CN117887813B

CN117887813B - Hybridization buffer solution and application

Info

Publication number: CN117887813B
Application number: CN202311603330.5A
Authority: CN
Inventors: 刘艳艳; 袁晓红
Original assignee: Beijing Stomatological Hospital
Current assignee: Beijing Stomatological Hospital
Priority date: 2023-11-28
Filing date: 2023-11-28
Publication date: 2024-07-12
Anticipated expiration: 2043-11-28
Also published as: CN117887813A

Abstract

The invention relates to the field of molecular detection, in particular to a hybridization buffer solution and application. Wherein, a hybridization buffer solution comprises an organic solvent, the organic solvent has a structure shown in a formula (I), wherein, R ₁ and R ₃ are alkyl, R ₂ is at least one of alkyl or hydrogen, and n is a natural number less than or equal to 6. The organic solvent is at least one of diglyme, triglyme, tetraglyme, hexaglyme, diethylene glycol ethyl methyl ether, diethylene glycol diethyl ether or dipropylene glycol dimethyl ether. The hybridization buffer solution containing the organic solvent with the structure shown in the formula (I) can reduce the contents of the size exclusion agent and formamide, reduce the viscosity of the hybridization buffer solution, shorten the denaturation time and the washing time, reduce the damage to the morphology of sample cells, enhance the signal intensity, reduce the background signal and improve the specificity of hybridization.

Description

Hybridization buffer solution and application

Technical Field

The invention relates to the field of molecular detection, in particular to a hybridization buffer solution and application.

Background

Techniques based on the principle of nucleic acid hybridization, such as in situ hybridization and targeted enrichment capture sequencing, are widely used in genetic variation detection and gene expression regulation research, and can provide key information beyond the prior art such as PCR and NGS, thus becoming gold standards for many kinds of genetic variation and cancer diagnosis.

The hybridization buffer provides a solution environment for the probe to hybridize, which is capable of substantially denaturing the intracellular DNA duplex at a temperature. The hybridization buffers currently in wide use all contain high concentrations of formamide, and typical hybridization buffers may consist of 50% -65% (v/v) formamide, 2X SSC (Saline Sodium Citrate) buffer, 10% (w/v) dextran sulfate, 100. Mu.g/mL salmon sperm DNA, or 0.3. Mu.g/uL COT-1DNA, etc. The formamide can effectively realize DNA double-chain denaturation under the complex environmental condition in the nucleus, but also obviously slows down the renaturation of the probe and the target sequence in the subsequent annealing (namely hybridization) step and prolongs the hybridization time due to the action of the formamide on a base binding site and a mechanism of interfering hydrogen bonding. Meanwhile, the high concentration of formamide can damage the morphology of cell morphology, cell nucleus structure or chromosome structure, so that the detection result is inaccurate. In addition, formamide is a compound harmful to the human body, and there is a certain risk during the operation. The addition of a large amount of dextran sulfate, which is another major component in the hybridization buffer, results in a high viscosity of the hybridization buffer, which has a significant impact on the diffusion of probe molecules during in situ hybridization. The rate limiting step of in situ hybridization is that the probe molecules diffuse from outside cells into the nuclei in the buffer, and dextran sulfate negatively affects the diffusion coefficient of the probe molecules in the buffer, which results in slow diffusion of the probe molecules, resulting in excessively long hybridization times.

The prior art discloses a hybridization composition and a method thereof using formamide with lower concentration, which can reduce the hybridization time and reduce the damage of cell morphology by increasing the concentration of salt ions and the concentration of an accelerator in a hybridization buffer solution and reducing the concentration of the formamide, but the viscosity of the hybridization buffer solution is increased due to the increase of the concentration of the accelerator, namely dextran sulfate, and the high-viscosity hybridization buffer solution can enable a treated sample to more easily retain tiny bubbles, so that a signal-free area is generated at the contact place of the sample and the bubbles, the signal intensity is reduced, and the washing time after hybridization is prolonged.

Disclosure of Invention

Therefore, the technical problem to be solved by the invention is to overcome the defect of high viscosity of the hybridization buffer solution in the prior art, so as to provide the hybridization buffer solution and application, which can shorten the hybridization time, improve the specificity, reduce the background signal, reduce the viscosity of the hybridization buffer solution and shorten the washing time after hybridization.

