WO2018084298A1

WO2018084298A1 - Method for rapidly detecting structural anomaly in chromosome, promoter, and kit including said promoter

Info

Publication number: WO2018084298A1
Application number: PCT/JP2017/039999
Authority: WO
Inventors: 雅彦前川
Original assignee: 株式会社Ｇｓｐ研究所
Priority date: 2016-11-04
Filing date: 2017-11-06
Publication date: 2018-05-11
Also published as: JP2018068268A

Abstract

The present invention provides a method for rapidly detecting a structural anomaly in a chromosome. In this method, a mixture is used for a labeled probe that bonds with a nucleic acid. Said mixture is dissolved in a liquid that contains: one type selected from the group consisting of betaine, carnitine, and trimethylglycine; or a combination thereof, i.e., two or three types simultaneously. The present design allows the time required for diagnosis to be markedly reduced because it is possible to stain targeted chromosomal DNA in an interphase cell and to perform detection such that the time required to determine whether or not there is a structural anomaly in a chromosome is reduced from 16-72 hours to within four hours.

Description

Method for rapidly detecting chromosomal structural abnormality, promoter and kit containing the same

The present invention relates to a method for detecting a structural abnormality of a chromosome, an accelerator, and a kit containing the same. More specifically, the present invention relates to a method for rapidly detecting a structural abnormality of a chromosome using a labeled probe that binds to a nucleic acid, a hybridization accelerator, and a kit containing the same.

It is known that structural abnormalities of chromosomes occur due to genetic diseases or cancer. There are several types of chromosomal structural abnormalities, such as chromosomes that are individually excess or lack, partial excess or lack of one chromosome, breaks, rings and chromosome rearrangements. Also among chromosomal rearrangements are translocations, dicentrics, inversions, insertions, amplifications and deletions. These chromosomal abnormalities can be detected by in situ hybridization.

Conventionally, for example, as described in Reference 1 below, a probe related to a gene translocation associated with a chromosomal structural abnormality was dissolved in a solution based on formamide and dextran sulfate and used for in situ hybridization. However, it took about 16-72 hours to stain the target chromosomal DNA in interphase cells and determine whether or not there is a structural abnormality of the chromosome.

Therefore, there has been a demand for a method, an accelerator, and a kit that can shorten the time required for detecting an abnormal chromosome structure by in situ hybridization.

Japanese Patent Application No. 2009-110602

The present invention has been made in view of the problems of the conventional examples as described above, and the purpose thereof is a labeled probe that binds to a nucleic acid in order to reduce time so that a genetic structural abnormality can be detected quickly. A method, an accelerator, and a method for detecting a structural abnormality of a chromosome using one or a combination selected from the group consisting of betaine, carnitine and trimethylglycine, ie, two or three simultaneously added hybridization solutions To provide a kit.

The present invention relates to a method for detecting a chromosomal abnormality, a hybridization solution in which one or a combination selected from the group consisting of betaine, carnitine and trimethylglycine is added to a mixture of a plurality of nucleic acid probes, that is, two or three at the same time. In order to solve the above-mentioned problem, the time required to stain the target chromosomal DNA in interphase cells and to determine whether or not there is a chromosomal structural abnormality is greatly reduced. is there.

That is, according to this specification, the following invention is provided.
(1) A method for detecting a genetic abnormality having an abnormal chromosome structure, wherein a plurality of nucleic acid probes are dissolved in a hybridization solution containing one or more selected from the group consisting of betaine, carnitine, and trimethylglycine. A method for detecting a chromosomal structural abnormality, characterized by hybridizing with the mixture obtained.

(2) The method according to (1), wherein polyethylene glycol is further added to the hybridization solution.
(3) A chromosomal structural abnormality characterized in that in (1) or (2), a target chromosomal DNA in an interphase cell is stained to quickly determine whether or not a chromosomal structural abnormality exists. How to detect.

