JPH06507318A - Chromosome structure and translocation detection methods - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Chromosome structure and translocation detection methods The present invention can target chromosomal DNA that may occur at any location in any chromosome. This invention relates to a method for detecting sites characterized by genetically significant rearrangements in a sequence.

The present invention further provides targeting first and second labeled probes in regions adjacent to said site. a method comprising applying the probe to a nucleic acid, wherein the probe is a chromosomal DNA of interest; Contains a DNA sequence that is complementary to the A sequence. One key element of the invention is the labeling The probe DNA then interacts with the first and second molecules by chemical diffusion. specifically associates with a second interdependent signal-generating moiety to generate a detectable signal. Is Rukoto. By addition of reagents, the first and second is induced to generate a signal, which is then optically detected to see if the signal is present. put out

The present invention also provides an alternative method for both reviving faded markers and confirming previous results. It also concerns the identification of fluorescent labels.

Background of the invention Chromosome structure is closely correlated with the mode and mechanism of gene expression in normal cell function. ing. Importantly, preservation of chromosome structure during cell division is preserved from cell to cell and This is necessary for the transmission of genetic information from generation to generation. However, Chromosome structure often undergoes changes, predicting problems in gene function.

Multiple chromosomal structural changes often occur simultaneously and are associated with many congenital genetic disorders and vines, which can cause degenerative diseases including some types of cancer. Such changes are total staining may take the form of the addition or absence of a body or the addition or absence of some chromosomes . Chromosomes can also be rearranged by translocations, where different chromosomal regions are joined together.

Hosts with other genetic defects including inversions, amplifications and complete deletions alone or The above defects occur jointly.

Some global chromosomal changes are detectable as diseases. addition of chromosomes or Absent changes can occur, for example, in Down syndrome (extra chromosome 21), Turner syndrome, etc. syndrome (deficiency of X chromosome in females) or Klinefelter syndrome (XXY chromosome deficiency) ) will cause Changes in some chromosomes can occur, for example, in chronic myeloid leukemia (C ML) and acute lymphocytic leukemia (ALL) (both chromosomes 9 and 22) It is caused by the presence of the so-called Philadelphia chromosome, which contains a translocation between the number chromosomes. believed to be able to cause

Karyotype analysis is currently used in the diagnosis of the aforementioned diseases. The karyotype is essentially the individual's The number and characteristics of chromosomes. Conventional karyotype analysis uses metaphase chromosomes and This is done by staining and visualizing characteristic patterns (called bands). be exposed. For example, ACT Cytogenetics Laboratory Manual, 2nd edition, page 222, Edited by Margaret J, Barch (1991), Raven Pres. s Ltd., New York, New York.

Such “banding analysis” allows for successful “metaphase elongation” of chromosomes from cultured cells. work with non-compromising cell types such as those present in some tumors because they are difficult to obtain (a problem that is particularly difficult to overcome when Karu. Band staining patterns (especially of abnormal chromosomes) are difficult to classify due to translocations. It would be difficult. Banding techniques are (a) intensive and time-intensive techniques performed by highly trained analysts; and (b) 3-15 methods depending on the particular defect. Changes in chromosomal regions of less than one base cannot be separated [Landegrene et al. et al., 5science, 242:229 (1988)]. It is indeed limited, as seen in other drawbacks. The present invention provide a way to overcome these limitations.

Recently, the aforementioned staining and banding techniques have been introduced into automated karyotyping systems. more facilitated, it is the burden of labor of a trained analyst (but not attentiveness) ). Such a system can detect chromosomes under the field of light microscopy. It is operated by identifying and organizing chromosomes based on their appearance.

Due to variable staining and chromosome bundling, trained cytogenetics The person must summarize all the results thus obtained. in this field In their current state, such systems are expensive and remain highly sophisticated to operate. We need trained analysts. Therefore, such analysis becomes easier and more efficient. It is hoped that this can be done more economically. The direct application of the present invention is to a simple optical microscope. It requires only a microscope and can be adapted to already available automation. Also, analysis The standards of training and judgment required of individuals are also significantly lowered.

Methods of karyotyping have improved, and recently in situ hybridization techniques and It takes the form of combined recombinant DNA methods. Purification D using the in-situ method Discrete nucleic acid probes obtained from NA “libraries” are used to probe specific locations on chromosomes. It has been used to create maps of locations. in fixed chromosomes or free probes. When DNA is “denatured” or in a form that has been disentangled from its normal duplex or double stranded form A hybridization process takes place. The resulting single paper nucleic acid probe array is stained Because it will only “renature” its exact complement in the body. , thus binding to a specific location. Typically such probes are labeled with a radioactive isotope. detected and localized in the chromosomes, where it can be visualized using autoradiography techniques. Wear.

Alternatively, if such probes are labeled with habten or antigen, localized using a fluorescent stain linked to the body and observed under a microscope that allows observation of fluorescence .

This latter method is known as fluorescence in situ hybridization (hereinafter FISH). (e.g. Gray et al., EPO Publication No. 0430402A2) checking).

Both of the above techniques have the advantage of being sequence-specific over chemical stains. . This applies to specific whole or partial chromosomes or specific genes on chromosomes. This means that knowledge of the corresponding DNA sequence will lead to high-resolution chromosomal analysis. I'm tasting it. Due to the potential risk of using hazardous radioactive materials, thousands of samples were In a processed clinical diagnostic environment, the use of radioactive probes has been eliminated.

This eliminates the need for band patterns for identification and for analyst training. Sequence-specific staining has many advantages, including less stringent conditions. I can't be there.

The major drawbacks that still remain are: (1) In cytochemical banding, etc. (2) Fluorescence in FISH cannot distinguish between translocations involving small regions of chromosomal material; For optically labeled probes, expensive fluorescence optics are required in the microscope, and (3) A unique technique that uses certain sensitive stains without disrupting chromosome morphology, which is necessary for karyotyping. It's the difficulty. Therefore, in situ methods have matured into routine and particularly useful clinical tools. In order to become such, it is necessary to address the above-mentioned drawbacks. The present invention does not have such limitations. An important new method to specifically and accurately label such chromosomal translocations In addition, the microscope requires only relatively simple phase optics.

DNA profiling is used to analyze DNA junctions caused by rearrangements, inversions, etc. Methods using probes are known in the art. The most common method is to use that method. It utilizes sequence-specific probes that require DNA sequence information for Its application is very limited. See, for example, the following references:

Carr, EPOO246864 has 3 Ds located complementary to the target sequence of interest. Using a NA partial hybrid probe, two probes are bonded to each other and have high thermal stability. A “spring” that produces a detectable signal or other distinguishable physical property in the form of stranded DNA. Discloses a method of forming a cut probe. But again this method applies First, you need to have accurate information about the area in question.

Weismann (U.S. Patent No. 4,710,465) may span between 20 and 2000 kilobases for use in locating genes in The preparation of a ligated fragment DNA probe is described. Same as before (, this method requires prior knowledge of the genetic region of interest before being applied, resulting in Not useful for analysis of color body structure.