In one aspect, the invention provides a hybridization buffer comprising an organic solvent having a structure according to formula (I):

wherein R ₁ and R ₃ are alkyl groups, R ₂ is at least one of alkyl groups and hydrogen groups, and n is a natural number less than or equal to 6.

The organic solvent is at least one of diglyme, triglyme, tetraglyme, hexaglyme, diethylene glycol ethyl methyl ether, diethylene glycol diethyl ether or dipropylene glycol dimethyl ether.

The volume concentration of the organic solvent in the hybridization buffer solution is 1-40%. Preferably, the volume concentration of the organic solvent in the hybridization buffer is 20-30%. More preferably, the mass concentration of the size-exclusion agent in the hybridization buffer is 0.05% -5%.

Also comprises hybridization promoter, buffer and salt solution.

The hybridization accelerator includes at least one of a size exclusion agent, a Denhardt solution, formamide, or DMSO.

Preferably, the size exclusion agent is at least one of polyvinylpyrrolidone, polysucrose, polyethylene glycol or dextran sulfate.

More preferably, the size exclusion agent of the hybridization buffer is dextran sulfate.

The buffer is at least one of citric acid, sodium citrate, disodium hydrogen phosphate, potassium dihydrogen phosphate and 4-hydroxyethyl piperazine ethane sulfonic acid. The concentration of the buffer is 1-50mM, and the pH value is 5-8.

The salt solution is at least one of sodium chloride solution, potassium chloride and magnesium chloride. The concentration of the salt solution is 300-1200mM.

Preferably, the hybridization promoter comprises the formamide at a volume concentration of 0-50%. More preferably, the formamide solution has a volume concentration of 5 to 20%

The pH value of the hybridization buffer solution provided by the invention is 3-10.

The hybridization buffer also includes a chelating agent, a detergent, and a blocking agent.

The chelating agent is at least one of EDTA or EGTA. The effect of background signal can be reduced by adding 1mM chelating agent, especially EDTA, to the hybridization buffer.

The detergent is at least one of sodium dodecyl sulfate, tween-20 and Triton X-100.

The blocking agent is at least one of salmon sperm DNA and Human COT1 DNA.

On the other hand, the hybridization buffer solution provided by the invention can be applied to in-situ hybridization technology and targeted enrichment capture sequencing technology.

The in situ hybridization technique and/or the targeted sequencing enrichment capture technique comprises the steps of denaturing the substance to be detected with the hybridization buffer containing a DNA probe, and hybridizing. The denaturation temperature is 73-95 ℃ and the denaturation time is 2-10min. The hybridization temperature is 37-45 ℃, and the hybridization time is 0.5-16h. The concentration of the DNA probe in the hybridization buffer solution is 0.1-100 ng/. Mu.L.

The technical scheme of the invention has the following advantages:

1. The hybridization buffer solution provided by the invention comprises an organic solvent, wherein R ₁ and R ₃ in the structure of the organic solvent are alkyl, R ₂ is at least one of alkyl or hydrogen, and n is a natural number less than or equal to 6. The hybridization buffer solution containing the organic solvent can reduce the contents of formamide and a size exclusion agent, reduce the obstruction to hydrogen bond pairing, and improve the diffusion coefficient, thereby shortening the hybridization time and the washing time, reducing the damage to the morphology of sample cells, enhancing the signal intensity, reducing the viscosity of the hybridization buffer solution, reducing the background signal and improving the specificity of hybridization.

2. The hybridization buffer solution provided by the invention has the volume concentration of the organic solvent of 1-40% and the mass concentration of the volume exclusion agent of 0.05% -5%. The invention reduces the content of the size exclusion agent in the hybridization buffer solution, so that the viscosity of the hybridization buffer solution is reduced, the diffusion of probe molecules in the hybridization buffer solution and the intracellular environment is facilitated, and the hybridization reaction kinetics is improved. In addition, the low viscosity of the hybridization buffer also facilitates penetration and diffusion of the hybridization buffer itself within tissues and cells, and it is difficult to generate or retain microscopic bubbles, avoiding leaving areas that cannot be denatured and hybridized due to inability to contact with the hybridization buffer. Meanwhile, the low viscosity of the hybridization buffer solution improves the washing efficiency after hybridization and shortens the washing time.

