CN112921074A - In-situ hybridization method for simultaneously detecting multiple loci of genome through combined colors - Google Patents
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Abstract
The invention relates to the field of application of molecular biology technology, in particular to an in-situ hybridization method for simultaneously detecting multiple loci of a genome through combined colors, which comprises the following steps: s1, preparing a hybridization probe: synthesizing a primer segment, and carrying out fluorescence labeling on the probe; the fluorescent label comprises a combined color fluorescent label; s2, hybridization: preparing a hybridization sheet, and carrying out hybridization reaction on the prepared hybridization sheet and a hybridization probe which is subjected to fluorescence labeling in S1; and S3, imaging and acquiring marking information. The in-situ hybridization method of the invention improves the accuracy of detection, greatly increases the number of detection sites and only needs one round of hybridization. At least five gene loci can be detected simultaneously, so that the detection efficiency is greatly improved, the operation is simpler, more convenient and faster, and the application range is expanded.
Description
Technical Field
The invention relates to the field of application of molecular biology technology, in particular to an in-situ hybridization method for simultaneously detecting multiple loci of a genome by combining colors, which is an application of a multicolor fluorescence in-situ detection method.
Background
In situ hybridization (in situ hybridization ISH) is a method that a nucleic acid probe with a known base sequence and a label is applied to specifically combine with nucleic acid to be detected in tissues and cells according to the base pairing principle to form a hybrid, then a detection system corresponding to the label is applied, a hybridization signal with fluorescence is formed in situ in the detected nucleic acid by a histochemistry or immunohistochemistry method, and the intracellular localization is carried out under a microscope or an electron microscope. The technology provides a means for researching the positioning of DNA and corresponding mRNA for coding various proteins and polypeptides in a single cell, and provides an effective tool for researching gene expression and related gene regulation in cells from a molecular level. Can be regarded as a revolutionary breakthrough in histochemistry or immunocytochemistry. In situ hybridization techniques have been applied to basic research such as genomic mapping (Gene mapping), transgene detection, Gene expression mapping (localization of Gene expression), mRNA arrangement and transport of nuclear DNA and RNA (arrangement and transport of mNA), replication (replication), and cell classification (Sorting of cells). Clinical studies have applications in Cytogenetics (Cytogenetics), Prenatal diagnosis (Prenatal diagnosis), diagnosis of neoplastic and infectious diseases, biological dosimetry (biological diagnosis) and etiologic diagnosis of virology, among others. With the continuous improvement of the preparation, labeling and basic operation of nucleic acid probes, new technologies are emerging, and it is believed that in the near future, in situ hybridization will be more widely applied to various disciplines, and provide new data for life science, and open up new fields.
In the process of occurrence and development of tumor diseases, abnormal changes of chromosomes, genes and the like are often found in tumor cells, including genetic variation, gene multicopy, chromosome aneuploidy and the like. FISH (Fluorescence in situ hybridization) can be used for prognosis or diagnosis reference of some tumors, such as cervical cancer, non-small cell lung cancer, breast cancer, bladder cancer and the like in solid tumors, and chronic myelogenous leukemia, multiple myeloma, chronic lymphocytic leukemia and the like in hematological tumors, by detecting abnormal changes of chromosomes and genes.
The FISH technique also has its limitations, firstly it can only use specific probe to detect known chromosome abnormality, secondly one probe can only detect one abnormality, for the simultaneous multiple abnormalities need to use multiple probes to detect, the cost will increase.