5tephenson (U.S. Pat. No. 4,681,840) is a so-called filler. Used to test for translocations in the Delphia chromosome (Ph' between chromosomes 9 and 22). Discloses a discontinuous DNA probe specific to human chromosome 22 for use in There is. This probe DNA is used for Southern blotting diagnostic applications; Due to its low complex-forming ability, it cannot be used for in situ chromosome analysis.

Methods for combining two different enzyme activities to produce a detectable signal are known in the art. has been known for a long time.

UI Iman (EPOO230768) is bound to the selected enzyme. The formation of so-called complementary specific binding pairs (or “sbp”), which Discloses a method for separating substances from a liquid medium, comprising determining the presence of aggregates. . An enzyme linked to sbp that interacts to produce a measurable signal or product sbp is detected by a signal generation system including a combination of. DNA-DNA or Although it has been described as an sbp capable of DNA-RNA hybridization, the chromosomal structure The application of interacting enzyme pairs for use with DNA probes for Not listed.

Similarly, Litman (U.S. Pat. No. 4,275,149) describes the antigen-antibody relationship. describes the use of enzyme-particles and enzyme-sbp complexes in Here the enzymes are selected to form a signal generating system. Litman's patent relies on the presence of the analyte as part of the detection and signal generation scheme; Assays have been described that utilize conjugated enzyme systems. however, There is no teaching or suggestion regarding the use of sbp in conjunction with labeled DNA probes. It has not been.

This book describes how to apply DNA probes in conjunction with immunology-based signal targeting techniques. Known in the field. Gray et al, (EPO application no. 90308718 ．． No. 7) is a “direct” fluorescent label containing highly complex DNA sequences from individual human chromosomes. Disclosed are methods and compositions for chromosome-specific staining using probes.

A specific application is the FISH treatment of two different radiated regions of the chromosome Detection of chromosomal translocations by microscopic detection of (color) fluorescent signals is required, but only However, the generation of fluorescence is not dependent on the presence of transposition sites.

Wiegant et al [Nucleic Ac1ds Res, 19° 3237 (1991)) is a nick transresponse method for producing fluorescently labeled human nucleic acid probes. discloses the use of fluorescent-dUTP in a fusion method. The probe is human It has been used for in situ hybridization of metaphase chromosomes, and also for fluorescent Also as a target for cellular immunological promotion using anti-fluorescent antibodies with photolabels act. However, regression using visible pigment production, as taught in this application, Nothing is taught regarding the enhancement of fluorescent signals.

Fluorescent labeling techniques solve some of the problems of karyotyping and are compatible with current banding methodologies. improvements, but still fall short of providing a simple conditional test for dislocations. First, it requires the microscope to be equipped with an expensive fluorescent device. Furthermore, using fluorescent labels The obvious disadvantage (versus visible dye labels) of such fluorescent signals is that It is an inevitable disappearance.

Combining discontinuous NA probes with immunological targeting and interacting enzymes Methods for generating such signals are known in the art. Taub (U.S. Patent No. 4,8 No. 20.630) is a concomitant that can enzymatically or physically interact to generate a signal. The use of discontinuous NA probes with labels is disclosed. Discontinuous DNA Lobes are used for the presence or absence of restriction sites or “biologically significant regions”. It is described as specifying the sequence to be analyzed. However, this The target sequence shown in their example is not a chromosome, and the DNA probe is vaguely High complex-forming capacity required for analysis of defined genetically important regions do not have. Additionally, the teachings of this patent require that "one or two" discontinuous marks be DNA probes have been identified, and “biologically important regions” have been identified for restriction mapping and is clearly operative in cases that are not already well characterized by sequence analysis. The general purpose of the present invention is to detect normal and altered regions of chromosome structure by signal generation. In this method, the in-situ technique is applied to Used in combination with regionally localized reagents.

It is an object of the present invention to combine in situ techniques with reagents that localize to said altered regions. Together, we developed a method to detect changes in chromosome structure through direct signal visualization. It is to provide.

A more specific object of the present invention is that regions of different chromosomes are linked and often associated with diseases. Provides a diagnostic method for chromosomal abnormalities such as translocations that occur or are thought to occur That's true.

The purpose of the present invention is to identify genes characterized by the occurrence of genetically significant transpositions in target chromosomal DNA sequences. detect the site where the site has been deleted (the site may occur at any location in any chromosome). This can be achieved by putting out. In the present invention, the following steps are applied = (a ,) the first protein in the first and second regions adjacent to the site of the target nucleic acid. lobe and a second probe, where the first probe is coupled substantially all of the chromosomal DNA having a first label and of said first contiguous region; contains DNA sequences of high and moderate complexing potential that are complementary to the A sequence and and the second probe can bind to the first region of the bound second target. and substantially all of the chromosomal DNA sequence of said second contiguous region. comprising complementary high and moderate complex-forming DNA sequences; Can be combined with a region.

(b) Two W-labeled products of (a) in the first and second mutually dependent signal generating units. The first interdependent signal generating moiety (ISPM) is contacted with an immunological means capable of specifically binding to said first label; said second interdependent signal; The developmental moiety (ISPM) is capable of specifically binding to said second moiety by immunological means. , where the first part and the second part interact by diffusion of a chemical substance. generates a detectable signal.

(C) A region where a genetically significant event has occurred by inducing the first and second regions. adding a reagent containing a chemical that can generate a detectable signal at the position; and (d) optically detecting the presence or absence of said signal;

The present invention provides methods, reagents, and compounds for in situ detection of chromosomal translocations. do. Reagents are used to detect most or all of the chromosomes or chromosomal regions to be detected. Essentially complementary to the region. High or medium unhybridized Contains a probe DNA sequence that has the ability to form a complex. Complex form of probe DNA Competency refers to the number of non-repeated bases in a sequence. such a professional The whole chromosome paint (or WCPs'+m Imagenetic s, POBox3011. Naperville, llinois 5056 6-7011), which is a covalent bond that can specifically react with immunological reagents. It has multiple labels attached together. The term “Whole Chromosome Paint” means multiple staining. one predetermined (i.e., preselected) staining of the body genome a probe adapted to contact or hybridize to a target, including a body; It refers to a probe composition such as the probe composition of the present invention. Typically, One WCP of the invention comprises one or more predetermined chromosomes. A second WCP is attached to the secondary WCP to allow indirect staining and subsequent detection of the area. Labeled probe DNA useful in the present invention has two essential parts: the polynucleotide. tide moiety and a chemically linked label moiety. Polynuc of probe DNA The leotide moieties are specifically stained with sufficient quality and juxtaposition to serve the purpose of the present invention. Plasmids that can properly localize (i.e., hybridize) to target sequences in the body , cosmids, phagemids, yeast artificial chromosomes (YACs) or other episomes It may be in the NA form as well as in the form of DNA fragments of large or small length.

Suitable probe DNAs are highly to moderately complementary to the chromosomal DNA sequence of interest. It is a whole chromosome paint containing DNA sequence fragments with the ability to form complexes.

The source of DNA sequences used in the present invention may be DNA isolated from a particular chromosome or It is well known to those skilled in the art that libraries of such NAs prepared by known methods are well known to those skilled in the art. However, it is not limited to this. The individual chromosomes from which DNA is isolated are Flow cytometry of micronuclear bodies or somatic DNA hybrids, or individual It can be prepared by a number of standard methods, including direct isolation from metaphase or diaphase cells.