3. The hybridization buffer solution provided by the invention further comprises a hybridization accelerator, a buffer and a salt solution, wherein the hybridization accelerator comprises formamide, the concentration of the salt solution is 300-1200mM, and the volume concentration of the formamide is 0-50%. The invention optimizes the content of each component in the hybridization buffer solution, enhances the signal intensity, improves the specificity and reduces the background signal.

4. The hybridization buffer solution provided by the invention reduces the content of formamide or does not use formamide, can still enhance the signal intensity, improve the specificity, reduce the background signal, and can avoid the harm of formamide to a user.

5. The hybridization buffer solution provided by the invention is applied to in-situ hybridization technology and targeted enrichment capture sequencing technology, can greatly reduce the dosage of probes, can improve the denaturation temperature and shorten the denaturation time, realizes more sufficient denaturation of a sample, and does not damage the chromosome structure of cells.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the description of the embodiments or the prior art will be briefly described, and it is obvious that the drawings in the description below are some embodiments of the present invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.

FIG. 1 is a fluorescence microscope lower image of the sample treated with the FISH probe reagent of examples 1-3 and comparative examples 1-2 of the present invention, wherein A1 is the image of the sample treated with the FISH probe reagent of comparative example 1 under the DAPI filter, B1 is the image of the sample treated with the FISH probe reagent of comparative example 1 under the Spectrum Orange filter, A2 is the image of the sample treated with the FISH probe reagent of comparative example 2 under the DAPI filter, B2 is the image of the sample treated with the FISH probe reagent of comparative example 2 under the Spectrum Orange filter, A3 is the image of the sample treated with the FISH probe reagent of example 1 under the DAPI filter, B3 is the image of the sample treated with the FISH probe reagent of example 1 under the Spectrum Orange filter, A4 is the image of example 2 under the Spectrum Orange filter, A5 is the image of the sample treated with the Spectrum Orange filter, and A3 is the image of the sample treated with the Spectrum Orange filter of example 5 under the sample treated with the FISH probe reagent of example 1 under the Spectrum Orange filter;

FIG. 2 is a fluorescence microscope image of samples treated with the FISH probe reagent prepared in example 3 and comparative example 1 according to the present invention at different temperatures;

FIG. 3 is a fluorescence microscopic image of the FISH probe reagent-treated sample prepared in example 8 and comparative example 3 of the present invention, wherein a1 is the image of the FISH probe reagent-treated sample prepared in comparative example 3 under the DAPI filter, b1 is the image of the FISH probe reagent-treated sample prepared in comparative example 3 under the Spectrum Orange filter, a2 is the image of the FISH probe reagent-treated sample prepared in example 8 under the DAPI filter, and b2 is the image of the FISH probe reagent-treated sample prepared in example 8 under the Spectrum Orange filter.

Detailed Description

The following examples are provided for a better understanding of the present invention and are not limited to the preferred embodiments described herein, but are not intended to limit the scope of the invention, any product which is the same or similar to the present invention, whether in light of the present teachings or in combination with other prior art features, falls within the scope of the present invention.

The specific experimental procedures or conditions are not noted in the examples and may be followed by the operations or conditions of conventional experimental procedures described in the literature in this field. The reagents or apparatus used were conventional reagent products commercially available without the manufacturer's knowledge.