In the current fluorescence in situ hybridization method, one hybridization sheet can realize fluorescence of at most three colors. In practice, DAPI is generally used to mark cell nuclei, so that for ordinary fluorescence microscopy, one hybridization plate can only simultaneously detect red light and green light, i.e., one hybridization plate detects two different genes or chromosomes. If more genes are hybridized, a hybridization sheet needs to be additionally added or other fluorescent colors need to be additionally prepared, so that the workload of operators is increased, and the detection cost is also increased. In addition, the conventional fluorescence in situ hybridization method usually uses one hybridization chip to detect only one target gene, and if a plurality of genes need to be detected simultaneously, multiple rounds of hybridization can be performed optionally. After the previous hybridization and detection of the same hybridization piece are finished, the hybridization and detection are carried out after the cleaning, and the aim of detecting various genes is fulfilled in such a circulating way. At present, the condition of incomplete cleaning generally exists in the process of multi-round hybridization, interference is brought to subsequent detection, the accuracy of a detection result is influenced, meanwhile, the time period of the multi-round hybridization is very long, and the gene detection efficiency is greatly reduced.
Disclosure of Invention
Accordingly, the present invention aims to provide an in situ hybridization method for simultaneous detection of multiple genomic loci by a combination of colors. The in-situ hybridization method of the invention improves the accuracy of detection, greatly increases the number of detection sites and only needs one round of hybridization. At least five gene loci can be detected simultaneously, so that the detection efficiency is greatly improved, the operation is simpler, more convenient and faster, and the application range is expanded.
The technical scheme of the invention is as follows:
an in situ hybridization method for simultaneous detection of multiple loci of a genome by a combination of colors, comprising the steps of:
s1, preparing a hybridization probe: synthesizing a primer segment, and carrying out fluorescence labeling on the probe;
s2, hybridization: preparing a hybridization sheet, and carrying out hybridization reaction on the prepared hybridization sheet and a hybridization probe which is subjected to fluorescence labeling in S1;
s3, imaging to obtain marking information;
the fluorescent label in S1 comprises a combination color fluorescent label.
Further, the hybridization probe preparation operation of S1 includes:
s11, carrying out PCR amplification on the segmented segments, and recovering an amplification product;
s12, carrying out in-vitro transcription on the amplification product recovered from S11, and collecting a transcription fragment;
s13, respectively carrying out reverse transcription on the transcription fragments of S12, wherein a reverse transcription system comprises dUTP with fluorescent modification, and recovering reverse transcription products.
Further, the amplification system in S11 is:
buffer：20μl；
primer fragment mixture (5 ng/. mu.l): 1 mul;
dNTP-mix(10mM)：1μl；
amplification primers MIX (10. mu.M/. mu.l): 4 mu l of the solution;
amplification enzyme: 1 mul;
ddH20：12μl。
further, the amplification procedure in S11 is: at 95 ℃ for 30 s; 30s at 60 ℃; 72 ℃ for 30 s; amplification was performed for 19 cycles.
Further, the reverse transcription system in S13 is:
5 × reverse transcription buffer: 5 mu l of the solution;
dNTP mix：2.8μl；
dUTP with fluorescent modification (1 mM): 2 mu l of the solution;
reverse transcription primer (20 μ M): 1 mul;
reverse transcriptase mixture: 2 mu l of the solution;
RNA：8.2μl。
further, the reverse transcription procedure in S13 is: 5min at 25 ℃; at 37 ℃ for 45 min; 85 ℃ for 5 min.
Further, the operation in S13 further includes purification of the single-stranded DNA hybridization probe: after completion of the inversion, 20. mu.l of 0.25M EDTA 0.5M NaOH buffer was added to the inversion system, and the reaction was carried out at 95 ℃ for 10 min.
Further, the dUTP with the fluorescent modification is dUTP with the fluorescent modification obtained by respectively carrying out combined modification on dUTP by using at least two basic color fluorescent modifiers.
