Preparation method and application of a fluorescent in situ hybridization probe specific to chromosomes of Centipedegrass

Abstract

The present invention provides a method for preparing a fluorescent in situ hybridization probe specific for chromosomes of Eremochloa chinensis and its application, and relates to the technical field of molecular biological detection. The present invention utilizes the published Eremochloa chinensis genome sequence to perform oligonucleotide sequence mining in non-repetitive DNA regions, constructs a probe library based on the obtained chromosome-specific single-copy non-overlapping oligonucleotide sequence, amplifies the probe library using an emulsion polymerase chain reaction method, and then obtains a single-stranded DNA probe by in vitro transcription and reverse transcription of the amplified product, and finally becomes a fluorescent in situ hybridization probe by fluorescently labeling the DNA probe. The oligonucleotide fluorescent probe and fluorescent in situ hybridization system provided by the present invention can be used for molecular cytogenetics problems such as the identification and karyotype analysis of chromosomes of Eremochloa chinensis and closely related species, chromosome DNA rearrangement, chromosome pairing and kinship analysis, and also provides important technical support for the cytological identification of distant hybridization and radiation-induced mutagenesis breeding of Eremochloa chinensis.

Description

Technical Field

[0001] The invention belongs to the technical field of molecular biological detection, and specifically relates to a preparation method of a chromosome-specific fluorescent in situ hybridization probe of Ervatamia oxyphylla and an application thereof. Background Art

[0002] Eremochloa ophiuroides (Munro) Hack. is an excellent warm-season C4 herbaceous plant belonging to the genus Eremochloa of the Poaceae family. It is widely distributed in the Yangtze River Basin and the areas to the south. It has good ornamental value and characteristics such as acid and aluminum resistance, barrenness resistance, few diseases and pests, and low maintenance level. It is a typical landscape and ecological multi-purpose grass species, so it has become the preferred grass species in the current urban landscaping and ecological environment construction applications in my country. In recent years, research on the creation of Eremochloa ophiuroides germplasm based on various radiation mutagenesis technologies has been carried out one after another. Based on this, more and more new variant germplasms of Eremochloa ophiuroides have been created, which provides important gene resources for the improvement of Eremochloa varieties, and also provides important tool materials for its chromosome biology and genomics research. In this process, the identification of new variant germplasms is particularly critical. Fluorescence in situ hybridization (FISH) technology is usually used in radiation mutagenesis of crops to identify and analyze the mutations at the chromosome level. However, to date, there have been no reports on the design and development of FISH probes and related fluorescence in situ hybridization techniques for Ervatamia oxyphylla.

[0003] FISH technology is the use of chromosome-specific probes to selectively display specific chromosomes or chromosome segments. It can achieve intuitive and accurate chromosome positioning of DNA sequences and precise detection of chromosome aberrations. It is one of the important technologies for in-depth research on the genome. With the increasing number of probe types used in FISH, FISH technology is not only used in plant cell genetics, but also widely used in the creation of new plant germplasm and breeding research. In FISH technology, the acquisition of chromosome-specific probes is the key to the application of this technology. In recent years, the development of FISH probes based on single-stranded oligonucleotides has been seen in several field economic crops. Because of its simple development, easy modification, low cost and high efficiency, oligonucleotide probes are expected to become a new generation of tools for biological chromosome identification and genome research.

[0004] Centipedegrass is a high-quality native grass species originating from my country, and plays an important role in my country's grass production. At present, the Grassland Research Center of the Institute of Botany, Chinese Academy of Sciences, Jiangsu Province, which is the supporting unit of the Engineering Technology Research Center for Warm-season Turfgrass Germplasm Innovation and Utilization of the State Forestry and Grassland Administration, not only has the world's richest germplasm resource bank of Centipedegrass, but also has completed the genome sequencing of the high-quality Centipedegrass variety "Ganbei Centipedegrass (No. E039)", which provides convenience for in-depth research on the chromosome biology and genomics of Centipedegrass. Summary of the invention