Other sources of such NA are prepared by standard methods and by traditions known to those skilled in the art. sources of material (American type cultures of human or other cloned genetic material) A specific chromosome D available from a collection (e.g. ATCC or other deposit) This is NA's library. Numerous chromosome libraries available from ATCC It is. Representative libraries are as follows: Human chromosome library ATCC number X 57750 X 57734 X 57752 X 57747 Y 57735 ATCC deposit is American Type Culture Collection, 12301° Parklawn River, Pockville, Maryland.

Available from. The present invention provides that such NA sequences may also be used in a variety of ways known to those skilled in the art. It is also contemplated that it may be synthesized in vitro by various enzymatic means. human staining a human chromosome-specific DNA library, which describes the preparation of a human chromosome-specific DNA library; See also the paper entitled Biotechnology 4:537 (1981). I want to be

The DNA used in the present invention can be prepared by methods known to those skilled in the art. isolated from the source of the material. Next, this DNA is sonicated and digested with restriction DNase. , limited house nuclease digestion and extraction of DNA through a small gauge needle. Numerous physical, chemical and or by enzymatic treatment into a heterogeneous mixture of fragments of various sizes. arising The mixture of DNA fragments has a size range of OO-500 base pairs (bp) in length. However, the average size of a suitable fragment is about 300 bp. for these operations As a result, a large number of DNA sequences are complementary to different parts of the chromosome to be detected. provided. Actually, there are not tens of thousands of NAs that are complementary to different parts of chromosomal DNA. However, thousands are provided.

To label these DNAs, the DNA fragments are first labeled with a number of techniques known to those skilled in the art. Chemical means (preferably carbon 4 (C-4) elemental amino acid of the nucleotide base cytosine) A moiety that is derivatized by a transamination reaction of a group and covalently bonded to an appropriate label. Create a DNA fragment with By derivatization, various moieties can be added to the C-4 position of this base. Noamine or diamine compounds are added (hydrazine, ethylenediamine, etc.) alkylene diamines having from 2 to 10 carbon elements, lysine or glutamine certain amino acids, peptides, ether derivatives such as (including, but not limited to, inorganic linker molecules). suitably The DNA fragment contains 5-25% transaminated cytosine residues.

The transaminated DNA sequence is covalently linked to the transaminated DNA sequence. Any compound or agent with a functional group that forms a bond with many labels. combined with habten in addition to any other functionality they may have. It can act as such. Habten is a compound that can be recognized and bound by antibodies. is a chemical species that is not so foreign to living organisms as to cause an immune response. Like that Examples of suitable labels include biotin (which also interacts with avidin and avidin-enzyme complexes). ). Phenyl and phenyl derivatives, caffeine and related compounds, fluorescein, rhodamine and other fluorescent species, mercury or other metals and A series of isoprenoids are listed, including carotenoids, sterols and steroids. However, it is not limited to these. Particularly interesting is Karubokki Cytetramethylrhodamine (CTMR, Molecular Probes.

Inc., Eugene, OR, catalog number C1171), ruboxyfluorescein (CFI, Molecular Probes, Inc. , available from Eugene, OR, catalog number C1311L Theophylline and dinitrophenyl (DNP), which in certain embodiments of the invention being discussed. Typically, transaminated DNA sequences are hyperfunctionalized. 60-80% of the transaminated sites are labeled by the reaction of the labeled labeled compound. .

Alternatively, previously hybridized DNA can be directly attached to a labeled probe. The label also targets the ISPM (enzyme)-antibody complex in the dual enzyme system described above. It acts as a vine. Directly labeled probes refer to a subsequent labeling step using a fluorescent label, etc. It is designed to stain or differentiate target DNA without any What you need Since it only means that it is such a sign or hubten, conventional technology Many of the markers described in can be used in such applications. such implementation In embodiments, peroxygen in combination with added hydrogen peroxide and a color-forming substance Sidase-type ISPM can be used with fluorescent labels, especially carboxytetramethylrhodamine (C TMR) and fluorescein and generally rhodamine, fluorescein, It can be used as a coloring system to target a series of substances including lanbelliferin. The enzyme-antibody conjugate system described above uses a “bleached” fluorescent label as well as a freshly prepared fluorescent label. Acts on optical labels. When applied in this way, antifluorescent labeling systems are Information for re-analysis or confirmation is obtained from the discolored and discolored “FISH-processed intermediate spread materials.” enable recovery. The method described here is also useful for FISH processing mid-term diffusers. It also provides a means of secondary analysis.

In situ hybridization The present invention provides a method for producing a variety of proteins specific for different chromosomes with distinct antigenically active markers. Concerning the use of robes. Such specifically labeled probes are useful for translocation and rearrangement. Hybridizes to chromosomes or chromosomal regions that are associated with each other. In particular, the present invention It leaves the natural chromosomal structure essentially intact and allows adjusted to preserve the physical relationships between the parts of the chromosome or between chromosomes and other cellular structures. “In situ transfer of these chromosome-specific probes to chromosomes from disrupted cells Regarding “thiohybridization”. Terminology “in situ thiohybridization” A hybridization is a probe contact or a hybrid produced between a probe and a target. or hybridization. This term “in situ hybridization” Dization is the denaturation and in situ hybridization of a probe to a target. including a hybrid or probe detection step that is performed after fusion. present invention In , the sample can be adhered as a layer onto the slide surface. this hive Targets of redization can be normal, diseased or malignant humans or others. the chromosomes of animal or plant cells, which undergo metaphase or meiosis or mitosis May be at any stage of division, including living or post-mortem tissues, organs or body fluids (e.g. offspring and egg cells, seeds, pollen, or zygotes, fetuses, chorion or amniotic cells or Embryonic cells, including other germinants: normal, immortalized or from long-term or short-term culture Transformed cells grown in vitro, between different cell types or Wakkanai hives or the differentiation stage of these cells); or, those skilled in the art Individual chromosomes or chromosomal portions or translocated, deleted or damaged chromosomes, which are known to those skilled in the art. Prokaryotic or other cloning vectors cloned in vitro by step comprising a library of such chromosomes propagated or amplified in includes (but is not limited to) semen, blood, hair or other samples including, but not limited to, chromosomes or regions of chromosomes in forensic samples that contain It's not like that.

Prior to hybridization, the labeled DNA sequence is preferably freed from non-specific hybridization. To block hybridization, an excess of the corresponding unlabeled D React with reassociated fractions of NA or unlabeled DNA. This blocking DNA is the entire genome It is used at a concentration of 1-10 micrograms per microliter of DNA and is preferred. The appropriate range depends on the chromosomes being hybridized. Blocking DNA is human placental DNA A or CotIDNA (CorIDNA is Life Technology es.

Gaithersburg, MD, Cat, #5279SA) at Probably. Briefly, CotlDNA condenses all human genomic DNA into less than 400 base pairs. prepared by mechanical shearing to a full average size. change this substance and then rehybridized for a sufficient time to generate a large fraction of highly repetitive DNA sequence duplexes. Breedize. A mixture of two and one paper DNA species is hybridized. A nucleic acid that specifically degrades single-stranded DNA into mono- and oligonucleotides. Treat with Rease S1. Undigested double-stranded Cotl was recovered from this mixture. The invention addresses the problem of translocations and the detection and identification of chromosomal regions that may be associated with translocations. Regarding. The present invention provides for the interoperability of two interdependent signal generating parts (ISPMs). This is done by generating a signal caused by the use of the signal.

