt, more preferably 30nt to 50nt, more preferably 35nt to 45nt, and still more preferably 40nt to 44 nt.

AboutSpecific nucleic acid sequences of the loop structure, one skilled in the art can consider the GC content, T, of the sequence based on the sequence of the target nucleic acid to be recognized_mValues, sequence specificity, and the like, as appropriate, for example, according to the method described in non-patent document 3, which is hereby incorporated by reference into the present disclosure. Preferably, the sequence identity between the loop structure and the reverse complement of the target nucleic acid is between 90% and 100%, preferably between 95% and 100%, preferably between 98% and 100%. Most preferably, the loop structure has 100% sequence identity to the reverse complement of the target nucleic acid, i.e., the loop structure is reverse complementary to the target nucleic acid.

The length and nucleic acid sequence of the arm structure are not particularly limited, and the design of the arm structure can be performed by referring to the method described in non-patent document 4, which is hereby incorporated by reference into the present disclosure. Preferably, the length of the arm structure is 4nt to 10nt, preferably 5nt to 9nt, preferably 6nt to 8nt, more preferably 7 nt.

In a preferred embodiment of the present disclosure, the hybridization probe may be a single-stranded nucleic acid represented by the following general formula (1):

5’-X₁X₂…X_mY₁Y₂…Y_nX’₁X’₂…X’_m-3’ (1)

wherein, X₁To X_m、Y₁To Y_n、X’₁To X'_mIt is meant any nucleotide, including, but not limited to,

from Y₁Y₂…Y_nThe indicated nucleotide strand segment is reverse complementary to the target nucleic acid,

from X₁X₂…X_mA nucleotide chain fragment represented by formula X'₁X’₂…X’_mThe nucleotide chain segments shown are complementary in reverse,

from Y₁Y₂…Y_nT wherein the nucleotide chain fragment shown is bonded to the target nucleic acid_mA value higher than that of X₁X₂…X_mA nucleotide chain fragment represented by formula X'₁X’₂…X’_mT in which the nucleotide chain fragment represented is bonded_mThe value of the one or more of,

nucleotide X₁Is conjugated with a fluorophore and nucleotide X'_mTo which a quencher group, or a nucleotide X, is conjugated₁Is conjugated with a quencher group and nucleotide X'_mOn which a fluorophore is conjugated,

m is an integer selected from 4 to 10, preferably 5 to 9, preferably 6 to 8, more preferably m is 7,

n is an integer selected from 20 to 60, preferably 25 to 55, preferably 30 to 50, preferably 35 to 45, more preferably 40 to 44.

Preferably, from Y₁Y₂…Y_nThe represented nucleotide chain fragment satisfies at least one of the following conditions: i) t is_mA value of not less than 70 ℃, ii) when the sample contains genomic nucleic acid, a value of Y₁Y₂…Y_nThe sequence of the represented nucleotide chain fragment is no more than 25nt in length from a similar sequence of a genomic non-target nucleic acid comprised in the sample, iii) does not comprise 6 or more consecutive repeats of nucleotides, iv) does not form secondary structures at a temperature equal to or higher than the hybridization temperature.

Preferably, from X₁X₂…X_mA nucleotide chain fragment represented by (A) and (B) X'₁X’₂…X’_mThe represented nucleotide chain fragment satisfies at least one of the following conditions: i) from X₁X₂…X_mA nucleotide chain fragment represented by formula X'₁X’₂…X’_mThe nucleotide chain fragment represented by (i) is a high GC content fragment (75% to 100%), ii) is represented by X₁X₂…X_mA nucleotide chain fragment represented by formula X'₁X’₂…X’_mT in which the nucleotide chain fragment represented is bonded_mA value in the range of 50 ℃ to 60 ℃, iii) from X₁X₂…X_mA nucleotide chain fragment represented by (A) and (B) X'₁X’₂…X’_mNone of the nucleotide chain fragments represented by Y₁Y₂…Y_nThe indicated nucleotide chain fragments form a secondary structure.

Preferably, the base in the fluorophore conjugated nucleotide is C (cytosine).

In a preferred embodiment of the present disclosure, the hybridization probe is a molecular beacon.

Fluorophores and quenchers

The reporter group of the hybridization probe is a recognizable label, preferably comprising a fluorophore and a quencher. Preferably, the emission maximum of the fluorophore is close to the absorption maximum of the quencher to obtain an optimal quenching effect.

Examples of fluorophores used in accordance with the present disclosure include, but are not limited to, fluorescein, texas red, nitrobenz-2-oxa-1, 3-oxadiazol-4-yl (NBD), coumarins, dansyl chloride, rhodamine, and the like. Preferred fluorophores are, for example, the Molecular sieves from Thermo Fisher

Alexa Fluor series of dyes sold under the trade name of (1), and the like.

Examples of quenchers for use according to the present disclosure include, but are not limited to, 4- (4' -dimethylaminophenylazo) benzoic acid (DABCYL), black hole quencher-1 (BHQ-1), black hole quencher-2 (BHQ-2), black hole quencher-3 (BHQ-3), and the like.

Target nucleic acid

A target nucleic acid is a nucleic acid or nucleic acid fragment to be recognized by the methods of the present disclosure. The target nucleic acid can be, for example, single-stranded or double-stranded DNA, including but not limited to genomic DNA, cDNA, and the like, or single-stranded or double-stranded RNA, including but not limited to messenger RNA, ribosomal RNA, microRNA, viral RNA, and the like.

Preferably the target nucleic acid is less than 4.9kb in length; more preferably, the target nucleic acid has a length of 3.3kb or less, still more preferably 2.5kb or less, and particularly preferably 2.0kb to 2.5 kb. Preferably, the target nucleic acid does not comprise a repetitive sequence. In the prior art, there is no precedent for successful labeling of repeat-free target nucleic acids less than 4.9kb in length. Furthermore, it is preferred that the target nucleic acid contains an enhancer sequence or a promoter sequence.

The methods of the present disclosure are generally applicable to labels that recognize multiple forms of a target nucleic acid, whether or not the target nucleic acid comprises a repetitive sequence. In the present disclosure, repetitive sequences are defined as identical units or symmetric fragments that occur multiple times in different locations or in the same broad range of locations in the genome, including but not limited to mild, moderate, highly repetitive sequences such as ALU families, centromere, telomeres, etc.; a non-repetitive sequence is defined as a sequence that is unique throughout the genome, without a repetitive sequence similar to it (in polyploids, such as diploids, non-repetitive sequences may occur in paired chromosomes, respectively). However, the advantages of the present disclosure become more apparent if used to identify target nucleic acids that do not comprise repetitive sequences. There are technical obstacles to labeling of short target nucleic acids without repetitive sequences in the prior art, and no report has been made on labeling of target nucleic acids without repetitive sequences having a length of less than 4.9 kb. When the length of the target nucleic acid not containing the repetitive sequence is short, for example, less than 4.9kb, the total number of hybridization probes bound to the target nucleic acid is small due to the length of the target nucleic acid, the sequence characteristics and the nucleic acid sequence length of each hybridization probe binding region, which results in a small number of reporter groups on the hybridization probes successfully bound to the target nucleic acid, and the total signal is weak, and thus it is difficult to distinguish from the background, which hinders further improvement of the recognition accuracy. The nucleic acid identification method of the present disclosure, in turn, successfully achieves high resolution imaging of non-repetitive target nucleic acid short fragments less than 4.9kb in length, as exemplified in examples 6, 11 below.

Contacting of self-quenching probes with samples

The method and conditions for contacting the sample with the hybridization probe are not particularly limited as long as the hybridization probe is allowed to bind to a target nucleic acid that may be present in the sample. For example, the sample and the self-quenching probe can be incubated at 70 ℃ to 80 ℃, preferably 73 ℃ to 77 ℃, more preferably 75 ℃, and then hybridized at 10 ℃ to 42 ℃, more preferably 12 ℃ to 38 ℃, more preferably 14 ℃ to 34 ℃, more preferably 16 ℃ to 34 ℃, more preferably 18 ℃ to 26 ℃, more preferably 22 ℃.

Means for identifying whether self-quenching probe binds to target nucleic acid

In the nucleic acid identification method of the present disclosure, the mode of identifying whether the self-quenching probe binds to the target nucleic acid can be appropriately selected depending on the characteristics of the sample, the self-quenching probe to be detected, and the target nucleic acid, and in accordance with the purpose of identification. For example, it can be read by a fluorescence spectrophotometer, or by fluorescence microscopy, such as ordinary fluorescence microscopy, confocal microscopy, or fluorescence super-resolution. When the reporter group includes a fluorophore and a quencher group, the wavelength used for excitation and identification of fluorescence may be appropriately selected depending on the nature of the fluorophore itself.