(4) In any one of the methods (1) to (3), staining a target chromosomal DNA in an interphase cell and quickly determining whether or not there is a structural abnormality in the chromosome. A method for detecting a structural abnormality of a characteristic chromosome.
(5) In in situ hybridization, a method of promoting hybridization by adding one or more selected from the group consisting of betaine, carnitine, and trimethylglycine to a hybridization solution.
(6) The method according to (5), wherein polyethylene glycol is further added to the hybridization solution.
(7) A hybridization promoter for in situ hybridization, containing one or more selected from the group consisting of betaine, carnitine, and trimethylglycine.
(8) The hybridization accelerator according to (7), wherein polyethylene glycol is further added to the hybridization solution.
(9) A kit comprising the hybridization accelerator according to (7) or (8).

Action

The present invention operates as follows.
Hybridization is promoted by adding one or more combinations selected from the group consisting of betaine, carnitine, and trimethylglycine to a mixture containing a plurality of nucleic acid probes, that is, two or three types at the same time, and interphase cells The time required for staining the target chromosomal DNA and determining whether or not there is a chromosomal structural abnormality can be greatly reduced. Actually, spots start to appear 15 minutes after the start of hybridization, and then increase after 60 minutes and 90 minutes. After 120 to 240 minutes, spots almost equal to those after 24 hours can be observed. On the other hand, when a control hybridization solution to which none of betaine, carnitine or trimethylglycine was added was used, no spots were observed until 240 minutes, and spots were first observed after 24 hours.

According to the present invention, the time required to stain a target chromosomal DNA in an interphase cell and determine whether or not a chromosomal structural abnormality exists is reduced.

FIG. 1 is a schematic diagram showing a state of hybridization between normal cells and abnormal cells. FIG. 2 is a diagram showing a change with time of the detection result of hybridization when the hybridization method of the present invention is used. FIG. 3 is a diagram showing a change with time of the detection result of hybridization when the control hybridization method is used.

The present invention relates to a method for increasing the efficiency of chromosome in situ hybridization, an accelerator, and a kit containing the same. The present invention is characterized in that the time required for hybridization can be shortened by adding one or more selected from the group consisting of betaine, carnitine, and trimethylglycine to a hybridization solution used for in に situ hybridization. is there.

The amount of betaine, carnitine, or trimethylglycine added to the hybridization solution of the present invention is not particularly limited as long as it promotes hybridization, but preferred lower concentrations are 10 μM or more, 100 μM or more, 1 mM or more, 10 mM or more, 50 mM or more. The upper limit concentration is preferably 1000 mM or less, 500 mM or less, or 300 mM or less.

The hybridization rate can be further improved by adding polyethylene glycol to the hybridization solution of the present invention. The polyethylene glycol is not particularly limited as long as hybridization is promoted. For example, polyethylene glycol 200, polyethylene glycol 4000, polyethylene glycol 6000, polyethylene glycol 8000, and the like are preferably used. In this case, the lower limit of the preferred concentration of polyethylene glycol to be added is 0.01% or more, 0.1% or more, 1% or more in terms of weight%, and the upper limit of the preferred concentration is 10% or less, % Or less and 2% or less. When polyethylene glycol is added together with betaine to the hybridization solution, it is preferable to add both at the same concentration (% by weight).

The hybridization solution used in the present invention is not particularly limited as long as it is a hybridization solution that can be used for in situ hybridization. For example, in standard hybridization, 50% formamide is often used. In the present invention, one or more selected from the group consisting of betaine, carnitine, and trimethylglycine are added to a standard hybridization solution. Further, polyethylene glycol may be added.

In the present invention, when formamide is added, it is preferable to use a concentration of 10% to 50%. The optimal formamide concentration to improve the hybridization rate is optimal in in situ hybridization experiments depending on other factors, i.e. the concentration of betaine, carnitine and / or trimethylglycine added, the concentration of polyethylene glycol, etc. The concentration can be determined.

When betaine, carnitine, and / or trimethylglycine is added, and when polyethylene glycol is further added, the preferred formamide concentration is not particularly limited as long as in situ hybridization is appropriately performed, but the upper limit of the preferred concentration is 50% or less, 40% or less, 35% or less, 30% or less, and the lower limit is 10% or more and 15% or more.