An interdependent signal generating moiety (ISPM) consists of a catalyst (usually an enzyme) and multiple groups. interaction when the substrate of one enzyme is the product of the other enzyme. Contains a combination of vine enzymes. The end products of such interactions are usually visible to the naked eye. visible pigments or light signals or reactions that interact with further added components A detectable signal such as a sexual chemical species. Numerous such enzyme substrates and The combination of 981).

In a preferred embodiment, the ISPM has two mutually dependent (i.e. conjugate) ISPMs. It is an enzyme (pled). Particularly interesting enzyme combinations include water peroxide colored substances (dyes or other (indicators) for which hydrogen peroxide can be used to oxidize. peroxide Examples of enzymes produced include glucose oxidase, galactose oxidase, aldehyde oxidase, xanthine oxidase, monoamine oxidase, Dihydroorotate dehydrogenase and L- and D-amino acid oxidizers Examples include, but are not limited to, peroxide-utilizing enzymes Examples include horseradish peroxidase, microperoxidase and catalase is included.

Horseradish peroxidase can be used in addition to or in conjunction with peroxide to It is of particular interest because it utilizes other compounds. In one embodiment The enzyme alkaline phosphatase converts 4-chloro-naphthyl-1-phosphate into Can be converted to 4-chloro-naphthol. Horseradish peroxidase and peracid Upon coupling hydrogen oxide, 4-chloro-naphthol is converted to an insoluble dark purple dye; Identify the problem area.

In other embodiments, horseradish peroxidase is a glucose oxidase. Combine with ze. 0. In the presence of glucose oxidase, β-D-glucose is converted into D-glucono-δ-lactone and hydrogen peroxide. horseradish peruo Oxidase uses hydrogen peroxide and combines with tetramethylbenzidine to produce a dark blue color. In this case as well, clarify the problem site.

In an embodiment of the invention, the aforementioned enzyme is a specific enzyme corresponding to a chromosome-specific probe. complexed with a foreign antibody. When said probe is brought into close proximity, and appropriate reagents is added, a signal visible to the naked eye is generated, which can be detected using a simple optical microscope. can be detected.

Since enzymes are convenient and reliable catalytic ISPMs, there are many (other) biochemical and biophysical systems will be used. formed in such a system The substances detected are typically detectable by well-known means and include fluorescent chromophores, chromophores, etc. , chemiluminescent groups, aromatic compounds and other compounds with properties that facilitate detection. is included.

In an embodiment of the invention, the compound dinitrophenyl (DNP), theophyl phosphorus, carboxytetramethylrhodamine (CTMR) or fluorescein. It is used for labeling DNA probes with a high degree of complex formation ability. single also is a conjugated enzyme containing glucose oxidase and horseradish peroxidase. In combination with elementary reactions, antibodies inhibit the activity of these enzymes by anti-theophylline, anti-di Nitrophenol, anti-CTMR or anti-fluorescein immunoglobulin (Ig G) to target the hybridized probe with. Such reagent enzymes are , they act dependently on each other in a conjugated system and independently in a single system, and the visualized Easily generate noise.

An important application of the invention is in chromosomal translocations, where regions of different chromosomes are linked. How to diagnose color abnormalities (often thought to cause or cause disease) It is to provide a stage. Examples of such diseases are chronic myeloid leukemia (CML) and and acute lymphocytic leukemia (ALL), both of which are so-called Philadelphia It is thought to occur due to the presence of the A chromosome (including translocations between chromosomes 9 and 22). is being given. The break point associated with this and other similar translocations is 150,000 salts. It is known that it occurs in a range larger than that of the group. This makes it widely usable. So-called specific DNA probes with their own such discrete probes cannot be used because it requires the microstructure of the cut point region to target the It is shown that.

The use of WCP in combination with the ISPM of the present invention does not exhibit such drawbacks.

Taking advantage of the wide range of effects of such highly complex-forming DNA probes in diagnostic applications For this purpose, the chromosome sample containing the target DNA must be isolated from the cells of interest (e.g. CML or (ALL) and fixed onto slides using the well-known in situ fixation method. Place it on a solid support. Next, the above labeled DNs corresponding to different chromosomal sequences Two of the A probes are hybridized to immobilized target DNA in the form of a chromosome, followed by Immunological reagents and signal-forming reactions with mutually dependent (coupled) enzymatic activities When treated with a reagent to catalyze the enzyme, the enzymes interact with each other only in the chromosomal region of the translocation. is placed near. Under a light microscope, the chromosome with the translocation is separated from the remaining unstained Appears to have colored or stained segments attached to segments and also corresponds to A complementary pattern appears on the chromosomes.

The use of ISPM conjugated enzymes was demonstrated in two different applications. In the first case, Generation of a detectable signal in a cytohybridization assay illustrates the use of ISPM methodology in WCP probe for chromosome 1 cells were labeled with dinitrophenyl and hybridized to normal lymphocyte metaphase extensions. Ta. After hybridization, add anti-dinitrophenyl goat IgG to the hybridizer. added to the labeled probe. It is then combined with horseradish peroxidase. The anti-goat IgG was reacted with the previously conjugated goat anti-dinitrophenyl antibody.

All prepared samples were then treated with alkaline phosphatase, 4-chloro-naphthol and and perfused with a solution containing hydrogen peroxide. The reaction causes strong black staining on chromosome 1. However, there was very little background staining of other chromosomes.

In a second application example, translocations between chromosomes 1 and 4 can be detected using the present invention. was detected. In this example, the cell line 5upB containing the chromosome 1-4 translocation 13 served as the chromosome source for the preparation of metaphase extensions. Two different whole chromosome pays (WCP) probe was used to detect translocations. WCP for chromosome 1 is Labeled with theophylline, the WCP for chromosome 4 is dinitrophenyl (DNP ). Horseradish peroxidase (HRP) is isolated by immunological means. Similarly, glucose oxidase (GOX) targets chromosome 4. targeted. In this example, a group containing the tetramethylbenzidine reagent is used. The coupled enzyme reaction was initiated by the addition of cose. Glucose oxidase produces guic acid Hydrogen oxide is a by-product of this reaction. Horseradish peroxidase is In the presence of hydrogen peroxide, many soluble colorless products are converted to insoluble dyes. This fruit In the example, tetramethylbenzidine, which is converted to a bright blue dye, is It was used in the assay as a substrate for oxidase. As a result, all of the observed metaphases Two chromosomes were stained. In each case, only part of the chromosome is stained The stained parts corresponded to each chromosome part derived from chromosome 1.

Elements of the invention are normally bleached by re-staining with the coupled enzyme coloring system of the invention. Amplify and restore fluorescent in situ hybridization (FISH)-labeled metaphase slides. It also provides a methodology for original or restoration. Such applications include, for example, Habtest for binding by immunological means of one or more ISPMs that have been The fluorescent label of the previously hybridized probe is used as a marker. the Such a method involves attaching a fluorescent label to a probe that has been previously hybridized. contact with a signal-generating moiety, wherein said moiety is derived from an antigen/antibody or an antibody/antibody pair. directed to the fluorescently labeled chromosome by immunological means; and A reagent containing a second substance is reacted with said moiety, thereby producing a colorless soluble substance. into an insoluble detectable signal, where said signal is in the visible light range and optically Detectable by means.