While a variety of means for identifying whether a self-quenching probe binds to a target nucleic acid are suitable for use in the nucleic acid identification methods of the present disclosure (e.g., the non-limiting examples set forth above), the advantages of the present disclosure are apparent if combined with fluorescence super-resolution imaging. When combined with a fluorescence super-resolution imaging method, the nucleic acid identification method of the present disclosure achieves an imaging resolution of 50nm or less, particularly 9nm, in the x-y direction and/or 40nm or less, particularly 22nm, in the z direction, if measured as standard deviation of single molecule repeat localization accuracy; or up to 50nm or less, in particular 22nm, in the x-y direction and/or up to 100nm or less, in particular 52nm, in the z direction, in full width at half maximum with a single molecule repeat positioning accuracy. This is a high resolution imaging that is difficult to achieve in the prior art, especially for non-repetitive target nucleic acid short fragments less than 4.9kb in length, and no high resolution imaging has been reported in the prior art.

The laser power used for excitation can be appropriately adjusted depending on the conditions such as the state of the sample and the type of fluorophore. Preferably, the laser power used during excitation is 0.80kW/cm²～2.20kW/cm². When the fluorophore is Alexa-647, the preferred laser power used for excitation is 0.80kW/cm²～2.00kW/cm²More preferably 0.90kW/cm²～1.45kW/cm²Further preferably 1.00kW/cm². With a preferred range of laser power at excitation, on the one hand the fluorophore can be excited so that a sufficiently strong fluorescence signal is obtained and on the other hand the progress of photobleaching of the fluorophore can be delayed.

The sampling frequency employed in identifying fluorescence may be appropriately adjusted according to the requirements for imaging quality, the performance of the imaging apparatus, and the like. The sampling frame frequency used for fluorescence discrimination is preferably 10Hz or more, more preferably 50Hz or more, still more preferably 60Hz or more, and still more preferably 85Hz or more. When fluorescence is identified, excitation light excitation power and sampling frame frequency in an optimal range are adopted, so that the imaging quality is improved, and high-resolution and low-noise images are obtained.

Embodiments of the present disclosure will be described in detail below with reference to examples, but those skilled in the art will appreciate that the following examples are only illustrative of the present disclosure and should not be construed as limiting the scope of the present disclosure. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.

EXAMPLE 1 preparation of cell sample (I)

Human SK-N-SH cells were infected with engineered pLL3.7-based lentiviral vectors. After infection, cells positive for EGFP expression were screened using flow cytometry (BD FACSAria SORP Cell Sorter). In MEM medium (Gibco) containing 10% fetal bovine serum, on coverslips (Fisherbrand)^TM Coverglass for Growth^TMCover Glasses, cat # 12-545-82) or human SK-N-SH cells not infected with lentivirus (negative control).

In positive cells, a viral RNA fragment of about 3.3kb in length of the lentiviral vector was reverse transcribed and integrated into the genome of SK-N-SH cells. The integrated 3.3kb fragment (SEQ ID NO:30) contains the EGFP-encoding sequence with expression promoted by the CMV promoter. One of the 3.3kb fragments, which is about 2.5kb in length, is identified as an exemplary target nucleic acid fragment in the identification method according to the present disclosure described in detail below (fig. 1 a). The genome of the cells as positive samples for recognition contained the above target nucleic acid fragments and had only 1 copy of the target nucleic acid fragment at each integration site. The cells as the negative samples for recognition do not contain the above-mentioned target nucleic acid fragments. The above properties of the positive and negative samples were confirmed by PCR (FIG. 1 b).

Example 2 design and Synthesis of hybridization probes MB1 to MB29

An exemplary hybridization probe provided in this example is a 56nt single-stranded oligonucleotide comprising a 42nt loop structure, and 7nt arm structures each flanking the loop structure. One end of the hybridization probe was conjugated to Alexa-647 as a fluorophore and the other end was conjugated to BHQ3 as a quencher.

29 hybridization probes were designed, designated MB1 through MB29, and synthesized by Life Technology. The sequences of the 29 hybridization probes are shown in the sequence Listing (SEQ ID NOS: 1 to 29). The sequence of the loop structure is reverse complementary to (i.e., identical to) a portion of the antisense strand of the target nucleic acid fragment in example 1, and the 29 hybridization probes can label the target nucleic acid fragment along the strand of the target nucleic acid through their respective loop structures. The sequences of the two arm structures in each probe are complementary to each other in reverse orientation. Thus, when the hybridization probe is not bound to the target nucleic acid fragment, the fluorescence of Alexa-647 is quenched by BHQ 3; when the hybridization probe binds to the target nucleic acid fragment, Alexa-647 of the probe may be excited to fluoresce and fluorescence is not affected by BHQ 3.

The sequence of the antisense strand of the target nucleic acid fragment of 3.3kb in example 1 is shown in FIG. 2, and the sequence fragments targeted by hybridization probes MB1 to MB29 in the target nucleic acid fragment are underlined. As can be seen, 29 hybridization probes can label the non-repetitive sequences of the target nucleic acid fragment. In addition, the sequence and loop structure T of 29 hybridization probes_mArm and arm structure T_mThe values are given in Table 1. In the probe sequence, the loop structures are indicated in uppercase letters and the arm structures are indicated in lowercase letters.

Table 1 sequences and parameters of hybridization probes for identifying target nucleic acid fragments in example 1

Example 3 specific binding of hybridization probes MB 1-MB 29 to target nucleic acids

The hybridization probe, the oligonucleotide (CS) complementary to the hybridization probe, and the oligonucleotide (NCS) not complementary to the sequence of the hybridization probe were dissolved in a buffer containing 50mM NaCl, 1mM EDTA, and 10mM Tris (pH 7.4).

80nM hybridization probes (MB 1-MB 29) were reacted with the corresponding CS (1600nM) or NCS (1600nM) in 2 XSSC, 50% formamide hybridization solution for 30min at room temperature. Thereafter, fluorescence emission readings were taken at 665nm using a spectrofluorometer with laser excitation at 647 nm. The reading for each hybridization probe is shown in FIG. 3 a. For each hybridization probe, the fluorescence emission reading after co-reaction with CS was significantly higher than the fluorescence emission reading after co-reaction with NCS, indicating specific binding of each hybridization probe to the target sequence.

80nM hybridization probe was co-reacted with the corresponding CS (1600nM) or NCS (1600nM) for 30min at different temperatures (42 ℃, 38 ℃, 34 ℃,30 ℃, 26 ℃, 22 ℃, 18 ℃, 14 ℃). Thereafter, fluorescence emission readings were taken at 665nm with laser excitation at 647 nm. The results are shown in FIG. 3 b.

In the prior art in situ hybridization based on non-self-quenched linear oligonucleotides, it is well known to those skilled in the art that as the hybridization temperature is lowered, the specific binding of the oligonucleotide to the target sequence and the non-specific binding to non-target sequences are simultaneously enhanced. In order to eliminate the adverse effect of non-specific binding, it is necessary to increase the hybridization temperature, for example, to select a temperature of 37 ℃ to 47 ℃ for hybridization. However, higher hybridization temperatures also negatively impact the strength of specific binding required. This is a dilemma faced in selecting hybridization temperatures.

In the present disclosure, however, as shown in fig. 3b, specific binding is enhanced as the hybridization temperature is decreased from 42 ℃ to 14 ℃; it was also found that non-specific binding unexpectedly decreased as the hybridization temperature was decreased. This indicates that in the present disclosure using hybridization probes, the dilemma of selecting hybridization temperatures is eliminated. Without being bound by any theory, it is believed that this phenomenon may be associated with the structure of the hybridization probe that protects the probe from interacting with NCS.