Further, the selected number of basic colors is N, N1 is the number of single color modifiable dutps (i.e., the number of single color encoding genes), N2 is the number of two color modifiable dutps (i.e., the number of two color encoding genes), N3 is the number of three color modifiable dutps (i.e., the number of three color encoding genes), and so on, N (N-1) is the number of (N-1) color modifiable dutps (i.e., the number of (N-1) color encoding genes), and Nn is the number of N color modifiable dutps (i.e., the number of N color encoding genes). N is the total number of dutps that can be modified by the N primary colors (i.e., the total number of genes encoded); n is a radical ofmaxThe maximum value of the total number of dutps that can be modified for the n primary colors (i.e., the maximum value of the number of genes encoded). The quantitative relationship is as follows:
carrying out combined marking according to actual needs, wherein N is the sum of N1 and at least one value of N2 to Nn; maximum value N of NmaxN1+ N2 … … + N (N-1) + Nn. Wherein C is a calculation formula of the combination number.
It should be noted that, theoretically, in the method of the present invention, the number n of basic colors may be any number, but due to the limitation of the current fluorescence modified dUTP raw material, the number of commonly used fluorescence modified dUTP raw materials is only 6, so that the maximum value of n here may be 6.
The number of dutps with fluorescent modification in the in situ hybridization method of the present invention corresponds to the number of loci that can be detected by the in situ hybridization method of the present invention, i.e., the number of genes that can be detected by the in situ hybridization method of the present invention is equal to N, wherein N is the sum of N1 and at least one value of N2 to Nn; maximum value N of Nmax＝N1+N2……+N(n-1)+Nn。
Further, the preparing of the hybrid sheet in the step S2 includes:
s21, preparing a hybrid sheet of the cells to be detected;
s22, the hybridization sheet is subjected to a hybridization reaction with a fluorescent probe.
Further, the S21 includes: collecting a cell sample to be detected, and cleaning; hypotonic suspension; fixing by using a fixing solution; smearing and drying; boiling slices; and (5) washing with alcohol.
Preferably, KCl (5.5g/L) is used as a suspension for hypotonic suspension, and the suspension is incubated at 37 ℃ for 30 min.
Preferably, the fixing operation of the fixing liquid includes: adding methanol/acetic acid stationary liquid into cells, uniformly blowing, centrifuging, and removing supernatant; repeating the operation for times; adding methanol/acetic acid stationary liquid for the third time, blowing uniformly, and fixing at room temperature for 10 min.
Preferably, the boiling operation comprises: the dried slide glass is placed in a 2XSSC solution and boiled in a water bath at 65 ℃ for 30 min.
Preferably, the alcohol washing operation includes: 70% -85% -100% alcohol gradient washing slide.
Further, the specific operation of S22 includes: adding hybridization solution containing fluorescent probe into the dried slide, sealing the slide (full sealing), and denaturing at 80 ℃ for 5 min; the pieces were placed in a wet box overnight at 37 ℃.
Preferably, in the hybridization solution, the volume of the fluorescent probe solution accounts for 10%; that is, 9. mu.l of the hybridization solution was added with 1. mu.l of the fluorescent probe.
Further, the preparing of the hybrid sheet in the step S2 further includes:
s23, dyeing and sealing: after hybridization, removing the mounting glue and the cover glass (soaking water when removing the cover glass to prevent cell abrasion when drying and taking the glass), and then placing in preheated 0.3% NP40/0.4XSSC for soaking for 4min at 65 ℃; after the washing with ddH2O, the cells were stained with 10. mu.l of aqueous DAPI solution for 1 min; then, after the mixture is processed by 100 percent alcohol for one time, the mixture is placed on a piece of mirror wiping paper to be dried; adding a drop of the sealing tablet, sealing and performing microscopic examination.
Further, the image forming operation of step S3 includes: and (3) photographing the hybridized slices of S2 by using a fluorescence microscope, respectively photographing red (564nm excitation), green (488nm excitation), magenta (594nm excitation) and blue (425nm excitation) channel pictures, and then merging the pictures to obtain the detected gene or chromosome information.