In the examples, comparative examples and experimental examples of the present invention: dextran sulfate (D8906), formamide (F9037), sodium citrate (S4641), polyvinylpyrrolidone, dextran, polyethylene glycol, triglyme, hexaglyme, diethylene glycol diethyl ether, dipropylene glycol dimethyl ether, diglyme, disodium hydrogen phosphate, citric acid, potassium dihydrogen phosphate, 4-hydroxyethylpiperazine ethanesulfonic acid were purchased from Sigma-Aldrich company; diethylene glycol ethyl methyl ether, tetraethylene glycol dimethyl ether (T819541), diethylene glycol diethyl ether (E808613) were purchased from microphone company; VECTASHIELD MOUNTING MEDIUM WITH DAPI (H1200) is from Vector Laboratories. In order to verify that the hybridization buffer solution of the invention can be applied to various probes, the invention selects a plurality of probes for verification, wherein the DNA probes in the examples 1-5 are CEP X/Y, and the concentration is 14 ng/. Mu.L, and are purchased from Abbott Molecular and coded as 07J20-050; the DNA probe of examples 6-11 was CEP 8, ready-to-use, at a concentration of 5 ng/. Mu.L, available from Abbott Molecular, code 07J22-008. The DNA probe in examples 12-18 was RET (10 q 11), purchased from amitraz, code F.01104-01.

Example 1

The embodiment provides a hybridization buffer solution, which comprises the following specific components in percentage by weight:

The hybridization buffer pH was adjusted to 6.5 by mixing, calculated as final concentration, formamide at a concentration of 10% by volume, triglyme at a concentration of 20% by volume, dextran sulfate at a concentration of 1% by mass, naCl at a molar concentration of 900mM, and sodium citrate at a molar concentration of 10 mM.

The DNA probe CEP X/Y was added to the hybridization buffer of this example at a final concentration of 1.17 ng/. Mu.L (1/12 of the original concentration) to prepare a FISH probe reagent.

Example 2

The hybridization buffer pH was adjusted to 6.5 by mixing, calculated as final concentration, formamide at a volume concentration of 10%, triglyme at a volume concentration of 25%, dextran sulfate at a mass concentration of 1%, naCl at a molar concentration of 900mM, and sodium citrate at a molar concentration of 10 mM.

Example 3

The hybridization buffer pH was adjusted to 6.5 by mixing, calculated as final concentration, formamide at a volume concentration of 10%, triglyme at a volume concentration of 30%, dextran sulfate at a mass concentration of 1%, naCl at a molar concentration of 900mM, and sodium citrate at a molar concentration of 10 mM.

Example 4

This example provides a hybridization buffer having the same specific composition and content as in example 2, except that the hybridization buffer was left to stand in a room temperature environment for 20 months.

Example 5

This example provides a hybridization buffer having the same specific composition and content as in example 3, except that the hybridization buffer was left to stand in a room temperature environment for 20 months.

Example 6

The hybridization buffer pH was adjusted to 6.5 by mixing the following substances, calculated as final concentration, triethylene glycol dimethyl ether at a volume concentration of 30%, dextran sulfate at a mass concentration of 1%, naCl at a molar concentration of 900mM, and sodium citrate at a molar concentration of 10 mM.

A DNA probe CEP 8 was added to the hybridization buffer of this example at a final concentration of 0.42 ng/. Mu.L (1/12 of the original concentration) to prepare a FISH probe reagent.

Example 7

The hybridization buffer pH was adjusted to 6.5 by mixing, calculated as final concentration, formamide at 5% by volume, triglyme at 30% by volume, dextran sulfate at 1% by mass, naCl at 900mM, sodium citrate at 10 mM.

Example 8

Example 9

The hybridization buffer pH was adjusted to 6.5 by mixing, calculated as final concentration, formamide at 20% by volume, triglyme at 30% by volume, dextran sulfate at 1% by mass, naCl at 900mM, sodium citrate at 10 mM.

Example 10

The hybridization buffer pH was adjusted to 6.5 by mixing, calculated as final concentration, formamide at a volume concentration of 10%, triglyme at a volume concentration of 30%, dextran sulfate at a mass concentration of 1%, naCl at a molar concentration of 600mM, and sodium citrate at a molar concentration of 10 mM.

Example 11

The hybridization buffer pH was adjusted to 6.5 by mixing, calculated as final concentration, formamide at a volume concentration of 10%, triglyme at a volume concentration of 30%, dextran sulfate at a mass concentration of 1%, naCl at a molar concentration of 1200mM, and sodium citrate at a molar concentration of 10 mM.

Example 12

The hybridization buffer pH was adjusted to 6.5 by mixing, calculated as final concentration, formamide at 45% by volume, triglyme at 5% by volume, dextran sulfate at 5% by mass, naCl at 900mM, sodium citrate at 10 mM.