[0005] In view of this, the purpose of the present invention is to provide a method for preparing a chromosome-specific fluorescent in situ hybridization probe of Ervatamia oxyphylla and its application. The prepared fluorescent in situ hybridization probe can be used to study molecular cytogenetics problems such as chromosome identification and karyotype analysis, chromosome DNA rearrangement, chromosome pairing, and kinship analysis of Ervatamia oxyphylla.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The invention provides a method for preparing a chromosome-specific fluorescent in situ hybridization probe of Ervatamia oxyphylla. The method comprises the following steps: constructing a probe library: selecting a non-repetitive DNA target region in an Ervatamia oxyphylla genome, continuously designing single-stranded oligonucleotide sequences that are completely complementary to the target region as candidate probes, adding polymerase chain reaction amplification primer sequences to both ends of the candidate probes, and synthesizing the target probe library through a gene chip; amplifying the target probe library: performing PCR amplification on the target probe library with the amplification primers; preparing library RNA: performing in vitro transcription on the amplification product to obtain library RNA corresponding to the target probe library; preparing a DNA probe: performing reverse transcription on the library RNA to obtain a single-stranded DNA probe; and preparing a fluorescent probe: performing fluorescent labeling on the obtained single-stranded DNA probe to obtain a fluorescent in situ hybridization probe.

[0008] Preferably, the candidate probes correspond to non-overlapping oligonucleotide fragment regions in the genome, and the distance between two adjacent oligonucleotide fragments is set to 3-50 nt.

[0009] Preferably, the candidate probe is 30 to 100 nt in length.

[0010] Preferably, the sequences of the polymerase chain reaction amplification primers are shown as SEQ ID NO.1 and SEQ ID NO.2.

[0011] Preferably, the prepared single-stranded DNA probe is fluorescently labeled with green 6-carboxyfluorescein at its 5' end.

[0012] The present invention also provides a chromosome-specific fluorescent in situ hybridization probe of Ervatamia oxyphylla prepared by the preparation method.

[0013] The present invention also provides an application of the centipedegrass chromosome-specific fluorescent in situ hybridization probe in centipedegrass chromosome identification or karyotype analysis.

[0014] Preferably, the identification or karyotype analysis method comprises the following steps: sample preparation: using the pressing method to prepare a chromosome slide of the metaphase phase of the cell division of the root tip of Ervatamia oxyphylla; FISH experiment; preparing FISH hybridization solution, dropping it onto the slide sample that has been denatured in advance for hybridization reaction, and finally counter-staining with DAPI; result identification: placing the slide after the hybridization reaction under a fluorescence microscope for observation and photography, and performing corresponding analysis on the FISH signal.

[0015] Preferably, the FISH hybridization solution is a 10 μL mixed solution system containing 1 μL of probe, 0.5 μL of salmon sperm DNA, 2 μL of 50% dextran sulfate, 2 μL of 20×SSC, and 4.5 μL of deionized formamide.

[0016] Preferably, the slide sample is denatured using a 0.15 equivalent NaOH / 70% ethanol solution.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] The present invention utilizes the published genome sequence of Ervatamia oxysporum to carry out oligonucleotide sequence mining of non-repetitive DNA regions, constructs a probe library based on the obtained chromosome-specific single-copy non-overlapping oligonucleotide sequence, adopts the emulsion polymerase chain reaction (PCR) method to amplify the probe library, and the amplified product is then transcribed and reversed in vitro to obtain a single-stranded DNA probe, and finally becomes a fluorescent in situ hybridization probe through a fluorescently labeled DNA probe. The fluorescent in situ hybridization probe obtained by the present invention is in situ hybridized on the chromosome of Ervatamia oxysporum mitosis metaphase, and after color development and counterstaining, the observation and photography of FISH signals are completed under a fluorescence microscope. The oligonucleotide fluorescent probe and fluorescent in situ hybridization technology system provided by the present invention can be used for molecular cytogenetics problems such as identification and karyotype analysis of chromosomes of Ervatamia oxysporum and closely related species, chromosome DNA rearrangement, chromosome pairing and kinship analysis, and also provides important technical support for cytological identification of distant hybridization and radiation-induced mutagenesis breeding of Ervatamia oxysporum. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 It is the electrophoresis detection result of the amplification product of the oligonucleotide (oligo) probe library after emulsion PCR amplification;

[0020] Figure 2These are the results of fluorescence in situ hybridization microscopy of metaphase mitosis of Ervatamia oxyphylla root tip cells based on a repetitive sequence probe. The scale bar in the figure is 5 μm.