The invention has the beneficial effects that:
at present, the fluorescence in situ hybridization is detected in three fluorescent colors, namely red, green and sky blue, wherein when the sky blue fluorescence is detected, a common fluorescence microscope cannot distinguish the colors, a corresponding optical filter is required to be equipped, the detection cost is increased, and the application scene is relatively limited. The method adopts the combined color in situ hybridization method, the fluorescence of multiple colors is used for marking the same gene locus, and the multiple colors simultaneously verify one gene or one chromosome, thereby realizing the purpose of simultaneously detecting multiple genes or multiple chromosomes by carrying out hybridization on one slide at one time, and particularly determining the number of the detected genes or chromosomes according to the existing fluorescence microscope. For example, a common microscope has red light, green light and blue light channels, and when hybridization detection is generally carried out, cell nuclei are stained by DAPI and observed to be blue light; thus, a common microscope can only detect two different genes or chromosomes, distinguished by red and green light, respectively. The probe is prepared by the method, imaging is carried out, three genes or chromosomes can be detected simultaneously, the detection efficiency is greatly improved, additional hybridization pieces do not need to be prepared, or a fluorescence channel does not need to be added, the detection cost is reduced, and the operation is more convenient. And the fluorescence microscope with four channels is provided, and six different genes or chromosomes can be detected on one slide at the same time in the actual operation process.
For a better understanding and practice, the invention is described in detail below with reference to examples.
Drawings
Fig. 1 is a schematic diagram of a single-color mark and a two-color mark in the case of 4 basic colors (n is 4). Where n is the number of primary colors. Ns (i.e., N1) is the single color marker gene factor, Nd (i.e., N2) is the double color marker gene factor, and N is the total number of marker genes.
Fig. 2 is a schematic diagram of labeling using the method of the present invention and the maximum total number of genes that can be detected, when 4 basic colors are taken as examples (n is 4).
FIG. 3 is a graph showing the results of multicolor fluorescence hybridization of THP-1 cells; wherein the site and marker colors are: MYC: CY3+ Alex 594; HER 2: alex488+ CY 3; TOP 2A: CY 3; TERC: alex 594; CCND 1: alex 488.
Detailed Description
The implementations described in the exemplary embodiments below are not intended to represent all implementations consistent with the present disclosure. Rather, they are merely examples of methods consistent with certain aspects of the present disclosure, as detailed in the appended claims.
The terminology used in the present disclosure is for the purpose of describing particular embodiments only and is not intended to be limiting of the disclosure. As used in this disclosure and the appended claims, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. It should also be understood that the term "and/or" as used herein refers to and encompasses any and all possible combinations of one or more of the associated listed items.
The specific techniques or conditions not indicated in the examples of this application are performed according to the techniques or conditions described in the literature in the field or according to the product description. The reagents or instruments used in the examples of the present application are not indicated by manufacturers, and are all conventional products available from commercial sources and the like.
The invention relates to an in situ hybridization method for simultaneously detecting multiple loci of a genome by combined colors, which comprises the following steps:
s1, preparing a hybridization probe: synthesizing a primer segment, and carrying out fluorescence labeling on the probe;
s2, hybridization: preparing a hybridization sheet, and carrying out hybridization reaction on the prepared hybridization sheet and a hybridization probe which is subjected to fluorescence labeling in S1;
s3, imaging to obtain marking information;
the fluorescent label in S1 comprises a combination color fluorescent label.
In some embodiments, the hybridization probe preparation procedure of S1 comprises:
s11, carrying out PCR amplification on the segmented segments, and recovering an amplification product;
s12, carrying out in-vitro transcription on the amplification product recovered from S11, and collecting a transcription fragment;
s13, respectively carrying out reverse transcription on the transcription fragments of S12, wherein a reverse transcription system comprises dUTP with fluorescent modification, and recovering reverse transcription products.