The first step in carrying out the method according to the present invention is to isolate chromosome-specific DNA. Isolating a sufficient amount of a particular chromosome type or chromosome subregion from which a staining composition is derived, and then extracting DNA from the isolated chromosome or chromosome subregion. The extracted DNA is used to form a DNA insert by cloning using standard genetic engineering techniques. This step can be performed, for example, by positional cloning.

DNA can be isolated from several sources. Chromosome-specific staining reagents can be prepared from plant or animal DNA. Important sources of animal DNA are mammals, particularly primates or rodents, in which primate sources are, for example, humans or monkeys, and rodent sources are, for example, rats or mice is there.

To search for a clone containing a specific gene region from a recombinant DNA library for each of the human chromosomes, see, for example, Human BAC library: construction and rapid screening. Gene 1997; 191: p69-79. The method can be used.

The DNA extracted from the selected chromosome type is amplified by cloning the DNA in a vector or growing the DNA in a cell line.

A single copy probe consists of a nucleic acid fragment that is complementary to a single copy sequence contained in the target region of the genome. In order to prepare such a probe, a DNA library formed by cloning a target region may be used. The clones in the library can include those that contain DNA whose entire sequence is a single copy, those that contain repetitive sequences, and those that have single copy sequences and portions of repetitive sequences. By selecting individual clones and pooling these clones containing only a single copy sequence, probes are obtained that specifically hybridize to the target region. For hybridization, a group of probes that bind to a series of continuous regions may be used.

Whether the hybridization method of the present invention is performed only with the target chromosome, that is, the specificity of the hybridization can be confirmed by testing by in situ hybridization, which is a known method. Clone DNA can be used as a probe if the nucleic acid fragment, dissolved in a solution containing betaine under appropriate hybridization conditions, binds to a specific single copy or repeat sequence as the desired target region.

In these methods, the hybridization rate of the same strand increases as the concentration of the complementary nucleic acid strand increases. Thus, when a probe mixture of nucleic acid fragments is denatured and incubated under conditions that permit hybridization, sequences present at high concentrations become duplexed more quickly than in other cases. Double-stranded nucleic acids in which probes that have not bound to the chromosomes are hybridized are then removed by washing. The rest can be used as probes. The partially hybridized mixture can be used as a probe, but the duplex sequence cannot bind to the target substance. The following method is a typical method of the population fixing method effective for forming the target-specific staining according to the present invention.

The double-stranded probe nucleic acid dissolved in the hybridization solution containing betaine is denatured and then incubated for a sufficient time for high-copy sequence under hybridization conditions to substantially become a double-stranded sequence. A hybridization mixture dissolved in a solution containing betaine is then applied to the sample. Remaining labeled-heavy chain copies of highly repeated sequences bind throughout the sample and form a weak and widely dispersed signal. The diverse binding of specific low copy sequences for the target region of the genome creates a specific signal that can be easily distinguished.

This method includes, for example, a probe having a concentration range of 1-10 ng / μl when a chromosome-specific library for chromosome 17 is used as a chromosome probe. A hybridization mixture obtained by dissolving a probe in a hybridization solution containing one or more selected from the group consisting of betaine, carnitine, and trimethylglycine can be used, for example, at 75 ° C. to denature the probe before being applied to the sample. And then incubated at 37 ° C. for 5, 15, 30, 60, 90, 120, 240 minutes and 24 hours. In this case, when a hybridization solution containing one or more selected from the group consisting of betaine, carnitine and trimethylglycine is used, a hybridization signal begins to appear 15 minutes after the start of hybridization, and after 90 minutes, After minutes, the number of signals increased, and after 240 minutes, hybridization signals almost equivalent to those obtained when hybridized for 24 hours were detected (FIG. 2). In contrast, when a hybridization solution containing neither betaine, carnitine, or trimethylglycine was used, no hybridization signal was detected 15 to 240 minutes after the start of hybridization, and the hybridization signal was not detected until 24 hours later. Was detected (FIG. 3).

The characteristic of “probe labeling” is that the probe bound to the target substance can be detected. The probe nucleic acid fragment can incorporate a labeling substance (for example, a labeled nucleotide) into a polynucleotide by a nick translation technique which is a standard genetic engineering technique.