[0021] Figure 3 These are the results of fluorescence in situ hybridization microscopy of metaphase mitosis of root tip cells of Centipedegrass based on oligonucleotide probes. The scale bar in the figure is 5 μm. DETAILED DESCRIPTION

[0022] The present invention provides a method for preparing a chromosome-specific fluorescent in situ hybridization probe of Centipedegrass, comprising the following steps:

[0023] Construction of probe library: Select the non-repetitive DNA target region in the genome of Ervatamia oxyphylla, continuously design single-stranded oligonucleotide sequences that are completely complementary to the target region as candidate probes, add polymerase chain reaction amplification primer sequences to both ends of the candidate probes, and obtain the target probe library through gene chip synthesis;

[0024] Amplifying the target probe library: performing PCR amplification on the target probe library with amplification primers added;

[0025] Prepare library RNA: perform in vitro transcription on the amplified product to obtain library RNA corresponding to the target probe library;

[0026] Preparing DNA probes: reverse transcribing the library RNA to obtain single-stranded DNA probes;

[0027] Preparation of fluorescent probes: The obtained single-stranded DNA probes are fluorescently labeled to obtain fluorescent in situ hybridization probes.

[0028] In the present invention, the candidate probes correspond to non-overlapping oligonucleotide fragment regions in the genome, and the distance between two adjacent oligonucleotide fragments is preferably set to 3-50 nt; more preferably 5 nt.

[0029] In the present invention, the length of the candidate probe is preferably 30 to 100 nt; more preferably 45 nt.

[0030] In the present invention, the sequences of the polymerase chain reaction amplification primers are shown in SEQ ID NO.1 and SEQ ID NO.2.

[0031] In the present invention, the prepared single-stranded DNA probe is preferably fluorescently labeled at its 5' end using green 6-carboxyfluorescein (6-FAM).

[0032] The present invention also provides a chromosome-specific fluorescent in situ hybridization probe of Ervatamia oxyphylla prepared by the preparation method.

[0033] The present invention also provides an application of the centipedegrass chromosome-specific fluorescent in situ hybridization probe in centipedegrass chromosome identification or karyotype analysis.

[0034] In the present invention, the method of identification or karyotyping comprises the following steps:

[0035] Sample preparation: The chromosome slides of the metaphase of the root tip cells of Ervatamia oxyphylla were prepared by the pressing method;

[0036] FISH experiment: Prepare FISH hybridization solution, add it to the pre-denatured slide sample for hybridization reaction, and finally counterstain with DAPI;

[0037] Result identification: Place the slide after hybridization reaction under a fluorescence microscope for observation and photography, and perform corresponding analysis on the FISH signal.

[0038] In the present invention, the chromosome slide preferably uses root tip cells, which are obtained by fixing the root tip tissue grown from the young runners of Eremochloa oleifera in water culture with Carnoy's fixative and dissociating with 45% acetic acid solution.

[0039] In the present invention, the FISH hybridization solution is preferably a 10 μL mixed solution system containing 1 μL probe, 0.5 μL salmon sperm DNA, 2 μL 50% dextran sulfate, 2 μL 20×SSC, and 4.5 μL deionized formamide.

[0040] In the present invention, the slide sample is denatured using a 0.15 equivalent NaOH / 70% ethanol solution.

[0041] In order to better explain the present invention and facilitate understanding, the present invention is described in detail below in conjunction with the accompanying drawings through specific embodiments. The experimental methods used in the following examples are conventional methods unless otherwise specified; the materials, reagents, etc. used are all commercially available unless otherwise specified.

[0042] Example 1

[0043] 1. Oligonucleotide probe development

[0044] (1) Based on the high-quality genome data of the high-quality centipedegrass variety "Ganbei centipedegrass (No. E039)" published by the Grassland Research Center of the Institute of Botany, Chinese Academy of Sciences, Jiangsu Province, the chromosome-specific single-copy sequence of the centipedegrass reference genome was discovered using the Chorus2 software to obtain a single-copy oligonucleotide library.

[0045] (2) Filtering of genomic sequences: All repetitive DNA sequences and DNA homopolymers (>6 nt) were filtered out to obtain single-copy oligonucleotide sequences.