The following examples describe the methods and reagents used to perform the aforementioned assays. Ru.

Example 1 Human chromosome-specific DNA probe Human chromosome-specific DNA probe VanDilla, M.A. etal (Biotechnology4; 537-552.1986) and as described by Lawre nce Livermore National Laboratories (LLNL) as a recombinant Furi library. these live Rally was amplified by growth in E. coli host strain. Purify the amplified phage , DNA was extracted and this DNA was digested with the restriction enzyme Hindm. insert DNA Purified from lambda vector DNA and transformed into plasmid vector pBS (Strateg ene, La Jolla, CA) into the Hindm site. Obtained E. coli strain DH5α (Bethesda Re5earch L ibraries, Gaitherburg, Maryland) did.

The plasmid library used in this example is ATCC #57738, 5775 3 and 57754 (chromosome 1); and ATCC number 57719.577 18.57745 and 57720 (chromosome 4). Library is 1 The cells were stored as frozen cells in ml portions. Seed storage for fermentation in these vials was used as the primary production source.

Bacteria were grown by fermentation. The seed stock obtained from ATCC was treated with ampicillin (20 0 micrograms/m+), and 8 g/l Bactodributon (Difco) , 5g/l Bacto yeast extract (Di f co), 15g/l Bacto agar (Difco) and 1.6 containing YT broth containing 5 g/l sodium chloride. % agar plates for 24 hours at 37°C. Collect cultured cells, 16g/l Bactodributon (Dirco), 10g/l Bacto yeast extract (Dirco) and 5 g/l of sodium chloride. 4 ml of 20% glycerol was added to the collected solution. E. coli cell culture vial Quickly freeze 5 ml of Q by submerging it in liquid nitrogen and keep it for 180 ml until use. Stored at °C.

350 ml of fermentation inoculum contains 13.2 g of seed culture/]Na2HPO4-7HzO : 3. Og/I KH2P 04.0.05g/lNaC1,1,0g/ lNH4Cl, 10.0g/Icasamino acid (Di f co); 0.03g/ lMg5O<, 0.004g/ICaC12・2Ht0, 3.0g/l glucose 0.025g/I Thiamine HCI, 0.0054g/1FecI, 0. 0004g/1ZnsO4, 0,0007g/ICoCIz, 0.0007g/ INazMoO4,o,oo.

8g/l Cu5Oa, 0.0002g/l) (, BOs and 0.0005g/l) in a 2 liter baffled shake flask in casamino acid medium containing lMn5o. The cells were prepared by culturing at pH 7 and 37°C in water. 350ml culture , 1% glucose, 13.2g/INazHPOt-7HzO13,Og/]K IhPO4,0,05g/lNaC1,1-Og/1NH4C1,10,0g/ l Casamino acid (Dirco), 0.03g/1Mg5O<　, 0.004g /lCaC112Hz0,0.025g/l Thiamine HCI, 0.0054g /lFeC1g, 0.0004g/I ZnSO4, 0,0007g/lCoC 1x, 0.0007g/INazMoOn, 0.0008g/] Cu5O n, 0.0002g/IH! Contains BO3 and 0.0005g/lMn5O 4.2 liters of fermentation medium was inoculated.

Immediately after fermentation is complete, bacterial cells are harvested using a Memplan cell concentrator and high-speed centrifugation. Collected. Fermented cell broth was passed through a 0.45 micron (μm) membrane filter ( 2 square feet) to concentrate from 5 liters to approximately 800 ml. Next, cell concentration Centrifuge the condensate in a refrigerated centrifuge at 7000Xg for 10 minutes, discard the supernatant, and remove the bacterial cells. The cell pellet was collected.

Plasmid DNA was extracted from bacterial cell pellets. cells, cell pellet volume 50 mM glucose (filter sterilized) in 3 volumes (in milliliters) of (M), 10 mM NaEDTA (pH 7,5-8,0) and 25mM Tris-HCI ( pH 8.0). 6 x M (in milliliters) amount of 0゜2　M　N　a　OH and 1% (W/V) sodium dodecyl sulfate (S After adding the solution containing DS), the cells were lysed by vigorously swirling and stirring. . When the solution becomes clear, add 55.5 ml of glacial acetic acid for a final volume of 500 ml. and 147° 4.5XM (ml) of a solution containing 5 grams of potassium acetate. When the quantities were thoroughly mixed, a woolly precipitate formed. Remove the supernatant from the woolly precipitate. Separate and centrifuge this supernatant at 7000Xg for 15 minutes to remove the remaining precipitate. did.

Add 1 volume of ethanol followed by centrifugation at 7000×g for 10 min to remove the supernatant. Precipitate the nucleic acids from the solution and collect the nucleic acid pellet in a total volume of Q, 54xM (in milliliters). resuspended in The nucleic acids are then dissolved in 1/2 volume of neutralized phenol and 1/2 volume of Extracted with loloform and precipitated with 2 volumes of ethanol. Nucleic acid is 0.3XM (Mi 50mM) Lis-HCl (pH 7,0) and 100mM acetic acid Resuspend in a solution of sodium. Then 1 of 0.77XM (in microliters) Add 0 mg/ml RNase (heat treated) and incubate for 30 min at room temperature or at 4°C. I left it overnight to digest. Next, 0.615XM (microliter) of protein A solution of Kase K (20 ml/ml) was added and incubated at 55° C. for 3 hours. D NA was extracted with 1/2 volume of neutralized phenol and 1/2 volume of chloroform; Precipitated with 2 volumes of ethanol.

Resuspend the DNA in 0.415 gM (in milliliters) of water and add 0.05XM in milliliters. liter of 5M NaCl and 0.155 gM ml of 50% (w/V ) Adding polyethylene glycol (PEG) (molecular weight 6000-8000), Incubate on ice water for 1 hour and pellet by centrifugation at 7000×g for 15 minutes. Ta. DNA was dissolved in 0.04XM ml of water and 1/10 volume of 3M sodium acetate. resuspend in 1/2 volume of neutralized phenol and 1/2 volume of chloroform. and precipitated with 2 volumes of ethanol. Purified DNA is 0.0476xM Resuspend in 1 ml of deionized water. DNA concentration determined by fluorimetry did.

Finally, the purified DNA was purified using Branson 5onifLer 450 (Danb about 300 base pairs by ultrasonication using Destroyed into small pieces. This size of the fragment is suitable for in situ hybridization. It has been empirically determined that this is the most suitable DNA probe for use in .

Reconstitute 4 milligrams of purified plasmid DNA prepared above in 2 ml water. Suspend and place in a dry ice/ethanol bath to prevent boiling during sonication. Ta. Insert the sonicator into the solution until the tip is 2-5 mm from the bottom of the tube. I sank the black tip. Ultrasonic treatment at 25-30 watts of power, 80% intermittently Work cycle (on 80% of the time, off 20% of the time) for 5 minutes provided. Following sonication, 0.2 ml of 3M sodium acetate (pH 5,5) and 4 ml of ethanol were added to precipitate the DNA. Sedimentation is 8000Xg The sample was collected by centrifugation for 5 minutes and dried under vacuum.