EXAMPLE 4 contacting of cell sample (I) with hybridization probes

When the cells on the coverslip in example 1 were grown to 80% confluence, they were fixed with 4% paraformaldehyde-PBS for 10min, soaked in PBS for 2min, treated with 1mg/mL sodium borohydride for 7min, and soaked in ddH₂And O2 min. And then, soaking the cells in 25% glycerol-PBS for 40-50 min, freezing with liquid nitrogen, unfreezing, and repeating the freeze-thaw cycle for 3 times. The cells were then treated with RNase A (100. mu.g/ml) at 37 ℃ for 1 hr. After washing with PBS, cells were infiltrated with PBS for 5 minutes. Cells were pre-warmed in 2 XSSC buffer at 75 ℃ for 5min, and then the 2 XSSC buffer was changed to 2 XSSC buffer, 80% deionized formamide and treatment continued at 75 ℃ for 3 min. Cells were then treated with cold ethanol infiltration (75% concentration, 90%, 100%) for 2min each. Cells were blocked overnight at room temperature (22 ℃) with 1.5% FISH blocking buffer (Roche), 50% formamide and 2 XSSC buffer. Cells were further blocked with 1.5% FISH blocking buffer (Roche), 50% formamide and 2 XSSC buffer in the new configuration at 42 ℃ until hybridization 3-6 hours before reaction with the hybridization probe. A mixture of the hybridization probes synthesized in example 2 (including all probes), 1.5% FISH blocking buffer, 50% formamide and 2 XSSC buffer was prepared at a total concentration of 714nM and the cells on each slide were hybridized with 14. mu.L of this mixture for 16-20 hours at 22 ℃. Finally, the cells were washed with a buffer containing 50% formamide and 2 XSSC for 40-50 min at room temperature and fixed with 4% paraformaldehyde-PBS for 5-10 min. The positive and negative test samples can be stored by soaking in 0.25 XSSC buffer at 4 ℃.

Example 5 optimization of the conditions for identification of cell sample (I)

The sample obtained in example 4 was identified by the following method. Imaging was performed by random optical reconstruction microscopy (STORM) using an inverted optical microscope (IX-71, Olympus) equipped with a 100 x oil immersion objective (UPlanSApo, n.a.1.40, Olympus). For Alexa-647 labeled on the hybridization probe, a 641nm laser is used to excite the Alexa647 state to radiate fluorescence, and the Alexa-647 is converted into a dark state and reactivated with a 405nm laser. Each transition from a light state to a dark state is registered as an on/off event.

Since the hybridization probe is labeled with the non-repetitive sequence of the target nucleic acid, the labeling density is relatively sparse, and the recognition of fluorescence may be interfered by cellular autofluorescence. In order to optimize the imaging conditions, the laser power and the sampling frame rate were adjusted, and multiple sets of experiments were performed, with the specific conditions of each set as shown in table 2. The false positive rate (FDR) is reported as the ratio of the number of on/off events recorded from negative samples to the number of on/off events recorded from positive samples under the same conditions. The average FDR under each condition is also shown in Table 2.

TABLE 2 sample STORM imaging conditions and false positive rates

As can be seen from the above table, the average false positive rates for conditions IX, X, XI, XII are relatively low and are the preferred conditions for STORM imaging with the hybridization probes of the present disclosure.

Further, for the experiments performed under conditions IX, X, XI, XII, the recorded change in the number of switching events over time was further observed, as shown in fig. 3 c. The x-axis in FIG. 3c represents the passage of time in units of 5 seconds; the y-axis represents the ratio of the number of bright molecules, and the number of bright molecules recorded in the first 5 seconds from the start of recording is scaled according to the number of bright molecules recorded in each 5 seconds during recording. As can be seen in FIG. 3c, the bright state molecular ratio decreases rapidly in the first 90 seconds and slowly in the subsequent 90 seconds. The proportion of the number of bright state molecules recorded throughout the imaging remained high when using condition XII compared to conditions IX, X, XI, indicating that condition XII contributes to a reduction of the permanent photobleaching effect and to a high quality image.

Example 6 identification of cell sample (I)

The positive samples prepared in example 4 were subjected to STORM imaging under condition XII of example 5. And (3) obtaining cluster distribution formed by repeated positioning of a plurality of fluorescent single molecules by performing light-dark conversion on Alexa-647. The 1378 localization profiles accumulated from 53 clusters are shown in fig. 4 a. The repeated positioning satisfies a Gaussian distribution with a full width at half maximum of 22nm in the transverse direction and 52nm in the axial direction, indicating that a resolution of about 20-30 nm is obtained in the x-y direction and a resolution of about 50-60 nm is obtained in the z direction (FIG. 4 b).

The STORM image reconstruction is performed according to the method described in non-patent document 5. Fig. 5 shows an image of an exemplary field of view of 3 nuclei. In FIG. 5, from top to bottom, the columns one, two, and three are images obtained by conventional optical microscopy, and it is seen that the fluorescence spots labeling the target nucleic acid cannot be distinguished. The fourth column is a super-resolution color image obtained by an STORM imaging method in the first, second and third middle green square frame areas, the fifth column is the enlargement of the white square frame area in the fourth column image, the morphology of the target nucleic acid in the super-resolution image is more clearly shown, and the number of normal molecules collected in the imaging process of the target nucleic acid is marked at the lower right corner. The color of the super-resolution color image represents the single molecule orientation in the z-direction (-350 to 350 nm). FIG. 6 shows intensity normalized distributions of the regions (i, ii, iii) where the red lines are located in the three target nucleic acid super-resolution structures obtained by the STORM imaging method in FIG. 5, showing that the imaging accuracy can resolve microstructures (i) at 44nm and individual structures (ii, iii) at 33-36nm in the structure. It can be seen that the method of the present disclosure is particularly advantageous for super-resolution imaging applications.

EXAMPLE 7 preparation of cell sample (II)

A fragment of about 3kb (mm9 mouse whole genome coordinate site, Chr6: 122612566-122615608, SEQ ID NO:66) is knocked out from a mouse CJ9 stem cell by using a CRISPR/Cas9 gene editing technology, and a homozygote knocked-out cell is obtained as a negative control after screening. In mES complete medium, on a coverslip (Fisherbrand)^TM Coverglass for Growth^TMCover Glasses, cat # 12-545-82) wild-type mouse CJ9 stem cells (positive cells) or homozygous knockout cells obtained by CRISPR/Cas9 technology as described above (negative control).

In positive cells, a fragment of about 2.5kb in length (mm9 mouse genome-wide coordinate site, Chr6:122612623-122615179) from chromosome 6 in the genome was identified as an exemplary target nucleic acid fragment (SEQ ID NO:65) in the following detailed identification method according to the present disclosure. The genome of the cell as a positive sample for recognition contains the above target nucleic acid fragment, and only 1 copy of the target nucleic acid fragment is present on chromosome 6 in the entire genome. Cells that were identified as negative samples did not contain the above-described target nucleic acid fragment, since a 3.3kb nucleic acid fragment containing a 2.5kb target nucleic acid had been knocked out by the CRISPR/Cas9 gene editing technique. The above properties of the positive and negative samples were confirmed by PCR (fig. 7).

Example 8 design and Synthesis of hybridization probes MB30 to MB63

An exemplary hybridization probe provided in this example is a 56nt single-stranded oligonucleotide comprising a 42nt loop structure, and 7nt arm structures each flanking the loop structure. One end of the hybridization probe was conjugated to Alexa-647 as a fluorophore and the other end was conjugated to BHQ3 as a quencher.

34 hybridization probes were designed, designated MB30 through MB63, and synthesized by Life Technology. The sequences of these 34 hybridization probes are shown in the sequence Listing (SEQ ID NOS: 31 to 64). Of these, the sequence of the loop structures of 24 hybridization probes (MB30, 31, 33, 35, 36, 37, 38, 39, 41, 42, 44, 45, 47, 49, 51, 52, 53, 55, 56, 58, 59, 61, 62, 63) was reverse-complementary to (i.e., identical to) a portion of the antisense strand of the target nucleic acid fragment in example 7, and the sequence of the loop structures of the remaining 10 hybridization probes (MB32, 34, 40, 43, 46, 48, 50, 54, 57, 60) was reverse-complementary to (i.e., identical to) a portion of the sense strand of the target nucleic acid fragment in example 7. The 34 hybridization probes can label the target nucleic acid fragments along the strands of the target nucleic acid by their respective loop structures. The sequences of the two arm structures in each probe are complementary to each other in reverse orientation. Thus, when the hybridization probe is not bound to the target nucleic acid fragment, the fluorescence of Alexa-647 is quenched by BHQ 3; when the hybridization probe binds to the target nucleic acid fragment, Alexa-647 of the probe may be excited to fluoresce and fluorescence is not affected by BHQ 3.

The sequence of the sense strand of the 2.5kb target nucleic acid fragment in example 7 is shown in FIG. 8, and the sequence fragments targeted by hybridization probes MB30 to MB63 in the target nucleic acid fragment are underlined or bolded. In the middle, underlining indicates that the hybridization probe targets the antisense strand of the target nucleic acid, plusBold indicates that the hybridization probe targets the sense strand of the target nucleic acid. As can be seen, 34 hybridization probes can label the non-repetitive sequences of the target nucleic acid fragments. The sequence and loop structure T of the 34 hybridization probes_mArm and arm structure T_mThe values are given in Table 3. In the probe sequence, the loop structures are indicated in uppercase letters and the arm structures are indicated in lowercase letters.