[0046] (3) Selection of specific oligonucleotide sequences: The single-copy oligonucleotide sequences obtained by preliminary filtering are mapped to the reference genome, and the oligonucleotide sequences that are mapped only to a single position in the reference genome are selected.

[0047] (4) Thermodynamic analysis: Primer3-py was used to calculate the hybridization Tm and hairpin Tm of each oligonucleotide, and only the oligonucleotide fragments with a difference between the hybridization Tm and the hairpin Tm greater than 10°C were retained for the next step of analysis.

[0048] (5) Further filtering: Through k-mer analysis, repeated oligonucleotide fragments are further filtered out, and finally oligonucleotide fragments with k-mer values ​​between 10% and 90% are retained; at the same time, the minimum distance between two adjacent oligonucleotide fragments is set to between 3-50nt, preferably to 5nt.

[0049] 2. Design and synthesis of oligonucleotide probe library

[0050] (1) Design of oligonucleotide probe library: Select chromosome 2 segment 83414-83505kb, chromosome 6 segment 603-673kb, chromosome 8 segment 59322-59414kb and 79501-79611kb, chromosome 9 segment 3792-3921kb and 63973-64067kb of the Centipedegrass genome. For the single copy oligonucleotide sequence of each segment, a candidate probe with a length of 30-100nt (preferably 45nt) that is completely complementary to the target region is designed every 3bp. Further, through BLAST comparison and scoring calculation, probes with non-specific Tm values ​​greater than 45°C are eliminated. Finally, non-overlapping oligonucleotide probes are selected, with about 200 probe sequences in each segment, totaling 1200 probe sequences. Its location information on the genome is shown in Table 1:

[0051] Table 1 Location information

[0052]

[0053]

[0054]

[0055]

[0056]

[0057]

[0058]

[0059]

[0060]

[0061]

[0062]

[0063]

[0064] (2) Synthetic probe library: The probe library is synthesized using gene chip technology, and the oligonucleotide probe library is obtained by primer amplification. The primer sequences are shown in Table 2:

[0065] Table 2 Primer sequences

[0066]

[0067] 3. Emulsion PCR amplification probe library

[0068] (1) Establishing an emulsion reaction system: According to the instructions of the Micellulia DNA Emulsion & Purification Kit of EURX Company, an aqueous phase system containing a probe library was prepared according to the proportions in Table 3, and the aqueous phase system was mixed with a mixed oil phase system containing three emulsion components.

[0069] Table 3 Emulsion reaction system

[0070]

[0071] (2) Perform emulsion PCR reaction: Divide the mixed solution into several 200 μL PCR tubes and perform amplification reaction on a PCR instrument according to the program in Table 4.

[0072] Table 4 Emulsion PCR reaction procedure

[0073]

[0074] (3) Purification of emulsion PCR products: After the PCR reaction is completed, collect the emulsion PCR products in all PCR tubes into a 2 mL centrifuge tube, add 1 mL of butanol, vortex mix, then add Orange-DX, vortex mix, and centrifuge. Remove the oil phase, pass the lower aqueous phase through the column, wash several times with washing solution, and finally recover with 100 μL of washing solution.

[0075] (4) Electrophoresis detection: The purified PCR product was detected by 2% agarose gel electrophoresis. If there was a specific amplification band at about 100 bp, it means that the emulsion PCR amplification was successful. Figure 1 .

[0076] 4. Prepare library RNA

[0077] The emulsion PCR product was transcribed in vitro using a transcription kit (MEGAscriptTMT7Transcription Kit) from Thermo Fisher Scientific to generate RNA, which was then purified and recovered using a Qiagen RNeasy Mini kit.

[0078] 5. Preparation of DNA probe

[0079] The reverse transcription kit SuperScript III First-Strand Synthesis SuperMix from Thermo Fisher Scientific was used to reverse transcribe the RNA product to obtain the reverse transcription product cDNA. RNase A was then added to the reverse transcription product DNA to eliminate RNA, and finally the Zymo Quick-RNA Miniprep kit was used to purify and recover the DNA.

[0080] 6. Fluorescently labeled DNA probe

[0081] The purified and recovered single-stranded DNA was sent to Thermo Fisher Scientific (Shanghai) and fluorescently labeled with 6-FAM at its 5' end.