Example 2 Bisulfite-catalyzed transamination of DNA D obtained by the method of Example 1 NA adds an amino group to the 04 carbon atom of the cytosine base by adding ethylenediamine. Transferred. This reaction is catalyzed by sodium bisulfite. hydrogen sulfite To prepare the salt buffer, add 1.7 ml of fuming HC to 1 ml of deionized water on ice. Add I slowly. Next, add 1 ml of new ethylene diamine (Si gma Log #E-4379) was added slowly on ice. After dissolving ethylenediamine, the solution Warm to room temperature and add 0.475 g of sodium metabisulfite (Aldrich solution). Talog #25,555-6) was added. Then fuming H until the pH reaches 7.0. CI was added slowly. Deionized water was added to a final volume of 5.0 ml. . 1 milligram of sonicated DNA was hooked to transfer amino groups to the DNA. 3ml H! resuspended in O. DNA by boiling at 100°C for 5 minutes was denatured and then quenched in an ice-water bath. Add 0.3 ml of this DNA solution to 2.7 ml Initiate the transamination reaction by adding to the bisulfite buffer of Incubated for 2 days at 37°C. DNA solution is 5-10 mmol Desalination was performed by conventional dialysis against sodium chloride (pH 8.0). After dialysis, transparent 0.3 ml of 3M sodium acetate (pH 5.5) was added to the precipitate. Aminated DN A was precipitated with 2.5 volumes of ethanol and centrifuged at 8000 x g for 10 min. It was later collected. The pellet was vacuum dried and rehydrated at a concentration of 3 mg/ml DNA. . This solution was stored at -80°C until use.

Example 3 Preparation of nitrophenyl derivatives of diamino acids 2.62 g of ε-amino-n - add caproic acid to 20 ml of water containing 40 mmol of sodium bicarbonate In this way, a 20 mM ε-amino-n-caproic acid solution was prepared. This melt Pour the solution into 20ml of Sanger's reagent (2,4-dinitro-fluorobenzene). It was mixed with M solution and left at room temperature for 1 hour. The mixture is then heated gently, causing the The liquid turned yellow and a small amount of dissolved sodium bicarbonate precipitated. A sufficient amount of this precipitate Redissolution by addition of concentrated HCI followed by standing at 4°C induced crystallization. crystal Collected by suction and washed with water, 4.2 g of yellow crystalline ε-dinitrophenylamino -n-caproic acid (DNP-NCA) was obtained.

DNP-NCA was esterified into 3-sulfo-N-hydroxysuccinimide as follows. It was activated by stealthization. 0.594g DNP-NCA, 0.46 8 g dicyclohexylcarbodiimide and 0.434 g 3-sulfo-N- Hydroxysuccinimide was stirred vigorously in 7 ml of dimethylformamide at room temperature overnight. Stirred. Thin layer chromatography shows that this reaction is more than 90% complete. More determined. The mixture was cooled to 0° C. and stirred for an additional hour. Then the mixture Filter and evaporate the yellow solution to a viscous yellow oil (which does not crystallize). Ta). When this oil is stirred with 50ml of ethanol, 0.996g of fine yellow A colored powder was obtained. This was collected by filtration and washed with ethanol. This compound is 6- N-(2,4-dinitrophenylamino)caproic acid-〇-(N-hydroxys succinimide)-3-sulfonate (sodium salt), hereinafter referred to as the object of the present invention. It is called 5-NHS-DNP for its purpose.

Example 4: DNAAll1 identification: DNP-derivatized ε-amino-n-caproic acid Example 1 Chromosome-specific DNA with an average length of about 300 bp prepared by the method of Example 2 ethylenediamine by a bisulfite-catalyzed transamination reaction as described in Derivatized. aminated DNA in a 5 ml plastic centrifuge tube (1 A solution of 00 μg total DNA) was evaporated under reduced pressure. Q, 5ml of 0. 2M3- (N-morpholino) propane sulfonic acid (MOPS) buffer and Shibarako 100 Microliter of 5-NHS-DNP (30mg/ml N,N-dimethyl Formamide) was added to the residue and the mixture was incubated at 25°C overnight. 60 μl of 3M sodium acetate (pH 5,5) followed by 1.5ml of water-cooled ether. The DNP-labeled DNA was precipitated by adding Kol and the mixture was incubated for at least 2 hours. Incubated at 0°C. Add this solution to 10. Centrifuge for 10 minutes at oooxg. Ta. The DNA pellet was washed twice with 5 ml of water-cold ethanol, and 100 μl of Dissolved in a sterile bottle. Two Cefazo Tux G-50 beds with a bed capacity of 0.8ml. A Recto D chromatography column was prepared. Add 50 μl of lysed pellet to each The mixture was applied to each column and centrifuged at 10,000 gg for 4 minutes. 10 μm purification D Dilute NA with 490 μl of 20 mM NaOH and determine optical density at 260 nm. and the DNA concentration was calculated. Dilute the purified DNA with water and add 100 mic/ml. A working concentration solution of DNA was prepared.

Example 5: Theophylline-8-N-(5-hydroxypentylamino)-5,1 6 grams of 8-bromotheophylline and 5.15 grams of 5-amino-1-pene A solution of tantanol in p-xylene (18 ml) was heated under reflux for 18 hours.

The reaction solution was allowed to cool and a solid/liquid mixture formed upon cooling to room temperature. .

The xylene was decanted off and the solids were washed with pentane (3 x 40 milliliter). Tor). The resulting solid was stirred in 20 milliliters of water for 30 minutes and filtered. Solid material was collected. Wash the collected solid material with water (3 x 20 ml) and dry. When dried, 3.73 grams of 8-(5-hydroxypentylamino)theophylline I got it. 1.12 grams of 8-(5-hydroxypentylamino)theophylline in anhydrous N, N-dimethylformamide (DMF) (15 ml) solution. , 0.516 grams of succinic anhydride and 0.2 grams of 4-dimethylaminopi Lysine (DMAP) was added. The mixture was stirred on a magnetic stirrer under anhydrous conditions at room temperature overnight. did. The precipitated colorless solid was collected by suction filtration, washed with dichloromethane and air-dried. and 1.20 grams of theophylline-8-N-(5-hydroxypentylamino )-0-succinic acid ester was obtained. This solid was boiled at a boiling point of 1:1 nitrogen/water. Recrystallization from the mixture and drying over calcium sulfate gave a colorless solid. 0. 725 grams of theophylline-8-N-(5-hydroxypentylamino)- Add 0 to a solution of 〇-cono monophosphate in anhydrous dimethylformamide (15 ml). ．． 414 grams of 3-sulfo-N-hydroxysuccinimide (Su1f o-NHS) was added. 0.488 grams dicyclohexyl carbodi A solution of DCC in anhydrous DMF (2 milliliters) was added to the above solution. profit The resulting mixture was stirred using a magnetic stirrer at room temperature for 16 hours. Water bath the reaction mixture After cooling for 1 hour, dicyclohexyl urea was removed by suction filtration. reduce filtrate Evaporation under pressure (2 torr, 30° C.) gave a viscous colorless oil. this oil The solid material was treated with 40 milliliters of absolute ethanol to precipitate a solid. solid The material was collected by filtration and dried over anhydrous calcium sulfate to yield 0.450 grams of te Ophiline-8-N-(5-hydroxypentylamino)-〇-succinoyl 〇’-(N’-(3-sulfosuccinimidyl)) ester (NH3-theof Irin) was obtained.