Table 3 sequences and parameters of hybridization probes for identifying target nucleic acid fragments in example 7

Example 9 specific binding of hybridization probes MB 30-MB 63 to target nucleic acids

The hybridization probe, the oligonucleotide (CS) complementary to the hybridization probe, and the oligonucleotide (NCS) not complementary to the sequence of the hybridization probe were dissolved in a buffer containing 50mM NaCl, 1mM EDTA, and 10mM Tris (pH 7.4).

80nM hybridization probes (MB 30-MB 63) were reacted with the corresponding CS (1600nM) or NCS (1600nM) in 2 XSSC, 50% formamide hybridization solution for 30min at room temperature. Thereafter, fluorescence emission readings were taken at 665nm using a spectrofluorometer with laser excitation at 647 nm. The reading for each hybridization probe is shown in FIG. 9 a. For each hybridization probe, the fluorescence emission reading after co-reaction with CS was significantly higher than the fluorescence emission reading after co-reaction with NCS, indicating specific binding of each hybridization probe to the target sequence.

80nM hybridization probe was co-reacted with the corresponding CS (1600nM) or NCS (1600nM) for 30min at different temperatures (46 ℃, 42 ℃, 38 ℃, 34 ℃,30 ℃, 26 ℃, 22 ℃, 18 ℃, 14 ℃). Thereafter, fluorescence emission readings were taken at 665nm with laser excitation at 647 nm. The results are shown in FIG. 9 b.

In the prior art in situ hybridization based on non-self-quenched linear oligonucleotides, it is well known to those skilled in the art that as the hybridization temperature is lowered, the specific binding of the oligonucleotide to the target sequence and the non-specific binding to non-target sequences are simultaneously enhanced. In order to eliminate the adverse effect of non-specific binding, it is necessary to increase the hybridization temperature, for example, to select a temperature of 37 ℃ to 47 ℃ for hybridization. However, higher hybridization temperatures also negatively impact the strength of specific binding required. This is a dilemma faced in selecting hybridization temperatures.

In the present disclosure, however, as shown in fig. 9b, specific binding is enhanced as the hybridization temperature is decreased from 46 ℃ to 14 ℃; it was also found that non-specific binding unexpectedly decreased as the hybridization temperature was decreased. This indicates that in the present disclosure using hybridization probes, the dilemma of selecting hybridization temperatures is eliminated. Without being bound by any theory, it is believed that this phenomenon may be associated with the structure of the hybridization probe that protects the probe from interacting with NCS.

EXAMPLE 10 contacting of cell sample (II) with hybridization probes

When the cells on the coverslip in example 7 grew to 80% confluence, they were fixed with 4% paraformaldehyde-PBS for 10min, soaked in PBS for 2min, treated with 1mg/mL sodium borohydride for 7min, soaked in ddH₂And O2 min. And then, soaking the cells in 25% glycerol-PBS for 40-50 min, freezing with liquid nitrogen, unfreezing, and repeating the freeze-thaw cycle for 3 times. The cells were then treated with RNase A (100. mu.g/ml) at 37 ℃ for 1 hr. After washing with PBS, cells were infiltrated with PBS for 5 minutes. Cells were pre-warmed in 2 XSSC buffer at 75 ℃ for 5min, and then the 2 XSSC buffer was changed to 2 XSSC buffer, 80% deionized formamide and treatment continued at 75 ℃ for 3 min. Cells were then treated with cold ethanol infiltration (75% concentration, 90%, 100%) for 2min each. Cells were blocked overnight at room temperature (22 ℃) with 1.5% FISH blocking buffer (Roche), 50% formamide and 2 XSSC buffer. Cells were further blocked with 1.5% FISH blocking buffer (Roche), 50% formamide and 2 XSSC buffer in the new configuration at 42 ℃ until hybridization 3-6 hours before reaction with the hybridization probe. A mixture of the hybridization probes synthesized in example 8 (including all probes), 1.5% FISH blocking buffer, 50% formamide and 2 XSSC buffer was prepared at a total concentration of 714nM and 14. mu.L of this mixture was usedThe material was hybridized with the cells on each slide for 16-20 hours at 22 ℃. Finally, the cells were washed with a buffer containing 50% formamide and 2 XSSC for 40-50 min at room temperature and fixed with 4% paraformaldehyde-PBS for 5-10 min. The positive and negative test samples can be stored by soaking in 0.25 XSSC buffer at 4 ℃.

Example 11 identification of cell sample (II)

The positive sample prepared in example 10 was subjected to STORM imaging under condition XII of example 5. And (3) obtaining cluster distribution formed by repeated positioning of a plurality of fluorescent single molecules by performing light-dark conversion on Alexa-647.

The STORM image reconstruction is performed according to the method described in non-patent document 5. Fig. 10 shows an image of an exemplary field of view of 3 nuclei. In FIG. 10, from top to bottom, columns one, two, and three are images obtained by conventional optical microscopy, and it is seen that the fluorescence spots labeling the target nucleic acid are not distinguishable. The fourth column is a super-resolution color image obtained by an STORM imaging method in the first, second and third middle green square frame areas, the fifth column is the enlargement of the white square frame area in the fourth column image, the morphology of the target nucleic acid in the super-resolution image is more clearly shown, and the number of normal molecules collected in the imaging process of the target nucleic acid is marked at the lower right corner. The color of the super-resolution color image represents the single molecule orientation in the z-direction (-350 to 350 nm). FIG. 11 shows intensity normalized distributions of the regions (i, ii, iii) where the red lines are located in the three target nucleic acid super-resolution structures obtained by the STORM imaging method in FIG. 10, exhibiting microstructures at 58nm (i) and 37nm (ii) apart from the individual structures at 25-34nm (iii) in the imaging-accurate energy-resolution structure. It can be seen that the method of the present disclosure is particularly advantageous for super-resolution imaging applications.

Having described embodiments of the present disclosure, the foregoing description is intended to be exemplary, not exhaustive, and not limited to the disclosed embodiments. Many modifications and variations will be apparent to those of ordinary skill in the art without departing from the scope and spirit of the described embodiments. The terminology used herein is chosen in order to best explain the principles of the embodiments, the practical application, or improvements made to the technology in the marketplace, or to enable others of ordinary skill in the art to understand the embodiments disclosed herein.