A DNA probe RET (10 q 11) was added to the hybridization buffer of this example at a final concentration of 0.4 ng/. Mu.L to prepare a FISH probe reagent.

Example 13

The following substances were mixed so as to contain formamide at a final concentration of 10% by volume, tetraglyme at a concentration of 20% by volume, polyethylene glycol at a concentration of 1% by mass, naCl at a molar concentration of 900mM, and disodium hydrogen phosphate at a molar concentration of 10mM, and the pH of the hybridization buffer thus prepared was adjusted to 8.

A DNA probe RET (10 q 11) was added to the hybridization buffer of this example at a final concentration of 0.1 ng/. Mu.L to prepare a FISH probe reagent.

Example 14

the pH of the hybridization buffer prepared by mixing formamide at a final concentration of 10%, diglyme at a volume concentration of 20%, polysucrose at a mass concentration of 5%, magnesium chloride at a molar concentration of 300mM and disodium hydrogen phosphate at a molar concentration of 50mM was adjusted to 10.

Example 15

the hybridization buffer prepared by mixing formamide at a final concentration of 10% by volume, diethylene glycol ethyl methyl ether at a volume concentration of 20% by volume, polyvinylpyrrolidone at a mass concentration of 5% by mass, potassium chloride at a molar concentration of 900mM and 4-hydroxyethyl piperazine ethane sulfonic acid at a molar concentration of 20mM was adjusted to pH 7.5.

A DNA probe RET (10 q 11) was added to the hybridization buffer of this example at a final concentration of 5 ng/. Mu.L to prepare a FISH probe reagent.

Example 16

The pH of the hybridization buffer prepared by mixing formamide at a final concentration of 10% by volume, diethylene glycol diethyl ether at a volume concentration of 20%, dextran sulfate at a mass concentration of 0.05%, naCl at a molar concentration of 900mM and potassium dihydrogen phosphate at a molar concentration of 25mM was adjusted to 3.

A DNA probe RET (10 q 11) was added to the hybridization buffer of this example at a final concentration of 10 ng/. Mu.L to prepare a FISH probe reagent.

Example 17

the hybridization buffer prepared by mixing formamide at a final concentration of 50% by volume, dipropylene glycol dimethyl ether at a volume concentration of 1%, polyvinylpyrrolidone at a mass concentration of 0.25%, naCl at a molar concentration of 900mM, and potassium dihydrogen phosphate at a molar concentration of 15mM was adjusted to pH 5.5.

A DNA probe RET (10 q 11) was added to the hybridization buffer of this example at a final concentration of 20 ng/. Mu.L to prepare a FISH probe reagent.

Example 18

The hybridization buffer prepared by mixing formamide at a final concentration of 10% by volume, hexaethyleneglycol dimethyl ether at a volume concentration of 40%, polyvinylpyrrolidone at a mass concentration of 0.25%, naCl at a molar concentration of 900mM, and potassium dihydrogen phosphate at a molar concentration of 15mM was adjusted to a pH of 5.5.

Comparative example 1

This comparative example provides a hybridization buffer, as a typical form of conventional formamide hybridization buffer, with the following specific composition and contents:

The following substances were mixed so as to contain formamide at a final concentration of 50% by volume, dextran sulfate at a mass concentration of 10%, naCl at a molar concentration of 300mM, sodium citrate at a molar concentration of 30mM, and salmon sperm DNA at a molar concentration of 100. Mu.g/mL.

The DNA probe CEP X/Y was added to the hybridization buffer of this comparative example at a final concentration of 1.17 ng/. Mu.L (1/12 of the original concentration) to prepare a FISH probe reagent.

Comparative example 2

This comparative example provides a hybridization buffer having the same specific composition and content as in example 1, except that the comparative example does not contain triglyme as an organic solvent, i.e., the hybridization buffer is formamide at a volume concentration of 10% based on the final concentration, dextran sulfate at a mass concentration of 1%, naCl at a molar concentration of 900mM, and sodium citrate at a molar concentration of 10 mM.