Example 5: DNA labeling - theophylline approximately aoo obtained by the method of Example 1 A chromosome-specific DNA probe for human chromosome 4 with an average length of bp was Ethylenediamine was prepared by a bisulfite-catalyzed transamination reaction as described in Section 2. It was derivatized with. Approximately 5% of the base was aminated. 1.5ml plasti Solution of aminated DNA (100 micrograms total DNA) in a microcentrifuge tube was evaporated under reduced pressure. 0.5 ml of 0.2M3-[N-morpholino]p ropanesulfonic acid (MOPS) buffer, then 100 microliters of NH3- Theophylline (26 mg/ml N, N-dimethylformamide ) was added to the residue and the mixture was incubated at 25° C. overnight. 50 microliters of 3M sodium acetate (pH 5°5) followed by 1.5 ml of water-cooled evaporation. Tanol was added and the mixture was incubated at -20°C for at least 2 hours.

The solution was centrifuged at 10,000xg for 10 minutes. 0.6ml of pellets After washing twice with 1 liter of water-cooled ethanol, dissolve in 100 microliters of sterile water. did. Two Sephadex G-50 Selects with bed capacity of 0.8 ml ToD chromatography column (5Ptime->3Prime, Inc.) was prepared. Apply 50 microliters of lysis pellet to each column and ,000×g for 4 minutes. 10 microliters of purified DNA Dilute with 0 microliters of 20mM NaOH and determine optical density at 260nm. to calculate the DNA concentration. Dilute the purified DNA with water and add 1ml per milliliter. A 100 microgram DNA working concentration solution was prepared.

Example 7: DNA label 5-(and-6)-carboxytetramethyl rhodami succinimidyl ester (CTMR) obtained by the method of Example 1. Chromosome-specific DNA profiles for human chromosomes 1 and 4 with an average length of 00 bp ethylene diamine with bisulfite catalyst as described in Example 2. It was derivatized with. Approximately 5% of the base was aminated. 1.5ml plasti Solution of aminated DNA (50 micrograms total DNA) in a microcentrifuge tube The solution was evaporated under reduced pressure. Add 377 microliters of 0.2M3- to this solution. Add [N-morpholino]propanesulfonic acid (MOPS) pH 7.4 buffer Ta. 22.8 microliters of CTMR (5-(and-6)-carboxylate) Tramethylrhodamine succinimidyl ester, N,N-dimethylformamide 50mM in decodylamide) was added to the transaminated DNA and the mixture was incubated at ~25°C overnight. The mixture was stirred for about 1.8 hours. Remove excess fluorescent chromophore from labeled DN by ethanol precipitation Separated from A. Dissolve the precipitated DNA pellet in sterile water, 28cm high, 1c inner diameter. m Sephadex G-25 column.

The desired fraction (column void volume) was eluted with water and dried to reduce the total volume.

Ethanol precipitation of the second DNA was performed to complete the purification.

The transaminated DNA probe obtained by the method of Example 2 was converted into 5-(and- 6)-Carboxyfluorescein combined with succinimidyl ester (CFI) I set it. After drying 50 micrograms of transaminated DNA, 377 micrograms of Resuspend in 200mM MOPS, pH 7.4. 22.8 microphone Lolitol's 50mM 5-(and-6) Carboquine Fluorescein Succini Midyl ester (CFI) N,N-dimethylformamide solution (150x molar excess) was added to the transaminated DNA. This reaction was carried out overnight at room temperature in the dark ( This proceeded by stirring (about 18 hours). First remove excess by ethanol precipitation. The fluorophore was separated from the labeled DNA. Resuspend the precipitate in water and place it at a height of 28 cm. It was passed through a Sephadex G-25 column with a diameter of 1 cm. desired fraction (column box) id volume) was eluted with water and dried to reduce the total volume. Ethano of the second DNA The purification was completed by precipitation. The absorption spectrum shows that 1.6% of the bases are labeled. 5upB13 (CML strain with multiple translocations, #10535) is Mic It was available from 　h　elie　LeBeau, University of Chicago. 8 cells 0% RPMI 1640 medium (Gi bco catalog #320-1875 ), 20% fetal bovine serum, 100 units of penicillin/streptomycin and and reseeding into a 25 cubic centimeter flask containing 10mM HEPES buffer. I planted a seed. Cell proliferation was monitored by counting and cells were re-cultured every 3 or 4 days. . When cells reach optimal concentration, 0.2 ml of 10 micrograms/milliliter Tutor Kolishimed (Gibco Catalog #890-1145-1) for each frame. Add to the flask and keep at 37°C for 1 hour in a 5% CO2 incubator. Ta. Cells were harvested by centrifugation and then reconstituted in 5 milliliters of 75 mM KCI. Suspended and held at 37°C for 10 minutes. Cells were collected by centrifugation again and 0. All but 2 milliliters of KCI supernatant was removed. Disperse the cells, Gradually fix in 10 ml of 3=1 methanol/acetic acid and place on ice for 15 min. held. Collect cells by centrifugation and add 5 ml of fresh methanol/acetic acid. and kept on ice for an additional 15 minutes. Collect cells and reconstitute in methanol/acetic acid. Suspended, added dropwise onto pre-cleaned glass slides and allowed to dry.

Example 10 In situ hybridization using standard protocol The coupling assay was tested by in situ hybridization. Target D NA was present at 5upB13 metaphase on glass microscope slides.

The slides were examined and positioned to contain the nucleus and metaphase extensions. diamond painting The glands outline the hybridization target region and identify markers. on the opposite side of the slide. on the slide before hybridization. Target DNA was dissolved in 70% formamide 10.3M NaCl/30mM sodium citrate. 2-10 minutes at 70°C. I made her have sex. After denaturation, the target DNA was placed in a 70% ethanol/water bath and vortexed. The lumamide solution was removed. The washing steps included cleaning the slides at 70%, 85% and 10%. Repeated by passing 0% ethanol (room temperature, 2 minutes each). hybridize The solution mixture was 50% formamide 10.3M NaCl/30mM citric acid. Consisting of sodium acid, pH 7.0. Human placental DNA as blocking DNA (2, 25 micrograms/10 microliters) was used. Theophylline label W CP1 and DNP-labeled WCP4 at a concentration of 10 nanograms/microliter each. It was added at a Total hybridization volume was 10 microliters. .