In some embodiments, the amplification system in S11 is:
buffer：20μl；
synthesis fragment mixture (5 ng/. mu.l): 1 mul;
dNTP-mix(10mM)：1μl；
amplification primers MIX (10. mu.M/. mu.l): 4 mu l of the solution;
amplification enzyme: 1 mul;
ddH20：12μl。
the amplification primer is an amplification primer aiming at the synthesized segment and can be designed by adopting the prior art means.
In some embodiments, the amplification procedure in S11 is: at 95 ℃ for 30 s; 30s at 60 ℃; 72 ℃ for 30 s; amplification was performed for 19 cycles.
In some embodiments, the reverse transcription system in S13 is:
5 × reverse transcription buffer: 5 mu l of the solution;
dNTP mix：2.8μl；
dUTP with fluorescent modification (1 mM): 2 mu l of the solution;
reverse transcription primer (20 μ M): 1 mul;
reverse transcriptase mixture: 2 mu l of the solution;
RNA：8.2μl。
in some embodiments, the reverse transcription procedure in S13 is: 5min at 25 ℃; at 37 ℃ for 45 min; 85 ℃ for 5 min.
In some embodiments, the operations in S13 further comprise purification of the single-stranded DNA hybridization probe: after completion of the inversion, 20. mu.l of 0.25M EDTA 0.5M NaOH buffer was added to the inversion system, and the reaction was carried out at 95 ℃ for 10 min.
In some embodiments, the dUTP with a fluorescent modification is a dUTP with a fluorescent modification that is a combination of modifications of dutps with at least two basic color fluorescent modifiers, respectively.
In some embodiments, the number of selected base colors is N, N1 is the number of monochrome modified dUTPs (i.e., the number of monochrome encoding genes), and N2 is bi-colorThe number of dUTPs modified (i.e., the number of genes encoding two colors), N3 the number of dUTPs modified by three colors (i.e., the number of genes encoding three colors), and so on, N (N-1) the number of dUTPs modified by (N-1) (i.e., the number of genes encoding (N-1)) colors, and Nn the number of dUTPs modified by N colors (i.e., the number of genes encoding N) colors. N is the total number of dutps that can be modified by the N primary colors (i.e., the total number of genes encoded); n is a radical ofmaxThe maximum value of the total number of dutps that can be modified for the n primary colors (i.e., the maximum value of the number of genes encoded). The quantitative relationship is as follows: the quantitative relationship is as follows:
carrying out combined marking according to actual needs, wherein N is the sum of N1 and at least one value of N2 to Nn; maximum value N of NmaxN1+ N2 … … + N (N-1) + Nn. Wherein C is a calculation formula of the combination number.
Referring to fig. 1, taking 4 basic colors as an example (n-4), the base factors of the single color mark and the two color mark are:
referring to fig. 2, taking 4 basic colors as an example (n is 4), the total number of genes that can be detected by the method of the present invention is:
in some embodiments, the preparing a hybrid sheet in step S2 includes:
s21, preparing a hybrid sheet of the cells to be detected;
s22, the hybridization sheet is subjected to a hybridization reaction with a fluorescent probe.
In some embodiments, the S21 includes: collecting a cell sample to be detected, and cleaning; hypotonic suspension; fixing by using a fixing solution; smearing and drying; boiling slices; and (5) washing with alcohol.
In some embodiments, the hypotonic suspension is suspended in KCl and incubated at 37 deg.C for 30 min.
In some embodiments, the fixation fluid fixation operation comprises: adding methanol/acetic acid stationary liquid into cells, uniformly blowing, centrifuging, and removing supernatant; repeating the operation for times; adding methanol/acetic acid stationary liquid for the third time, blowing uniformly, and fixing at room temperature for 10 min.
In some embodiments, the sheeting operation comprises: the dried slide glass is placed in a 2XSSC solution and boiled in a water bath at 65 ℃ for 30 min.
In some embodiments, the alcohol wash operation comprises: 70% -85% -100% alcohol gradient washing slide.