Comparative example 3

This comparative example provides a hybridization buffer as a form of the disclosed formamide hybridization buffer, with the following specific composition and amounts:

The following substances were mixed so as to contain, as calculated in terms of final concentration, formamide at a volume concentration of 15%, dextran sulfate at a mass concentration of 20%, naCl at a molar concentration of 600mM, sodium citrate at a molar concentration of 10mM, and pH was adjusted to 6.2.

The DNA probe CEP 8 was added to the hybridization buffer of this comparative example at a final concentration of 0.42 ng/. Mu.L (1/12 of the original concentration) to prepare a FISH probe reagent.

Application example 1

Washing a slide glass of peripheral blood cells in a metaphase of mitosis with a 2 XSSC buffer solution for 2 times at room temperature for 3 minutes each time, sequentially dehydrating the washed slide glass in 70%, 85% and 100% ethanol for 1min respectively, and airing;

The treated samples were denatured with 10. Mu.L of the FISH probe reagents prepared in examples 1-5 and comparative examples 1-2, respectively, at 73℃for 10min, hybridized at 37℃for 16h, the incubated samples were washed at 65℃in 2 XSSC for 10min, then washed at room temperature in 2 XSSC for 3 min, the washed samples were dehydrated sequentially in 70%, 85%, 100% ethanol for 1min, and then dried in the air, and the slides were mounted with 10. Mu.L of DAPI-containing anti-fluorescence quenching mounting agent to prepare FISH slides.

Application example 2

The treated samples were denatured with 10. Mu.L of the FISH probe reagent prepared in examples 6 to 18 at 95℃for 2min, hybridized at 45℃for 2h, the incubated samples were washed in 65℃2 XSSC for 10min, then washed in room temperature 2 XSSC for 3 min, the washed samples were dehydrated in 70%, 85% and 100% ethanol for 1min, and dried in the air, and the slides were blocked with 10. Mu.L of DAPI-containing anti-fluorescence quenching blocking agent to prepare FISH slides.

Application example 3

Washing a slide glass of peripheral blood cells in a metaphase of mitosis with a2 XSSC buffer solution for 2 times and 3 minutes each time at room temperature, sequentially dehydrating the washed slide glass in 70%, 85% and 100% ethanol for 1min respectively, and airing;

The above treated samples were denatured with 10. Mu.L of the FISH probe reagents prepared in comparative examples 1 and 3, respectively, at 73℃and 78℃and 82℃for 10min, hybridized at 37℃for 16h, the incubated samples were washed in 65℃2 XSSC for 10min, and then washed in room temperature 2 XSSC for 3 min, the washed samples were dehydrated in 70%, 85% and 100% ethanol for 1min, dried in the air, and slides were mounted with 10. Mu.L of DAPI-containing anti-fluorescence quenching mounting agent to prepare FISH slides.

Application example 4

the treated samples were denatured with 10. Mu.L of the FISH probe reagents prepared in comparative example 3 and example 8, respectively, at 82℃for 10min, hybridized at 37℃for 16h, the incubated samples were washed in 2 XSSC at 65℃for 10min, then washed in 2 XSSC at room temperature for 3 min, the washed samples were dehydrated in 70%, 85% and 100% ethanol for 1min in order, and dried in the air, and the slides were sealed with 10. Mu.L of DAPI-containing anti-fluorescence quenching sealing agent to prepare FISH slides.

Experimental example 1

The FISH slides prepared in application examples 1 and 2 were observed under a 63 x oil microscope using a Zeiss Axiovert200 fluorescence microscope equipped with DAPI, FITC, cy single color filters within two days after staining. Two trained observers evaluated fluorescence signal intensity, specificity and background signal, scored as described in the cell/tissue section scoring guidelines. The results are shown in tables 1-3.

Cell/tissue section scoring guidelines: signal intensity was evaluated on a scale of 0-5, where 0 indicates no signal and 5 indicates a strong signal. Signal specificity on a scale of 0-5, where 0 indicates no distinction between specific and non-specific signals and 5 indicates no non-specific signal. Between 0 and 5 scores, there may be a 1/2 scale, i.e., the scale between adjacent scores is 1/2.

Signal strength was scored according to the grading definition on the 0-5 scale:

0. indicating no visible signal;

1. the signal is weak;

2. the signal is weak but can be identified;

3. The signal is clear;

4. indicating that the signal is strong;

5. Indicating that the signal is very strong;

the signal strength scoring system allows for the use of 1/2 stages.