染色体 Prior to in situ hybridization, the chromosomes are treated with a reagent that removes the protein. For example, proteins can be removed from chromosomes by using FFPE FISH Pretreatment kit 2 (sold by GSP Laboratory) and processing according to the manual attached to the kit.

After the chromosomal DNA has been denatured, the degeneration factors are typically removed prior to application of the probe mixture. Here, formamide and heat are the main denaturing factors, and their removal is conveniently accomplished by several washes with solvents. Such solvents are often cooled, such as the 70% and 100% cold ethanol series. The composition of the denaturing material can be adjusted as appropriate for in situ hybridization, either by adding other components or washing with an appropriate solvent. The probe and target nucleic acid are denatured simultaneously by application of the hybridization mixture and subsequent appropriate heating.

The physicochemical conditions of the chromosomal DNA and probe atmosphere during the time the probe mixture is applied are related to hybridization conditions or annealing conditions. The best conditions for a particular application can be adjusted by controlling a number of factors, including the following: Concentration of components, incubation time of chromosomes in the probe mixture, concentration of nucleic acid fragments that make up the probe mixture, complexity, and length. In general, hybridization conditions should be close enough to the melting temperature (Tm) to minimize non-specific binding. On the other hand, such conditions are not as stringent as reducing the exact hybridization of complementary sequences below detection levels or requiring excessive long incubation times.

The concentration of nucleic acid in the hybridization mixture is an important factor. Such concentrations must be high enough so that sufficient hybridization of each chromosomal binding locus occurs within a reasonable time (5-240 minutes). Concentrations higher than those required to obtain a sufficient signal should be avoided, thereby minimizing non-specific binding. An important practical constraint on the concentration of nucleic acids in the probe of the probe mixture is solubility. There is an upper limit on fragment concentration, eg, unit length of nucleic acid per unit volume, which can be retained in solution and hybridizes effectively.

Selection of a clone library containing the HER2 gene DNA fragments from a human chromosome-specific library can be obtained from an independently constructed BAC library.

A number of recent studies have revealed the presence of structural and numerical chromosomal abnormalities that are useful in diagnosing the phenotype of a particular disease and provide clues to the genetic nature of the disease itself. Today's progress to reveal specific abnormalities in new types of tumors is limited by the difficulty of creating representative high-quality banded metaphase spreads for cytogenetic analysis. These problems stem from the fact that many human tumors are difficult or impossible to grow in incubators. Therefore, it is usually difficult to obtain dividing cells. Even if the cells can grow in the incubator, there is a considerable risk that the growing cells are not representative of the tumor population. This difficulty also hinders the application of existing genetic knowledge to clinical diagnosis and prognosis.

The present invention overcomes these limitations by allowing rapid detection of specific structural and numerical abnormalities in the interphase nucleus. These anomalies are detected as described above. As the genetic nature of specific malignancies is increasingly well known, interphase assessment analysis can be made more specific by selecting hybridization probes that target genetic disorders. . Specific structural abnormalities on selected chromosomes can be detected by the use of an exact hybridization probe. The use of these probes allows diagnosis of specific disease phenotypes. They can be used to follow the reduction and reappearance of malignant cells during treatment. In such applications, interphase analysis is particularly important because there may be a small number of cells that may be present and they may be difficult or impossible to stimulate and mitose.

Processes associated with loss of duplication and deletion, gene amplification and heterozygosity can also be rapidly detected in the metaphase and interphase using the techniques of the present invention. Such a process is associated with an increase in the number of different tumors.

Example 1
Breast cancer tissue HER2 gene and chromosome 17 marker probe Probes of HER2 gene (white circle in Fig. 1) and chromosome 17 marker (black circle in Fig. 1) that have been used to analyze gene translocation associated with chromosomal abnormalities (Patent No. 5554008) was used to analyze the genetic structure (numerical) abnormalities of the HER2 gene and chromosome 17 marker produced in breast cancer cells. Normal cells can detect two white signals of the HER2 gene and two black signals of the chromosome 17 marker, but when they become breast cancer cells, the number of both signals changes (Fig. 1).