In some embodiments, the specific operation of S22 includes: adding hybridization solution containing fluorescent probe into the dried slide, sealing the slide (full sealing), and denaturing at 80 ℃ for 5 min; the pieces were placed in a wet box overnight at 37 ℃.
In some embodiments, the volume ratio of the hybridization solution to the fluorescent probe solution is: 10:1, i.e., 9. mu.l of the hybridization solution plus 1. mu.l of the fluorescent probe.
In some embodiments, the preparing a hybrid sheet in step S2 further comprises:
s23, dyeing and sealing: after hybridization, removing the mounting glue and the cover glass (soaking water when removing the cover glass to prevent cell abrasion when drying and taking the glass), and then placing in preheated 0.3% NP40/0.4XSSC for soaking for 4min at 65 ℃; after the washing with ddH2O, the cells were stained with 10. mu.l of aqueous DAPI solution for 1 min; then, after the mixture is processed by 100 percent alcohol for one time, the mixture is placed on a piece of mirror wiping paper to be dried; adding a drop of the sealing tablet, sealing and performing microscopic examination.
In some embodiments, the imaging operation of step S3 includes: and (3) photographing the hybridized slices of S2 by using a fluorescence microscope, respectively photographing red (564nm excitation), green (488nm excitation), magenta (594nm excitation) and blue (425nm excitation) channel pictures, and then merging the pictures to obtain the detected gene or chromosome information.
Embodiment one in situ hybridization method for simultaneous detection of multiple loci of a genome by a combination of colors of the present invention an in situ hybridization method for simultaneous detection of multiple loci of a genome by a combination of colors of the present invention comprises the steps of:
s1: hybridization probes are prepared.
S11, synthesizing short sequence primers by Nanjing Kingsler, Shanghai Biotech, American Integrated DNA Technologies (IDT) and other companies, carrying out PCR amplification on the synthesized single-stranded primers, and recovering amplification products; the amplification system is as follows:
PCR buffer (Beijing Jiang alliance): 20 mu l of the mixture;
synthesis fragment mixture (5 ng/. mu.l): 1 mul;
dNTP-mix(10mM)：1μl；
amplification primers MIX (10. mu.M/. mu.l): 4 mu l of the solution;
taq amplification enzyme (Beijing Jiang Co.): 1 mul;
ddH20：12μl；
the amplification procedure was: at 95 ℃ for 30 s; 30s at 60 ℃; 72 ℃ for 30 s; amplification was performed for 19 cycles.
S12, carrying out in vitro transcription on the amplification product recovered in S11, and collecting a transcription fragment.
S13, carrying out reverse transcription on the transcription fragment of S12, wherein a reverse transcription system comprises dUTP with fluorescent modification; after the inversion is finished, 20 mul of 0.25M EDTA 0.5M NaOH buffer is added into the inversion system, and the reaction is carried out for 10min at the temperature of 95 ℃; and then the reverse transcription product is recovered by a kit.
The reverse transcription system is as follows:
5 × reverse transcription buffer (Nanjing Novozam corporation): 5 mu l of the solution;
dNTP mix：2.8μl；
dUTP (1mM) with fluorescent modification (Thermo Co., USA): 2 mu l of the solution;
reverse transcription primer (20 μ M): 1 mul;
reverse transcriptase mix (Nanjing Novozam Co.): 2 mu l of the solution;
RNA：8.2μl。
the reverse transcription procedure was: 5min at 25 ℃; at 37 ℃ for 45 min; 85 ℃ for 5 min.
In the S13, five oncogene loci are labeled with three color fluorescence-modified dutps CY3 (red), Alex488 (green), and Alex594 (magenta), specifically:
site 1: the MYC amplification detection probe is double-labeled by red fluorescence (CY3) and magenta fluorescence (Alex 594);
site 2: the HER2 amplification detection probe is double-labeled by red fluorescence (CY3) and green fluorescence (Alex 488);
position 3: the TOP2A amplification detection probe is a single label of red fluorescence (CY 3);
position 4: the TERC amplification detection probe is a fuchsin fluorescence (Alex594) single label;
position 5: the CCND1 amplification detection probe is a single label of green fluorescence (Alex 488).