Signal specificity was scored according to the grading definition on the 0-5 scale: 0 means that the specific signal is completely indistinguishable in intensity from the non-specific signal; 1 indicates that the specific signal is not easily recognized as compared to the non-specific signal; 2 indicates that a specific signal can be recognized as compared to a non-specific signal;

3 indicates that the specific signal is easily recognized compared to the non-specific signal;

4 indicates that the nonspecific signal is much weaker than the specific signal;

5 represents no identifiable non-specific signal;

The signal specificity scoring system allows the use of 1/2 stages.

Scoring the background signal according to a hierarchical definition on a scale of 0-5:

0. indicating that no background signal is visible;

1. Indicating that there is a little background signal;

2 indicates that there is some background signal;

3 represents a medium background signal;

4 indicates a higher background signal;

5 indicates a very high background signal;

the background signal scoring system allows for the use of 1/2 stages.

TABLE 1 fluorescence Signal Strength, specificity and background Signal scoring of FISH slides made with hybridization buffers provided in examples 1-5 and comparative examples 1-2

Group of	Average signal strength	Average specificity	Average background signal
				Example 1	2 1/2	2 1/2	2 1/2
Example 2	3	3	2
				Example 3	3 1/2	3	1 1/2
Example 4	2 1/2	2	21/2
				Example 5	3	2 1/2	2
Comparative example 1	2	2	2 1/2
				Comparative example 2	1/2	0	3 1/2

It can be seen that comparative example 1 shows better performance in terms of average signal intensity, average specificity or average background signal compared with example 3, and that the inventive example shows better performance than the conventional hybridization buffer using formamide buffer, and that comparative example 2 does not produce a recognizable signal and has higher background signal compared with example 1, demonstrating that the low content of formamide and the low content of dextran sulfate cannot achieve technical effects without adding organic solvent.

Referring to FIG. 1, the hybridization result of comparative example 1 is that 1 target signal with higher intensity and 4 nonspecific signals with weaker intensity exist in each cell nucleus, and the nonspecific signals have a certain interference on the target signal recognized by observers. In the hybridization results of example 3, the target signal intensity was high, but the non-specific signal intensity was extremely low, and the recognition was not disturbed.

From a comparison of the data of example 4 with the data of example 2 and the data of example 5 and example 3 in Table 1, it is clear that the hybridization buffer provided by the present invention can still maintain a higher signal intensity after being left at room temperature for 20 months.

TABLE 2 fluorescence Signal Strength, specificity and background Signal scoring of FISH slides made with hybridization buffers provided in examples 6-11

Group of	Average signal strength	Average specificity	Average background signal
				Example 6	2	2	3
Example 7	3	3	2
				Example 8	3 1/2	3 1/2	1 1/2
Example 9	3 1/2	3	1 1/2
				Example 10	2 1/2	2 1/2	2
Example 11	3 1/2	3 1/2	1 1/2

As can be seen from the data of examples 6-9, the signal specificity of the hybridization buffer increased and the background signal decreased with increasing formamide concentration over a range. As is clear from the data of examples 8 and 10 to 11, the fluorescence intensity generated by the hybridization buffer gradually increased and the background signal gradually decreased as the salt concentration was increased within a certain range.

TABLE 3 fluorescence Signal Strength, specificity and background Signal scoring of FISH slides made with hybridization buffers provided in examples 12-18

As can be seen, the DNA probe prepared from the hybridization buffers provided by the invention is a FISH probe reagent of RET (10 q 11), which has better average signal intensity and average specificity, and the average background signal is reduced.

The data relating to fluorescence signal intensity, specificity and background signal scoring of FISH slides prepared by the prepared hybridization buffers of examples 1-18 demonstrate that the hybridization buffers provided by the present invention can be used with a variety of probes.

Experimental example 2

The FISH slide prepared in application example 3 was observed under a 63 x oil microscope using a Zeiss Axiovert200 fluorescence microscope equipped with DAPI, FITC, cy single color filters within two days after staining. The results of chromatin structure in FISH slides are shown in table 4 and fig. 2.