[0082] Example 2 Fluorescence in situ hybridization (FISH)

[0083] 1. Preparation of chromosomes from the root tip of Centipedegrass

[0084] (1) Select young runners of Ervatamia oxyphylla, cut them into three-node segments, wrap them with sponges and insert them into the holes of a foam board (hole diameter 1.5-2.0 cm), then place them in a plastic bucket (diameter 15 cm, depth 30 cm) filled with distilled water and culture them at room temperature.

[0085] (2) When the roots grow to 1.5-2.0 cm, cut several roots and collect them in a 1.5 mL centrifuge tube (with a hole punched in the cap of the centrifuge tube in advance), add 1-2 drops of distilled water to ensure that the root tip is moistened; place the centrifuge tube in a nitrous oxide tank and fill it with nitrous oxide until the pressure reaches 0.8-1.2 MPa, and treat for 1.5 hours.

[0086] (3) Take out the centrifuge tube, add Carnoy's fixative (V glacial acetic acid: V ethanol = 1:3), place on ice for 10 min, and then rinse with distilled water three times.

[0087] (4) Remove the root tip and dissociate it in 45% acetic acid for 3-5 min. Cut off the root cap and then cut the root tip meristem onto a glass slide. Add a drop of 45% acetic acid and cover with a coverslip. Tap gently to disperse the cells. Bake evenly with the outer flame of an alcohol lamp and press the slide after the mist has dispersed. Observe under a phase contrast microscope and store the slides with good chromosome morphology, complete number and uniform dispersion in a -80°C refrigerator.

[0088] 2. Hybridization test

[0089] (1) Take out the sample slide from the freezer, remove the cover slip, immerse it in 0.15N NaOH / 70% ethanol solution for denaturation treatment at room temperature for 5 minutes, then dehydrate it in 70%, 70%, and 99% ethanol at room temperature for 5 minutes each. After taking it out, blow dry it with an ear bulb or air dry it before using it for in situ hybridization.

[0090] (2) Prepare hybridization solution. The hybridization solution system is 10 μL, including 1.0 μL of probe, 0.5 μL of salmon sperm DNA (10 mg mL -1 ), 2.0 μL dextran sulfate (50%), 2.0 μL 20×SSC, 4.5 μL deionized formamide. The reaction solution was prepared on ice, then incubated at 16° C. for 2 h in a thermal cycler, and stored at -20° C. for later use.

[0091] (3) Add the prepared hybridization solution onto the sample slide, 10 μL of hybridization solution per slide, cover with a cover slip and place in a moist aluminum box in a 37°C incubator for hybridization for more than 2 h.

[0092] (4) After hybridization, wash off the coverslip with 2×SSC, then place the slide in 2×SSC solution for 5–8 min, rinse once with clean water, blow dry, and finally add 10 μL of DAPI to each slide, cover with a coverslip, and restain for at least 30 min.

[0093] (5) Place the slide under an Olympus BX60 fluorescence microscope for observation, and take pictures using the CCD (camera system) equipped with the microscope and save them.

[0094] 3. Result identification

[0095] (1) Perform the first fluorescence in situ hybridization (FISH) using 5S rDNA and 18S rDNA repeat sequence probes and observe and photograph. Figure 2 As shown; then after the signal was eluted, a second FISH was performed on the same slide using the newly developed oligonucleotide probe set. The results are shown in Figure 3 shown.

[0096] (2) According to the FISH signals of the repetitive sequence probe and the oligonucleotide probe set, it was found that two pairs of chromosomes in the centipede chromosomes showed strong 5S rDNA (red) and 18S rDNA (green) hybridization signals, which also confirmed that the centipede is diploid. Through oligonucleotide probe hybridization, it can also be clearly seen that there are clear FISH signals (green) on the four pairs of chromosomes. At the same time, combined with the information of the designed oligonucleotide sequence in the corresponding chromosome segment, the corresponding chromosome can be accurately identified, that is, the probe FISH signal produced in this embodiment can accurately identify chromosomes 2, 6, and 9 of centipede; in particular, the two green signals of the long arm of chromosome 8 hybridize to chromosome 4 where 18SrDNA is located, so the chromosome is a chromosome translocation of the long arm 8 and the long arm 4 (8L-4L). The signal distribution characteristics of the 5SrDNA and 18S rDNA repetitive sequence probes and the newly developed oligonucleotide probes on the centipede chromosomes are as follows:

[0097] Chromosome 2: Oligonucleotide dot signals are distributed at the end of the long arm, corresponding to the chromosomes of the normal genome.