Sequence listing

<110> Ni Yanxiang

<120> method for accurately identifying nucleic acid

<130> 6519-170019I

<160> 66

<170> PatentIn version 3.5

<210> 1

<211> 56

<212> DNA

<213> Artificial sequence

<220>

<223> sequence of hybridization Probe MB1

<400> 1

cggcctggcc tccccgcatc gataccgtcg acctcgatcg agacctagac aggccg 56

<210> 2

<211> 56

<212> DNA

<213> Artificial sequence

<220>

<223> sequence of hybridization Probe MB2

<400> 2

cgtgcgctac gtcccttcgg ccctcaatcc agcggacctt ccttcccgcg cgcacg 56

<210> 3

<211> 56

<212> DNA

<213> Artificial sequence

<220>

<223> sequence of hybridization Probe MB3

<400> 3

cggcacgtgg gcactgacaa ttccgtggtg ttgtcgggga aatcatcgtc gtgccg 56

<210> 4

<211> 56

<212> DNA

<213> Artificial sequence

<220>

<223> sequence of hybridization Probe MB4

<400> 4

cggccgattg ccaccacctg tcagctcctt tccgggactt tcgctttcct cggccg 56

<210> 5

<211> 56

<212> DNA

<213> Artificial sequence

<220>

<223> sequence of hybridization Probe MB5

<400> 5

cgggcagggc cgcgactcta gatcataatc agccatacaa ttcgtcgagc tgcccg 56

<210> 6

<211> 56

<212> DNA

<213> Artificial sequence

<220>

<223> sequence of hybridization Probe MB6

<400> 6

cggcgcacgt gctgctgccc gacaaccact acctgagcac ccagtccgct gcgccg 56

<210> 7

<211> 56

<212> DNA

<213> Artificial sequence

<220>

<223> sequence of hybridization Probe MB7

<400> 7

cgtcgggtgc agctcgccga ccactaccag cagaacaccc ccatcggcgc ccgacg 56

<210> 8

<211> 56

<212> DNA

<213> Artificial sequence

<220>

<223> sequence of hybridization Probe MB8

<400> 8

cgcgggtaag gtgaacttca agatccgcca caacatcgag gacggcagca cccgcg 56

<210> 9

<211> 56

<212> DNA

<213> Artificial sequence

<220>

<223> sequence of hybridization Probe MB9

<400> 9

cggtgcgcca caacgtctat atcatggccg acaagcagaa gaacggcatc gcaccg 56

<210> 10

<211> 56

<212> DNA

<213> Artificial sequence

<220>

<223> sequence of hybridization Probe MB10

<400> 10

cgaccggcac catcttcttc aaggacgacg gcaactacaa gacccgcgcc cggtcg 56

<210> 11

<211> 56

<212> DNA

<213> Artificial sequence

<220>

<223> sequence of hybridization Probe MB11

<400> 11

cgcggctcac atgaagcagc acgacttctt caagtccgcc atgcccgaaa gccgcg 56

<210> 12

<211> 56

<212> DNA

<213> Artificial sequence

<220>

<223> sequence of hybridization Probe MB12

<400> 12

cgctcggacc ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc cgagcg 56

<210> 13

<211> 56

<212> DNA

<213> Artificial sequence

<220>

<223> sequence of hybridization Probe MB13

<400> 13

cgctggctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcg ccagcg 56

<210> 14

<211> 56

<212> DNA

<213> Artificial sequence

<220>

<223> sequence of hybridization Probe MB14

<400> 14

cggacgcgcc catcctggtc gagctggacg gcgacgtaaa cggccacaag cgtccg 56

<210> 15

<211> 56

<212> DNA

<213> Artificial sequence

<220>

<223> sequence of hybridization Probe MB15

<400> 15

cgcgcgtacc ggtcgccacc atggtgagca agggcgagga gctgttcaca cgcgcg 56

<210> 16

<211> 56

<212> DNA

<213> Artificial sequence

<220>

<223> sequence of hybridization Probe MB16

<400> 16

cgccgtgagc ttcgaattct gcagtcgacg gtaccgcggg cccgggatcc acggcg 56

<210> 17

<211> 56

<212> DNA

<213> Artificial sequence

<220>

<223> sequence of hybridization Probe MB17

<400> 17

cgcgtcggtg aaccgtcaga tccgctagcg ctaccggact cagatctcgc gacgcg 56

<210> 18

<211> 56

<212> DNA

<213> Artificial sequence

<220>

<223> sequence of hybridization Probe MB18

<400> 18

cgcggtcgca aatgggcggt aggcgtgtac ggtgggaggt ctatataagg accgcg 56

<210> 19

<211> 56

<212> DNA

<213> Artificial sequence

<220>

<223> sequence of hybridization Probe MB19

<400> 19

cgagggctca acgggacttt ccaaaatgtc gtaacaactc cgccccattg ccctcg 56

<210> 20

<211> 56

<212> DNA

<213> Artificial sequence

<220>

<223> sequence of hybridization Probe MB20

<400> 20

cgggcgtcaa gtctccaccc cattgacgtc aatgggagtt tgttttggca cgcccg 56

<210> 21

<211> 56

<212> DNA

<213> Artificial sequence

<220>

<223> sequence of hybridization Probe MB21

<400> 21

cgtcggcggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg ccgacg 56

<210> 22

<211> 56

<212> DNA

<213> Artificial sequence

<220>

<223> sequence of hybridization Probe MB22

<400> 22

cgggtccggg actttcctac ttggcagtac atctacgtat tagtcatcgg gacccg 56

<210> 23

<211> 56

<212> DNA

<213> Artificial sequence

<220>

<223> sequence of hybridization Probe MB23

<400> 23

cggcagccca cttggcagta catcaagtgt atcatatgcc aagtacgccg ctgccg 56

<210> 24

<211> 56

<212> DNA

<213> Artificial sequence

<220>

<223> sequence of hybridization Probe MB24

<400> 24

cgctggggac gtatgttccc atagtaacgc caatagggac tttccattgc ccagcg 56

<210> 25

<211> 56

<212> DNA

<213> Artificial sequence

<220>

<223> sequence of hybridization Probe MB25

<400> 25

cggtgcctaa cttacggtaa atggcccgcc tggctgaccg cccaacgacg gcaccg 56

<210> 26

<211> 56

<212> DNA

<213> Artificial sequence

<220>

<223> sequence of hybridization Probe MB26

<400> 26

cgtggcgtta cggggtcatt agttcatagc ccatatatgg agttccgcgc gccacg 56

<210> 27

<211> 56

<212> DNA

<213> Artificial sequence

<220>

<223> sequence of hybridization Probe MB27

<400> 27

cgagccgatt acagggacag cagagatcca gtttggttag taccgggccc ggctcg 56

<210> 28

<211> 56

<212> DNA

<213> Artificial sequence

<220>

<223> sequence of hybridization Probe MB28

<400> 28

cgtcgcgatt agtgaacgga tcggcactgc gtgcgccaat tctgcagacc gcgacg 56

<210> 29

<211> 56

<212> DNA

<213> Artificial sequence

<220>

<223> sequence of hybridization Probe MB29

<400> 29

cgcgcagcag ggatattcac cattatcgtt tcagacccac ctcccaaccc tgcgcg 56

<210> 30

<211> 3243

<212> DNA

<213> Artificial sequence

<220>

<223> target nucleic acid fragment inserted in cell sample (I)