Denature the hybridization mixture at 70°C for 5 minutes and remove the target DNA from the slide. added to the area containing. Place the coverslip onto the hybridization mixture. The edges were sealed with rubber cement. Place the slide in a humidified chamber and Hybridization was allowed to proceed overnight at 37°C. Hybridization After cleaning, remove the coverslip and soak the slide in 50% formamide 10.3M N ac1/3mM sodium citrate pH 7.0 at 45°C 3 times (5 times each) (min). Next, each slide was heated to 45°C with 1/3 m of 0.3 M NaC. 5 min in M sodium citrate and 0.1 M sodium phosphate 10.1% Washed in NP40 (PN buffer) for 5 minutes. The slides were then placed in PN buffer at room temperature. Washed twice in liquid (2 minutes each wash). Slides were soaked in PNM buffer (5% delipidized and dried). Anti-dinitrophenol diluted 1:250 in PN buffer (with milk) (Rabbit) for 20 minutes. Slides were placed in PN buffer at room temperature. Washed three times for 2 minutes each. The slides were then exposed to glucose oxidase-anti-rabbit complex. Incubate for 20 minutes in the coalescent and wash again three times in PN buffer. lastly Slides were incubated in horseradish peroxidase-antitheophylline complex for 20 minutes. Incubate three final times in PN buffer (with fresh buffer for each wash step). (used) was washed.

Example 11 Color development and results Commercially available Vecct o r TMB (tetramethylbenzidine) peroxy A modified Dase kit was used for color development. In particular, remove hydrogen peroxide from substrate preparation. 100 microliters of 1M glucose was added instead. prepared and ha Slides containing hybridized metaphase extensions were incubated with chromophore reagent for 1 hour. The cells were incubated, washed briefly with a gentle stream of distilled water, and then air-dried.

TMB-treated metaphase extensions were counterstained with freshly prepared filtered Giemsa for 5 min. Ta. Slides were washed with a gentle stream of distilled water. The slide is then exposed to air or nitrogen. Dry by spraying. The stained metaphase extensions were examined under a microscope.

Among all metaphases observed, at least two chromosomes were labeled.

In each case, only part of the chromosome is labeled, and the labeled part is one part of each chromosome. It corresponded to the part derived from the number chromosome. In some middle-period works, there is a third dye. The chromoplasts were detectably labeled. In each case, this third chromosome is stained #1 It was a normal copy of the body.

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Claims (10)

[Claims] 1. genetic in the target chromosomal DNA sequence that can occur at any location in any chromosome A method for detecting sites characterized by significant dislocation occurrence, the method comprising: the first probe and the second region adjacent to said site of the target nucleic acid; and a second probe, wherein the first probe is attached to the bound first label. and is homologous to substantially all of the chromosomal DNA sequences in the first adjacent region. containing complementary high and moderate complex-forming DNA sequences; and the second probe has a second label attached thereto; and is complementary to substantially all of the chromosomal DNA sequences in the second adjacent region. comprising DNA sequences of high and moderate complex-forming ability that bind to said second region. sell; (b) combining the labeled product of step (a) with first and second interdependent signal-generating moieties; wherein said first interdependent signal generating moiety (ISPM) is in contact with an immune system. capable of specifically binding to said first label by chemical means, and said second label being dependent on each other. The signal generating moiety (ISPM) specifically binds to said second label by immunological means. and the first part and the second part are mutually bonded to each other by diffusion of a chemical substance. (c) inducing said first and second parts; to generate a detectable signal at the site of the genetically significant event. adding reagents containing chemicals; and (d) optically detecting the presence or absence of said signal. 2. The immunological binding means is in combination: (1) conjugated to a first ISPM; a first antibody capable of immunologically binding to the first label, and a second ISPM; a second antibody that is conjugated and is capable of immunologically binding to a second label; or (2) a first conjugated to the first ISPM and capable of immunologically binding to the first label; a second antibody capable of immunologically binding to a second label, and a second ISPM. a third antibody conjugated to and capable of immunologically binding to said second antibody; or (3) a first antibody capable of immunologically binding to a first label, conjugated to a second ISPM; a second antibody capable of immunologically binding to a second label; and a first ISPM. a third antibody conjugated to a third antibody capable of binding to the first antibody; , and the first and second ISPs such that the immunological means generates a signal. A target DN as indicated in claim 1, characterized in that M is juxtaposed A method for detecting the occurrence of genetically significant rearrangements in an A sequence. 3. said first label and said second label are not identical and each: a) a number of xane Chin or lower alkyl substituted xanthine derivatives; b) with one to three nitro groups; a substituted phenyl; and c) a fluorescent compound; 3. The method of claim 2, wherein the method is selected from one of the three groups consisting of: 4. The first and second ISPMs contain a chemical substance essential for generating a detectable signal. is a coupled enzyme system in which a first enzyme can interact with a second enzyme by diffusion of Genetically significant rearrangements in the target DNA sequence, such as those listed in Section 3. How to detect. 5. There are two probes, each corresponding to a site throughout an individual chromosome. A high-complexity whole stain consisting essentially of chromosome-specific labeled DNA fragments. 2. The method of claim 1, wherein the method is body paint (WCP). 6. the first probe is whole chromosome paint, and the second probe DN A yeast autonomous chromosome (Y The method according to claim 1, which is an AC) clone or a cosmid clone. 7. said target DNA sequence is removed from a biological source of interest, and said DNA is It may be in the form of whole nuclei, chromosomes or fragments thereof, naked DNA or fragments thereof, Here, such DNA preserves distinct chromosomal or nuclear morphology identification. the DNA is immobilized on the slide, or the DNA is naked. Claim 1, wherein the fractionation and separation means are attached to a solid substrate after being applied. Detection and occurrence of genetically significant rearrangements in target DNA sequences, such as those shown in and how to locate it. 8. Enhances signal from fluorescently labeled, in situ hybridized chromosomes or a method of replacing the fluorescent label, wherein the fluorescent label is bleached or otherwise Undetectable and below: (a) The fluorescently labeled part of the fluorescently labeled hybridized chromosome is used as the signal generating part. wherein the signal-generating moiety is in contact with an antigen/antibody or an antibody/antibody pair. and (b) a first or second fluorescently labeled chromosome; reacting a reagent containing a second substance with the signal generating moiety, thereby A colorless soluble substance is converted into an insoluble detectable signal, where said signal is in the visible light range. and detectable by optical means. 9. a fluorescently labeled image, wherein the fluorescent label is carboxytetramethylrhodamine; Claims that enhance or replace signals from in situ hybridized chromosomes The method described in paragraph 8. 10. Occurrence of translocations in chromosomal DNA sequences that can occur at any location in any chromosome A method for detecting junctions arising from a target chromosomal DNA sequence, It is in the form of a period extension and is as follows: (a) The first probe and the second probe for the target chromosome are the first and second regions on the side where the first probe is attached to the A whole chromosome paint labeled with filin and hives into the first region of the target. the second probe contains a DNA sequence that can be redized, and the second probe is a dinitrophenyl a whole-chromosome paint labeled with and capable of hybridizing to said second region Contains DNA sequences; (b) The theophylline label was conjugated with horseradish peroxidase anti-theophylline. The dinitrophenyl label was first treated with rabbit anti-dinitrophenyl. , then contacted with glucose oxidase anti-rabbit conjugate; (c) glucose and tetramethylbenzidine to induce and dye said first and second moieties. adding a reagent capable of generating a detectable signal at the junction site of the chromosomal translocation; and (d) determining the presence or absence of said signal under an optical microscope; A method consisting of