Hybridization solution to which betaine was added (based on betaine 300 mM, dextran sulfate 20%, formamide 30%, sodium chloride 600 mM, trisodium citrate dihydrate 60 mM, 0.1% Tween-20, 0.1% SDS % Means weight%, and so on) and conventional hybridization solutions based on formamide and dextran sulfate (20% dextran sulfate, 30% formamide, 600 mM sodium chloride, trisodium citrate dihydrate) And a solution based on 60 mM, 0.1% Tween-20, and 0.1% SDS). The probe was heated on a hot plate at 75 ° C. for 5 minutes to denature, then incubated at 37 ° C. for 5, 15, 30, 60, 90, 120, 240 minutes and 24 hours. Wash 2X SSC / 0.3% Np-40 at room temperature for 5 minutes, 2X SSC / 0.3% Np-40 at 72 ° C ± 1 ° C for 1-2 minutes, 2X SSC at room temperature for 5 minutes, and compare with 1500ng / ml DAPI Counterstain Staining was performed by chromosomal and nuclear DNA staining. A comparison of changes over time in the hybridization signal is shown in FIGS. When betaine was added, a hybridizing signal started to appear after 15 minutes, and after 120 minutes, a hybridization signal comparable to that after 240 minutes and 24 hours was detected. On the other hand, when hybridization was performed using a conventional solution based on formamide and dextran sulfate without addition of betaine, it was high after 60 minutes, 90 minutes, 120 minutes, and 240 minutes. The hybridization signal was hardly detected, and the signal was detected for the first time after 24 hours. From the above results, it can be seen that the addition of betaine to the hybridization solution promotes the hybridization reaction about 6 to 12 times.
More specifically, in the case of the conventional method in which betaine is not added, a fluorescence signal indicating hybridization was observed 24 hours after the start of hybridization after the addition of a probe, but 5 to 240 minutes after the start of hybridization. Then, no fluorescence of hybridization was observed. However, in the method of the present invention to which betaine was added, fluorescence emission was observed 5 minutes after the start of hybridization, and fluorescence having the same intensity as that after 24 hours was observed 15 minutes later. From 90 minutes to 120 minutes after the start of hybridization, approximately the same number of fluorescent signals as 24 hours later were observed. In addition, there were many parts where the fluorescence was clear and the outline of the fluorescent part was clearly visible as compared with the group without addition of betaine.

Further, in-situ hybridization was performed using a hybridization solution obtained by adding polyethylene glycol to the betaine-added hybridization solution, and the hybridization signal was examined by visual inspection. Compared with the case where a hybridization solution containing only betaine was used. In the case of using a hybridization solution to which betaine + polyethylene glycol was added, a stronger signal was obtained in a shorter time. This is considered to mean that the hybridization reaction is further promoted by adding both betaine and polyethylene glycol.

The present invention can be used in the medical industry, the inspection industry, and the like.

Claims

A method for detecting a genetic abnormality having an abnormal chromosomal structure, wherein a plurality of nucleic acid probes each labeled with a different label are one or more selected from the group consisting of betaine, carnitine, and trimethylglycine. A method for detecting a structural abnormality of a chromosome, wherein hybridization is performed using a mixture dissolved in a hybridization solution.
The method according to claim 1, wherein polyethylene glycol is further added to the hybridization solution.
3. The method for detecting a chromosomal structural abnormality according to claim 1 or 2, wherein the target chromosomal DNA in the interphase cell is stained to quickly determine whether or not a chromosomal structural abnormality exists.
The method according to any one of claims 1 to 3, wherein the target chromosomal DNA in the interphase cells is stained to quickly determine whether or not there is a structural abnormality in the chromosome. A method to detect structural anomalies.
In in situ hybridization, a method of promoting hybridization by adding one or more selected from the group consisting of betaine, carnitine, and trimethylglycine to a hybridization solution.
The method according to claim 5, wherein polyethylene glycol is further added to the hybridization solution.
A hybridization promoter for in situ hybridization containing one or more selected from the group consisting of betaine, carnitine, and trimethylglycine.
The hybridization accelerator according to claim 7, wherein polyethylene glycol is further added to the hybridization solution.
A kit comprising the hybridization accelerator according to claim 7 or 8.