<400> 30

tctaggtctc gatcgaggtc gacggtatcg atgcggggag gcggcccaaa gggagatccg 60

actcgtctga gggcgaaggc gaagacgcgg aagaggccgc agagccggca gcaggccgcg 120

ggaaggaagg tccgctggat tgagggccga agggacgtag cagaaggacg tcccgcgcag 180

aatccaggtg gcaacacagg cgagcagcca aggaaaggac gatgatttcc ccgacaacac 240

cacggaattg tcagtgccca acagccgagc ccctgtccag cagcgggcaa ggcaggcggc 300

gatgagttcc gccgtggcaa tagggagggg gaaagcgaaa gtcccggaaa ggagctgaca 360

ggtggtggca atgccccaac cagtgggggt tgcgtcagca aacacagtgc acaccacgcc 420

acgttgcctg acaacgggcc acaactcctc ataaagagac agcaaccagg atttatacaa 480

ggaggagaaa atgaaagcca tacgggaagc aatagcatga tacaaaggca ttaaagcagc 540

gtatccacat agcgtaaaag gagcaacata gttaagaata ccagtcaatc tttcacaaat 600

tttgtaatcc agaggttgat tatcgataag cttgatatcg aattgtacct agtggaaccg 660

gaacccttaa acatgtataa cttcgtataa tgtatgctat acgaagttat taggtccctc 720

gacgaattgt atggctgatt atgatctaga gtcgcggccg ctttacttgt acagctcgtc 780

catgccgaga gtgatcccgg cggcggtcac gaactccagc aggaccatgt gatcgcgctt 840

ctcgttgggg tctttgctca gggcggactg ggtgctcagg tagtggttgt cgggcagcag 900

cacggggccg tcgccgatgg gggtgttctg ctggtagtgg tcggcgagct gcacgctgcc 960

gtcctcgatg ttgtggcgga tcttgaagtt caccttgatg ccgttcttct gcttgtcggc 1020

catgatatag acgttgtggc tgttgtagtt gtactccagc ttgtgcccca ggatgttgcc 1080

gtcctccttg aagtcgatgc ccttcagctc gatgcggttc accagggtgt cgccctcgaa 1140

cttcacctcg gcgcgggtct tgtagttgcc gtcgtccttg aagaagatgg tgcgctcctg 1200

gacgtagcct tcgggcatgg cggacttgaa gaagtcgtgc tgcttcatgt ggtcggggta 1260

gcggctgaag cactgcacgc cgtaggtcag ggtggtcacg agggtgggcc agggcacggg 1320

cagcttgccg gtggtgcaga tgaacttcag ggtcagcttg ccgtaggtgg catcgccctc 1380

gccctcgccg gacacgctga acttgtggcc gtttacgtcg ccgtccagct cgaccaggat 1440

gggcaccacc ccggtgaaca gctcctcgcc cttgctcacc atggtggcga ccggtggatc 1500

ccgggcccgc ggtaccgtcg actgcagaat tcgaagcttg agctcgagat ctgagtccgg 1560

tagcgctagc ggatctgacg gttcactaaa ccagctctgc ttatatagac ctcccaccgt 1620

acacgcctac cgcccatttg cgtcaatggg gcggagttgt tacgacattt tggaaagtcc 1680

cgttgatttt ggtgccaaaa caaactccca ttgacgtcaa tggggtggag acttggaaat 1740

ccccgtgagt caaaccgcta tccacgccca ttgatgtact gccaaaaccg catcaccatg 1800

gtaatagcga tgactaatac gtagatgtac tgccaagtag gaaagtccca taaggtcatg 1860

tactgggcat aatgccaggc gggccattta ccgtcattga cgtcaatagg gggcgtactt 1920

ggcatatgat acacttgatg tactgccaag tgggcagttt accgtaaata ctccacccat 1980

tgacgtcaat ggaaagtccc tattggcgtt actatgggaa catacgtcat tattgacgtc 2040

aatgggcggg ggtcgttggg cggtcagcca ggcgggccat ttaccgtaag ttatgtaacg 2100

cggaactcca tatatgggct atgaactaat gaccccgtaa ttgattacta ttaataacta 2160

actagagcgg gcccggtact aaccaaactg gatctctgct gtccctgtaa taaacccgaa 2220

aattttgaat ttttgtaatt tgtttttgta attctttagt ttgtatgtct gttgctatta 2280

tgtctactat tctttcccct gcactgtacc ccccaatccc cccttttctt ttaaaattgt 2340

ggatgaatac tgccatttgt ctgcagaatt ggcgcacgca gtgccgatcc gttcactaat 2400

cgaatggatc tgtctctgtc tctctctcca ccttcttctt ctattccttc gggcctgtcg 2460

ggtcccctcg gggttgggag gtgggtctga aacgataatg gtgaatatcc ctgcctaact 2520

ctattcacta tagaaagtac agcaaaaact attcttaaac ctaccaagcc tcctactatc 2580

attatgaata attttatata ccacagccaa tttgttatgt taaaccaatt ccacaaactt 2640

gcccatttat ctaattccaa taattcttgt tcattctttt cttgctggtt ttgcgattct 2700

tcaattaagg agtgtattaa gcttgtgtaa ttgttaattt ctctgtccca ctccatccag 2760

gtcgtgtgat tccaaatctg ttccagagat ttattactcc aactagcatt ccaaggcaca 2820

gcagtggtgc aaatgagttt tccagagcaa ccccaaatcc ccaggagctg ttgatccttt 2880

aggtatcttt ccacagccag gattcttgcc tggagctgct tgatgcccca gactgtgagt 2940

tgcaacagat gctgttgcgc ctcaatagcc ctcagcaaat tgttctgctg ctgcactata 3000

ccagacaata attgtctggc ctgtaccgtc agcgtcattg acgctgcgcc catagtgctt 3060

cctgctgctc ccaagaaccc aaggaacaaa gctcctattc ccactgctct tttttctctc 3120

tgcaccactc ttctctttgc cttggtgggt gctactccta atggttcaat ttttactact 3180

ttatatttat ataattcact tctccaattg tccctcatat ctcctcctcc aggtctgaag 3240

atc 3243

<210> 31

<211> 56

<212> DNA

<213> Artificial sequence

<220>

<223> sequence of hybridization Probe MB30

<400> 31

cgcctgggtt taaacttgta aaactccagt cgtgggctaa actgtctggc caggcg 56

<210> 32

<211> 56

<212> DNA

<213> Artificial sequence

<220>

<223> sequence of hybridization Probe MB31

<400> 32

cgctcggcta ctgtgttcac ggacaggtgc cagcccagtg acccagagcc cgagcg 56

<210> 33

<211> 56

<212> DNA

<213> Artificial sequence

<220>

<223> sequence of hybridization Probe MB32

<400> 33

cgccggtccc agggcagtgc taggtttcgg gagacaactg aaagggaata ccggcg 56

<210> 34

<211> 56

<212> DNA

<213> Artificial sequence

<220>

<223> sequence of hybridization Probe MB33

<400> 34

cggcgtgcct ggagcccatc tcagtctgcc agcgattctt ttcaaacaac acgccg 56

<210> 35

<211> 56

<212> DNA

<213> Artificial sequence

<220>

<223> sequence of hybridization Probe MB34

<400> 35

cgggctgtct tgtagcaact catagatctg caaatacatg aacctgtggc agcccg 56

<210> 36

<211> 56

<212> DNA

<213> Artificial sequence

<220>

<223> sequence of hybridization Probe MB35

<400> 36

cgggtcggcc gctccctgga tagcgatgaa tccttcagcc tactgtccac gacccg 56

<210> 37

<211> 56

<212> DNA

<213> Artificial sequence

<220>

<223> sequence of hybridization Probe MB36

<400> 37

cggctgcagg aggccaagcg ccttgtccgc tccgcaccac ccacccatcg cagccg 56

<210> 38

<211> 56

<212> DNA

<213> Artificial sequence

<220>

<223> sequence of hybridization Probe MB37

<400> 38

cgtccgggcg ggtggtgata aaccggtgca cctcgcggcc tcccgaattc cggacg 56

<210> 39

<211> 56

<212> DNA

<213> Artificial sequence

<220>

<223> sequence of hybridization Probe MB38

<400> 39

cggctcgcag gaggtcccaa ctgtccttac tcacagttca ttccaagccc gagccg 56

<210> 40

<211> 56

<212> DNA

<213> Artificial sequence

<220>

<223> sequence of hybridization Probe MB39

<400> 40

cggtgcggaa aaggccttcc tagcccaagg gtgggaatgt taaggctggc gcaccg 56

<210> 41

<211> 56

<212> DNA

<213> Artificial sequence

<220>

<223> sequence of hybridization Probe MB40

<400> 41

cggtggcgac ctaggagagc ccagtcaccc atctgtgtga ggtttcccag ccaccg 56

<210> 42

<211> 56

<212> DNA

<213> Artificial sequence

<220>

<223> sequence of hybridization Probe MB41

<400> 42

cgtgcgcctc tgacacctct gggtcttgca aaccgggtgc tccacatatg cgcacg 56

<210> 43

<211> 56

<212> DNA

<213> Artificial sequence

<220>

<223> sequence of hybridization Probe MB42

<400> 43

cgtcgggata gatttggaat taaccgtaca gctggcatgg cagtgttggc ccgacg 56

<210> 44

<211> 56

<212> DNA

<213> Artificial sequence

<220>

<223> sequence of hybridization Probe MB43

<400> 44

cgtgccgtct gaccttcctc ttgctaatcc accttgaaga tctcacatcc ggcacg 56

<210> 45

<211> 56

<212> DNA

<213> Artificial sequence

<220>

<223> sequence of hybridization Probe MB44

<400> 45

cgtcgcggac ctttcagcaa gcaacccttc aactgtgagt tgctagttac gcgacg 56

<210> 46

<211> 56