TABLE 4 chromosome structure under different denaturation conditions of FISH slide glass prepared in application example 3

Referring to FIG. 2, the sample cells in the FISH slide glass prepared by the hybridization buffer solution provided in comparative example 1 show abnormal chromosome structure morphology under the denaturation condition of 78 ℃ and 82 ℃ and are marked as 'burr' -shaped and 'cavitation' -shaped defects, and focusing is difficult when observed by a microscope. Therefore, in the case of a sample which is difficult to hybridize such as FFPE or amniotic fluid cells, hybridization is often poor at a conventional denaturation temperature (73 ℃ and the like), one of the reasons is insufficient denaturation of the target sequence. Increasing the denaturation temperature or extending the denaturation time is one of the methods to ameliorate the lack of denaturation of such samples. However, conventional hybridization buffers, like the buffer of comparative example 1, contain high concentrations of formamide, which cannot withstand the denaturation at higher temperatures, and above 73 ℃, formamide damages chromatin structure, compromising the shape of the fluorescent signal, exposing its inapplicability. The FISH slide prepared with the hybridization buffer provided in example 3 was complete in chromatin at different temperatures, demonstrating that increasing the denaturation temperature did not have a significant effect on the hybridization buffer of this example.

Experimental example 3

The procedure and method were the same as in experimental example 1, except that the sample was a FISH slide prepared in application example 4, and the evaluation results of fluorescence signal intensity, specificity and background signal are shown in fig. 3 and table 5.

TABLE 5 fluorescence signal intensity, specificity and background signal scoring of FISH slides prepared by application example 4

Group of	Average signal strength	Average specificity	Average background signal
				Example 8	3 1/2	3	1 1/2
Comparative example 3	3	2	2

Referring to FIGS. 3 and 5, it can be seen that the hybridization buffer prepared in example 8 of the present invention can cause the slide to exhibit higher signal intensity, higher specificity and lower background than comparative example 3 at a denaturation temperature of 82 ℃.

Experimental example 4

The hybridization buffers prepared in example 8 and comparative example 3 were tested for viscosity using a micro-viscometer, and the viscosity was substituted into Stokes-Einstein formula to calculate the diffusion coefficient, and the calculation method of the diffusion coefficient was found in Joana Lima et al at Biotechnology and bioengineering.2020; 117:3212-3223.

After detection and combination of the data in Table 5, the hybridization buffer viscosity of example 8 was 3.89 mPas, the diffusion coefficient of the double-stranded DNA probe of 40bp length in the hybridization buffer was calculated to be 5.38X10 ^-11m²/s, the average signal intensity was 3.5, the average specificity was 3, and the average background signal was 1.5; comparative example 3 the viscosity of the hybridization buffer was 64.2 mPas, and the diffusion coefficient of the double-stranded DNA probe of 40bp length in the hybridization buffer was calculated to be 3.26X10 ^-12m²/s, average signal intensity 3, average specificity 2, average background signal 2. It is known that the use of triglyme, an organic solvent, can reduce the amount of dextran sulfate, thereby reducing the viscosity of the hybridization buffer, and thus increasing the diffusion coefficient of the probe in the hybridization buffer, without reducing the signal intensity or specificity or increasing the background signal even if the amount of dextran sulfate is reduced.

It is apparent that the above examples are given by way of illustration only and are not limiting of the embodiments. Other variations or modifications of the above teachings will be apparent to those of ordinary skill in the art. It is not necessary here nor is it exhaustive of all embodiments. While still being apparent from variations or modifications that may be made by those skilled in the art are within the scope of the invention.

Claims

1. The application of the hybridization buffer solution in preparing a detection reagent for fluorescence in situ hybridization is characterized in that the hybridization buffer solution comprises the following specific components in percentage by weight:

The following were mixed and the hybridization buffer was made to contain, calculated as final concentration: formamide with the volume concentration of 10%, triglyme with the volume concentration of 30%, dextran sulfate with the mass concentration of 1%, naCl with the molar concentration of 900mM and sodium citrate with the molar concentration of 10mM, and the pH value of the prepared hybridization buffer solution is adjusted to 6.5.