The probe prepared by the method is stable and can be applied to various in-situ hybridization preparation slides. Such as paraffin sections, cell drops, etc.
S2, hybridization: preparing a hybridization sheet, and carrying out hybridization reaction on the prepared hybridization sheet and the hybridization probe which is subjected to fluorescence labeling in S1.
S21, preparing a hybrid sheet of the cells to be detected;
the fluorescence in situ hybridization of the invention can be performed by using paraffin sections or cell dropping sheets, and the embodiment adopts a human leukemia THP-1 cell line as a hybrid cell.
The specific operation comprises the following steps:
1) collecting THP-1 cells, and washing the cells once with PBS;
2) centrifuging to remove supernatant, suspending the cells with 5ml of hypotonic KCL, and bathing at 37 deg.C for 30 min;
3) adding 1ml of methanol/acetic acid stationary liquid, blowing and beating uniformly, centrifuging, and removing supernatant;
4) adding 1ml of methanol/acetic acid stationary liquid, blowing and beating uniformly, centrifuging, and removing supernatant;
5) adding 1ml methanol/acetic acid stationary liquid, blowing and beating uniformly, and fixing for 10min at room temperature;
6) dripping 10 μ l of the fixed cell suspension on a glass slide, and drying in an oven for 20 min;
7) boiling the slide in 2XSSC solution in water bath at 65 ℃ for 30 min;
8) washing the slide with 70% -85% -100% alcohol gradient;
9) air-drying with ethanol, adding hybridization solution (9 μ l hybridization solution and 1 μ l fluorescent probe), sealing (sealing completely),
denaturation at 80 deg.C for 5 min;
10) putting the slices into a wet box, and hybridizing at 37 ℃ overnight;
11) after hybridization, removing mounting glue and slide (soaking water when cover glass is removed to prevent cell abrasion when slide is taken out by drying), preheating 0.3% NP40/0.4XSSC, and soaking for 4min at 65 ℃;
12) after the single-pass treatment in ddH2O, the cells were stained in 10. mu.l of an aqueous solution of DAPI for 1 min;
13) after the 100% alcohol is used for one time, putting the glass cleaning paper on the glass cleaning paper for drying;
14) adding a drop of the sealing tablet, sealing and performing microscopic examination.
And S3, imaging and acquiring marking information.
The hybridized slices of S2 were photographed by fluorescence microscope, and red (564nm excitation), green (488nm excitation), magenta (594nm excitation), and blue (425nm excitation) channel pictures were taken, where the blue channel is the channel of nuclear dye DAPI; image merge is then performed to obtain the detected gene or chromosome information, see FIG. 3.
Analysis of results
As shown in FIG. 3, in THP-1 leukemia cell line, there were only two signal points at TOP2A, CCND1, and TERC sites, indicating that the three oncogenes are structurally normal and no chromosomal alteration has occurred, while the MYC and HER2 sites have 3 signal points indicating that there is a disruption or chromosomal translocation of the MYC and HER2 genes in THP-1 cells.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention.
Claims (9)
1. An in situ hybridization method for simultaneous detection of multiple genomic loci by a combination of colors, comprising the steps of:
s1, preparing a hybridization probe: synthesizing a primer segment, and carrying out fluorescence labeling on the probe;
s2, hybridization: preparing a hybridization sheet, and carrying out hybridization reaction on the prepared hybridization sheet and a hybridization probe which is subjected to fluorescence labeling in S1;
s3, imaging to obtain marking information;
the fluorescent label in S1 comprises a combination color fluorescent label.