<212> DNA

<213> Artificial sequence

<220>

<223> sequence of hybridization Probe MB45

<400> 46

cgcgcgtaag ggcaaactgg cgacagtaaa cataattagc tgacccttca cgcgcg 56

<210> 47

<211> 56

<212> DNA

<213> Artificial sequence

<220>

<223> sequence of hybridization Probe MB46

<400> 47

cgtggcgggc ctttgagata gcagattgcc gacggacttt atttcctttc gccacg 56

<210> 48

<211> 56

<212> DNA

<213> Artificial sequence

<220>

<223> sequence of hybridization Probe MB47

<400> 48

cgcgggtgcc agccttaact cctgcaaagt taagcccaca gtacttgaaa cccgcg 56

<210> 49

<211> 56

<212> DNA

<213> Artificial sequence

<220>

<223> sequence of hybridization Probe MB48

<400> 49

cggtcgccgt gctccgtctc ctcgagccaa caaaggaaga aataattaag cgaccg 56

<210> 50

<211> 56

<212> DNA

<213> Artificial sequence

<220>

<223> sequence of hybridization Probe MB49

<400> 50

cgccgtgcca gcaggacagg tgtcatgtcc tcctcagcaa aagcttcaac acggcg 56

<210> 51

<211> 56

<212> DNA

<213> Artificial sequence

<220>

<223> sequence of hybridization Probe MB50

<400> 51

cgtgggcaga gcccgggaag atggaaaacc agtttcctcc ggtctctgcg cccacg 56

<210> 52

<211> 56

<212> DNA

<213> Artificial sequence

<220>

<223> sequence of hybridization Probe MB51

<400> 52

cggtcggcag ccttgtgctt cgcgtgtgct gttggagcga tttcgttaac cgaccg 56

<210> 53

<211> 56

<212> DNA

<213> Artificial sequence

<220>

<223> sequence of hybridization Probe MB52

<400> 53

cgtgcggccg atcttatgtc acctcacctg aggacccagt ttgatcattc cgcacg 56

<210> 54

<211> 56

<212> DNA

<213> Artificial sequence

<220>

<223> sequence of hybridization Probe MB53

<400> 54

cgctgcggaa tagtactttt caccatgggt aaacttgaac ccaggtctgc gcagcg 56

<210> 55

<211> 56

<212> DNA

<213> Artificial sequence

<220>

<223> sequence of hybridization Probe MB54

<400> 55

cgcggtgggc tgtacagggc agtgaagagc cgtgaggctg agcatggagc accgcg 56

<210> 56

<211> 56

<212> DNA

<213> Artificial sequence

<220>

<223> sequence of hybridization Probe MB55

<400> 56

cgctgggcca cacatctgca cccttgcctt gtaaacatct ggcataagac ccagcg 56

<210> 57

<211> 56

<212> DNA

<213> Artificial sequence

<220>

<223> sequence of hybridization Probe MB56

<400> 57

cgtcggctga agggacagac acttaaaggg tcttactgcc cagatacttg ccgacg 56

<210> 58

<211> 56

<212> DNA

<213> Artificial sequence

<220>

<223> sequence of hybridization Probe MB57

<400> 58

cggtgccggg aagtcactct gcagttcatt cctggtgata ggatggttgg gcaccg 56

<210> 59

<211> 56

<212> DNA

<213> Artificial sequence

<220>

<223> sequence of hybridization Probe MB58

<400> 59

cgggtgcaac aggatagata ttggtgactg aggttggaag acaggcctgg cacccg 56

<210> 60

<211> 56

<212> DNA

<213> Artificial sequence

<220>

<223> sequence of hybridization Probe MB59

<400> 60

cgggctccat gtcttctgtg gtgtccaatt ctgttctcat gcatttgcag agcccg 56

<210> 61

<211> 56

<212> DNA

<213> Artificial sequence

<220>

<223> sequence of hybridization Probe MB60

<400> 61

cggcgtcggt aaatggtacc caggcccttc cggccatctc agctctctgg acgccg 56

<210> 62

<211> 56

<212> DNA

<213> Artificial sequence

<220>

<223> sequence of hybridization Probe MB61

<400> 62

cgcggtctct gaggccggaa ggagacatcg caagcatctt agagtgcagg accgcg 56

<210> 63

<211> 56

<212> DNA

<213> Artificial sequence

<220>

<223> sequence of hybridization Probe MB62

<400> 63

cgggtccaac ttccatattg atttcacaat tcttctggga gtagctctcg gacccg 56

<210> 64

<211> 56

<212> DNA

<213> Artificial sequence

<220>

<223> sequence of hybridization Probe MB63

<400> 64

cgctggcatt ctctctgctt atccaattat cctcaggtat gaaagaggag ccagcg 56

<210> 65

<211> 2556

<212> DNA

<213> mouse (Mus musculus)

<400> 65

gtttaaactt gtaaaactcc agtcgtgggc taaactgtct ggagcaggtt ctgtctctta 60

ctgtccaaga tttctcaatg ctcgtgggtc tggaagaaag agttctgcct actgtgttca 120

cggacaggtg ccagcccagt gacccagagc aatgtaggcc cattcccttt cagttgtctc 180

ccgaaaccta gcactgccct ggggctgcct gctcagcacc ctcacctctc tcccctggag 240

cccatctcag tctgccagcg attcttttca aacaaaggtt tctactgtgg tttgaatgtg 300

aaacgtcccc ccacaggttc atgtatttgc agatctatga gttgctacaa gaaggctggc 360

cgctccctgg atagcgatga atccttcagc ctactgtcca ggaagtgaac cggcttcaga 420

ctgagaccaa agtgccactc ccaaggtgaa ctgggcagga ggccaagcgc cttgtccgct 480

ccgcaccacc cacccatccc ccttgcagct gggcctcttt gatgctgctg ctgagagtct 540

atagcgggtg gtgataaacc ggtgcacctc gcggcctccc gaattcagct tctggagaca 600

atccactcgc tagaaacaag ggcgctttcc ccaggaggtc ccaactgtcc ttactcacag 660

ttcattccaa gccctaacac agccctaata aaccagtctg ctcttcacct cccagaagag 720

aaaaggcctt cctagcccaa gggtgggaat gttaaggctg ggggataaca gcgcatttgc 780

tgtggagtac tgttcctaaa aagccgtggg aaacctcaca cagatgggtg actgggctct 840

cctaggtccc ctctgctggc tttttttcct ctgacacctc tgggtcttgc aaaccgggtg 900

ctccacatat accggggatc cgctgtgaca cccccaccac caccacctgt gaccgtgtta 960

gtgactgaat ttaccttcca tagatttgga attaaccgta cagctggcat ggcagtgttg 1020

gggcagacaa ggagtcgggg agatgtgaga tcttcaaggt ggattagcaa gaggaaggtc 1080

agactttcaa actgagacct ttcagcaagc aacccttcaa ctgtgagttg ctagttagtt 1140

aaaaatgaac ccaaagggca actggggcct taaagtggcc acaaagctaa agggcaaact 1200

ggcgacagta aacataatta gctgaccctt cctggcccaa agctaacaca aacttaaagg 1260

aaataaagtc cgtcggcaat ctgctatctc aaaggcccct cctgagttgg ggccagcctt 1320

aactcctgca aagttaagcc cacagtactt gaatgcagtt aattatttct tcctttgttg 1380

gctcgaggag acggagcacg tttttgaccc ccaccctcac ccccaggctc acatgagccc 1440

cagcaggaca ggtgtcatgt cctcctcagc aaaagcttca atgcagagac cggaggaaac 1500

tggttttcca tcttcccggg ctctgcagcc ttgtgcttcg cgtgtgctgt tggagcgatt 1560

tcgttaaaag cccctttagc ctttcttctg gactctttgg gttttttttg tctgagaagg 1620

tgctgcgggg gggggggggc cctccccgat cttatgtcac ctcacctgag gacccagttt 1680

gatcattcag taaggatagt acttttcaaa aaaaagaaag aaagaaagaa agaaagaaag 1740

aaagaaagaa agaaagaaag aaagaaagaa agaaagaaag aaaagaatag tacttttcac 1800

catgggtaaa cttgaaccca ggtctgtctc catgctcagc ctcacggctc ttcactgccc 1860

tgtacagcct acaccccagc ccgcccacac atctgcaccc ttgccttgta aacatctggc 1920

ataagaacat caaatgtttc ataaacttga gtccagctac agcatctcaa ccttgaaggg 1980

acagacactt aaagggtctt actgcccaga tacttttaaa aaattgaggc ttgcaaccat 2040

cctatcacca ggaatgaact gcagagtgac ttcccatttt ccaagagtgg caacaggata 2100

gatattggtg actgaggttg gaagacaggc ctgatttcct catctgacca ggtaagcact 2160

cagacagagg tgtgacttac agaaccccat gtcttctgtg gtgtccaatt ctgttctcat 2220

gcatttgcaa atgtgaatgt gaggagccag tgcaggagga gcaggcagag agctgagatg 2280

gccggaaggg cctgggtacc atttacccag gcacataagt ctgaggccgg aaggagacat 2340

cgcaagcatc ttagagtgca gtcctttggt cttgaatttc tcacttctct gggactgtat 2400

cctttttccg aacttccata ttgatttcac aattcttctg ggagtagctc tctacttctc 2460

ccaaggtcat ctatccctga gtgcccactt tctcacttct ctgccctcta ccgaattctc 2520

tctgcttatc caattatcct caggtatgaa agagga 2556

<210> 66

<211> 3043

<212> DNA

<213> mouse (Mus musculus)