[0098] Chromosome 4: 18S rDNA signals are mainly distributed in the middle of the long arm or near the centromere. In addition, the two oligonucleotide signals of the long arm of chromosome 8 hybridize to the long arm of chromosome 4, so it can be inferred that a chromosomal translocation occurred between the long arms of chromosome 8 and chromosome 4.

[0099] Chromosome 5: 5S rDNA signal is mainly distributed in the middle of the long arm.

[0100] Chromosome 6: Oligonucleotide dot signals are distributed at the end of the short arm, corresponding to the chromosomes of the normal genome.

[0101] Chromosome 9: There are oligonucleotide dot hybridization signals distributed at the proximal ends of the long and short arms, corresponding to chromosome 9 of the genome.

[0102] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing a chromosome-specific fluorescent in situ hybridization probe of Centipedegrass, characterized in that: The steps include: Construction of probe library: Select the non-repetitive DNA target region in the genome of Ervatamia oxyphylla, continuously design single-stranded oligonucleotide sequences that are completely complementary to the target region as candidate probes, add polymerase chain reaction amplification primer sequences to both ends of the candidate probes, and obtain the target probe library through gene chip synthesis; Amplifying the target probe library: performing PCR amplification on the target probe library with amplification primers added; Prepare library RNA: perform in vitro transcription on the amplified product to obtain library RNA corresponding to the target probe library; Preparing DNA probes: reverse transcribing the library RNA to obtain single-stranded DNA probes; Preparation of fluorescent probes: fluorescently labeling the obtained single-stranded DNA probe to obtain a fluorescent in situ hybridization probe; The genome data of the high-quality centipedegrass variety "Ganbei centipedegrass No. E039" published by the Grassland Research Center of the Institute of Botany, Chinese Academy of Sciences in Jiangsu Province was used as the reference genome of centipedegrass; The non-repetitive DNA target region in the genome of the centipede grass includes chromosome segment 2 83414-83505kb, chromosome segment 6 603-673kb, chromosome segment 8 59322-59414kb and 79501-79611kb, chromosome segment 9 3792-3921kb and 63973-64067kb of the centipede grass genome; the location information of the target region on the genome is as follows:

2. The preparation method according to claim 1, characterized in that: The candidate probes correspond to non-overlapping oligonucleotide fragment regions in the genome, and the distance between two adjacent oligonucleotide fragments is set to 3-50 nt.

3. The preparation method according to claim 1, characterized in that: The candidate probe has a length of 30 to 100 nt.

4. The preparation method according to claim 1, characterized in that: The sequences of the polymerase chain reaction amplification primers are shown in SEQ ID NO.1 and SEQ ID NO.

2.

5. The preparation method according to claim 1, characterized in that: The prepared single-stranded DNA probe was fluorescently labeled at its 5' end using green 6-carboxyfluorescein.

6. A chromosome-specific fluorescent in situ hybridization probe for Ervatamia oxyphylla prepared by the preparation method according to any one of claims 1 to 5.

7. Use of the centipedegrass chromosome-specific fluorescent in situ hybridization probe according to claim 6 in centipedegrass chromosome identification or karyotype analysis.

8. The use according to claim 7, characterized in that: The method of identification or karyotyping comprises the following steps: Sample preparation: The chromosome slides of the metaphase of the root tip cells of Ervatamia oxyphylla were prepared by the pressing method; FISH experiment: Prepare FISH hybridization solution, add it to the pre-denatured slide sample for hybridization reaction, and finally counterstain with DAPI; Result identification: Place the slide after hybridization reaction under a fluorescence microscope for observation and photography, and perform corresponding analysis on the FISH signal.

9. The use according to claim 8, characterized in that: The FISH hybridization solution is a 10 μL mixed solution system containing 1 μL of probe, 0.5 μL of salmon sperm DNA, 2 μL of 50% dextran sulfate, 2 μL of 20×SSC, and 4.5 μL of deionized formamide.

10. The use according to claim 8, characterized in that: The slide samples were denatured using a 0.15 N NaOH / 70% ethanol solution.

Images