2. The in situ hybridization method for simultaneous detection of genomic multiple sites by a combination of colors as claimed in claim 1, wherein the hybridization probe preparation procedure of S1 comprises:
s11, carrying out PCR amplification on the segmented segments, and recovering an amplification product;
s12, carrying out in-vitro transcription on the amplification product recovered from S11, and collecting a transcription fragment;
s13, respectively carrying out reverse transcription on the transcription fragments of S12, wherein a reverse transcription system comprises dUTP with fluorescent modification, and recovering reverse transcription products.
3. The in situ hybridization method for simultaneous detection of multiple genomic loci according to claim 2, wherein the amplification system in S11 is:
buffer：20μl；
5 ng/. mu.l of primer fragment mixture: 1 mul;
10mM of dNTP-mix: 1 mul;
10 μ M/μ l amplification primer mix: 4 mu l of the solution;
amplification enzyme: 1 mul;
ddH20：12μl；
the amplification procedure in S11 is: at 95 ℃ for 30 s; 30s at 60 ℃; 72 ℃ for 30 s; amplification was performed for 19 cycles.
4. The in situ hybridization method for simultaneous detection of genomic multiple sites by a combination of colors according to claim 2, wherein the reverse transcription system in S13 is:
5 × reverse transcription buffer: 5 mu l of the solution;
dNTP mix：2.8μl；
1mM dUTP with fluorescent modification: 2 mu l of the solution;
reverse transcription primer (20 μ M): 1 mul;
reverse transcriptase mixture: 2 mu l of the solution;
RNA：8.2μl；
the reverse transcription procedure in S13 is: 5min at 25 ℃; at 37 ℃ for 45 min; 85 ℃ for 5 min.
5. The in situ hybridization method for simultaneous detection of multiple genomic sites by a combination of colors as claimed in claim 2, wherein the operations in S13 further comprise purification of single stranded DNA hybridization probe: after the completion of the reverse transcription, 20. mu.l of 0.25M EDTA 0.5M NaOH buffer was added to the reverse transcription system, and the reaction was carried out at 95 ℃ for 10min, followed by recovering the reverse transcription product.
6. The in situ hybridization method for simultaneous genomic multisite detection by a combination of colors according to claim 2 wherein said dUTP with fluorescent modification comprises dUTP with fluorescent modification by a combination of modifications with at least two basic color fluorescent modifiers.
7. The method of claim 6, wherein the total number of dUTPs having fluorescent modifications is N, which is the sum of N1 and at least one value selected from N2 to N;
wherein,
n is the number of selected primary colors, N1 is the number of mono-color modifiable dUTPs, N2 is the number of modified dUTPs of the bi-color label, N3 is the number of modified dUTPs of the tri-color label, and so on, N (N-1) is the number of modified dUTPs of the (N-1) color label, and Nn is the number of modified dUTPs of the N-color label.
8. The in situ hybridization method for simultaneous detection of multiple genomic sites according to claim 1, wherein the preparation of the hybridization sheet in step S2 comprises:
s21, preparing a hybrid sheet of the cells to be detected: collecting a cell sample to be detected, and cleaning; performing warm bath on KCl suspension at 37 ℃ for 30 min; fixing by using a fixing solution; smearing and drying; boiling slices; gradient washing with alcohol;
s22, carrying out hybridization reaction on the hybridization sheet with a fluorescent probe: adding hybridization solution containing a fluorescent probe into the dried slide, sealing the slide, and performing denaturation at 80 ℃ for 5 min; putting the slices into a wet box, and reacting at 37 ℃;
in the hybridization solution, the volume of the fluorescent probe solution accounts for 10%;
and S23, dyeing and sealing.
9. The in situ hybridization method for simultaneous detection of genomic multiple sites by a combination of colors according to claim 1, wherein the imaging operation of step S3 comprises: the hybridized pieces of S2 are photographed by a fluorescence microscope, and then the images are combined to obtain the information of the detected gene or chromosome.
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