<400> 66

cagcacgtgc gccggcacac acagacacac ccacaacaat aataatcaat atatattagt 60

ttaaacttgt aaaactccag tcgtgggcta aactgtctgg agcaggttct gtctcttact 120

gtccaagatt tctcaatgct cgtgggtctg gaagaaagag ttctgcctac tgtgttcacg 180

gacaggtgcc agcccagtga cccagagcaa tgtaggccca ttccctttca gttgtctccc 240

gaaacctagc actgccctgg ggctgcctgc tcagcaccct cacctctctc ccctggagcc 300

catctcagtc tgccagcgat tcttttcaaa caaaggtttc tactgtggtt tgaatgtgaa 360

acgtcccccc acaggttcat gtatttgcag atctatgagt tgctacaaga aggctggccg 420

ctccctggat agcgatgaat ccttcagcct actgtccagg aagtgaaccg gcttcagact 480

gagaccaaag tgccactccc aaggtgaact gggcaggagg ccaagcgcct tgtccgctcc 540

gcaccaccca cccatccccc ttgcagctgg gcctctttga tgctgctgct gagagtctat 600

agcgggtggt gataaaccgg tgcacctcgc ggcctcccga attcagcttc tggagacaat 660

ccactcgcta gaaacaaggg cgctttcccc aggaggtccc aactgtcctt actcacagtt 720

cattccaagc cctaacacag ccctaataaa ccagtctgct cttcacctcc cagaagagaa 780

aaggccttcc tagcccaagg gtgggaatgt taaggctggg ggataacagc gcatttgctg 840

tggagtactg ttcctaaaaa gccgtgggaa acctcacaca gatgggtgac tgggctctcc 900

taggtcccct ctgctggctt tttttcctct gacacctctg ggtcttgcaa accgggtgct 960

ccacatatac cggggatccg ctgtgacacc cccaccacca ccacctgtga ccgtgttagt 1020

gactgaattt accttccata gatttggaat taaccgtaca gctggcatgg cagtgttggg 1080

gcagacaagg agtcggggag atgtgagatc ttcaaggtgg attagcaaga ggaaggtcag 1140

actttcaaac tgagaccttt cagcaagcaa cccttcaact gtgagttgct agttagttaa 1200

aaatgaaccc aaagggcaac tggggcctta aagtggccac aaagctaaag ggcaaactgg 1260

cgacagtaaa cataattagc tgacccttcc tggcccaaag ctaacacaaa cttaaaggaa 1320

ataaagtccg tcggcaatct gctatctcaa aggcccctcc tgagttgggg ccagccttaa 1380

ctcctgcaaa gttaagccca cagtacttga atgcagttaa ttatttcttc ctttgttggc 1440

tcgaggagac ggagcacgtt tttgaccccc accctcaccc ccaggctcac atgagcccca 1500

gcaggacagg tgtcatgtcc tcctcagcaa aagcttcaat gcagagaccg gaggaaactg 1560

gttttccatc ttcccgggct ctgcagcctt gtgcttcgcg tgtgctgttg gagcgatttc 1620

gttaaaagcc cctttagcct ttcttctgga ctctttgggt tttttttgtc tgagaaggtg 1680

ctgcgggggg gggggggccc tccccgatct tatgtcacct cacctgagga cccagtttga 1740

tcattcagta aggatagtac ttttcaaaaa aaagaaagaa agaaagaaag aaagaaagaa 1800

agaaagaaag aaagaaagaa agaaagaaag aaagaaagaa aagaatagta cttttcacca 1860

tgggtaaact tgaacccagg tctgtctcca tgctcagcct cacggctctt cactgccctg 1920

tacagcctac accccagccc gcccacacat ctgcaccctt gccttgtaaa catctggcat 1980

aagaacatca aatgtttcat aaacttgagt ccagctacag catctcaacc ttgaagggac 2040

agacacttaa agggtcttac tgcccagata cttttaaaaa attgaggctt gcaaccatcc 2100

tatcaccagg aatgaactgc agagtgactt cccattttcc aagagtggca acaggataga 2160

tattggtgac tgaggttgga agacaggcct gatttcctca tctgaccagg taagcactca 2220

gacagaggtg tgacttacag aaccccatgt cttctgtggt gtccaattct gttctcatgc 2280

atttgcaaat gtgaatgtga ggagccagtg caggaggagc aggcagagag ctgagatggc 2340

cggaagggcc tgggtaccat ttacccaggc acataagtct gaggccggaa ggagacatcg 2400

caagcatctt agagtgcagt cctttggtct tgaatttctc acttctctgg gactgtatcc 2460

tttttccgaa cttccatatt gatttcacaa ttcttctggg agtagctctc tacttctccc 2520

aaggtcatct atccctgagt gcccactttc tcacttctct gccctctacc gaattctctc 2580

tgcttatcca attatcctca ggtatgaaag aggaataaaa ataaataatt ttaaaaagac 2640

atgaaaaagt gtgtggctgg ctataggtca actattcatt tctttttaaa agatttattt 2700

gcagccgggc gtggtggcat acacctttaa tcccaacact cgggaggcag aggcaggcag 2760

atttctgagt tcgaggccag cctggtctac aaagtgagtt ccaggatagc cagagctaca 2820

cagagaaacc ctgactcaaa aaaacaaaaa caaacaaaaa aaagagagag ggttttcatt 2880

tttgttttga aattatgata taattacaac tttttccatt tcctccaaac tctcccatat 2940

accccacctt tctacctttt aaattcatgg cctcttttaa aataactgtt attacacgca 3000

tattatattc ttaaatatga ccttctcggt ctgtatgatg tca 3043

Claims (10)

1. A method for identifying a nucleic acid, comprising the steps of:

a. the sample is brought into contact with the hybridization probe,

and

c. identifying whether the hybridization probe binds to the target nucleic acid;

wherein the hybridization probe comprises a reporter group and the hybridization probe differs in state in two cases, thereby generating an identifiable signal by the reporter group: (i) the hybridization probe is in a free state, (ii) the hybridization probe is bound to the target nucleic acid;

step a of the identification method is carried out according to the following method: incubating the sample with the hybridization probe at a temperature of 73 ℃ to 77 ℃ and then hybridizing at a temperature of 14 ℃ to 22 ℃;

the target nucleic acid is less than 4.9kb in length, the target nucleic acid does not contain a repeat sequence;

step c of the identification method is carried out by fluorescence microscopy, wherein the fluorescence microscopy is STORM super-resolution imaging;

wherein the hybridization probe comprises a binding region and a dissociation region, the dissociation region self-hybridizes when the hybridization probe is in a free state, and the reporter group does not emit an identifiable signal; when the hybridization probe is specifically combined with the target sequence segment of the target nucleic acid through the combination region, the dissociation region is dissociated, and the reporter group emits an identifiable signal;

wherein the reporter group comprises a fluorophore and a quencher group, and the identifiable signal is fluorescence;

wherein the hybridization probe comprises a loop structure serving as the binding region, and two arm structures serving as the dissociation region, wherein the two arm structures are located on both sides of the loop structure, respectively;

wherein, the length of the ring structure is 40 nt-44 nt, and the length of the arm structure is 7 nt.

2. The identification method according to claim 1, characterized in that it comprises the steps of:

a. the sample is brought into contact with the hybridization probe,

b. eluting hybridization probes that are not bound to the target nucleic acid, and

c. identifying whether the hybridization probe binds to the target nucleic acid.

3. The method for identifying according to claim 1, wherein the fluorophore is Alexa-647 and the quencher is BHQ 3.

4. The method of any one of claims 1 to 3, wherein the sequence identity between the loop structure and the target sequence fragment of the target nucleic acid is between 90% and 100%.

5. The method of identifying according to any one of claims 1 to 3, wherein the sequence identity between the loop structure and the target sequence fragment of the target nucleic acid is between 95% and 100%.

6. The method of identifying according to any one of claims 1 to 3, wherein the sequence identity between the loop structure and the target sequence fragment of the target nucleic acid is between 98% and 100%.

7. The method according to claim 1 or 2, wherein the target nucleic acid has a length of 3.3kb or less.

8. The method according to claim 1 or 2, wherein the target nucleic acid has a length of 2.5kb or less.

9. The identification method according to claim 1 or 2, wherein step a of the identification method is performed according to the following method: the sample is incubated with the hybridization probes at a temperature of 75 ℃ and then hybridized at a temperature of 22 ℃.

10. Use of a hybridization probe in fluorescence microscopy imaging of a target nucleic acid, wherein the hybridization probe comprises a reporter group and the hybridization probe differs in state in two cases, whereby an identifiable signal is generated by the reporter group: (i) the hybridization probe is in a free state, (ii) the hybridization probe is bound to the target nucleic acid;

incubating the nucleic acid and the hybridization probe together at a temperature of 73-77 ℃ and then hybridizing at a temperature of 14-22 ℃;

the fluorescence microscopic imaging is STORM super-resolution imaging;

wherein the hybridization probe comprises a binding region and a dissociation region, the dissociation region self-hybridizes when the hybridization probe is in a free state, and the reporter group does not emit an identifiable signal; when the hybridization probe is specifically combined with the target sequence segment of the target nucleic acid through the combination region, the dissociation region is dissociated, and the reporter group emits an identifiable signal;

wherein the reporter group comprises a fluorophore and a quencher group, and the identifiable signal is fluorescence;

wherein the hybridization probe comprises a loop structure serving as the binding region, and two arm structures serving as the dissociation region, wherein the two arm structures are located on both sides of the loop structure, respectively;

wherein, the length of the ring structure is 40 nt-44 nt, and the length of the arm structure is 7 nt.

Images