CN111411146B - Method for detecting and quantifying single-copy extrachromosomal DNA or RNA - Google Patents
Method for detecting and quantifying single-copy extrachromosomal DNA or RNA Download PDFInfo
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- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
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- C12Q1/6841—In situ hybridisation
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Abstract
The invention provides a method for accurately, effectively, stably and practically detecting the single copy number and the single copy position of extrachromosomal gene sequences and viral genes in cells, which comprises the following steps: (1) On a metaphase of a chromosome of a target cell or a cell smear, three primary probes are hybridized and combined with a target gene sequence; (2) hybridization binding using a secondary probe; the secondary probe is a nucleic acid sequence capable of specifically combining with two immediately adjacent primary probe sequences in the three primary probes; (3) adding an amplification probe to bind to the secondary probe; (4) Hybridization binding of the labeled probe bound to the amplification probe; (5) performing microscopic examination under a fluorescence microscope.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a method for detecting and quantifying single copy extrachromosomal DNA or RNA.
Background
Tumors are caused by activation of protooncogenes or loss of oncogene function. Recent studies have found that a large number of genes related to cancer are not on the chromosome, but rather are shed from the chromosome, and become a small circular DNA of several hundred Kb to Mb in size, which is called extrachromosomal DNA (ecDNA). Extrachromosomal DNA is widely present in tumor cells, and the copy number of the extrachromosomal DNA is often high, and the copy number of RNA of the corresponding gene is also high, and the extrachromosomal DNA is expected to become an important marker of tumor heterogeneity and also an important marker of tumor gene therapy and drug resistance along with dynamic changes of cell replication or drug therapy.
Extrachromosomal DNA is found not only in blood but also in other body fluids including pleural effusions. Extrachromosomal DNA is not only associated with tumors, but also may be a mechanism of gene amplification within eukaryotic cells to address crisis, so extrachromosomal DNA may also be present in other cells to address crisis, and its importance and application size may be very broad.
In conventional methods of chromosomal karyotype plus extrachromosomal DNA analysis, in cells that do not grasp the metaphase, substantially no extrachromosomal DNA is detected and the likelihood of analysis in small numbers of circulating tumor cells is much less likely. The DNA microscopy technology cannot meet the accurate requirements of the quantity and the position of DNA because the detection signal of single copy DNA/RNA is not strong enough. The same problem is encountered with in situ detection of viral RNA or viral DNA in virally infected cells.
Thus, there is a great need in the art for an accurate, efficient, simple and feasible method for detecting the single copy number and location of extrachromosomal DNA and RNA in a cell.
Disclosure of Invention
Aiming at the defects and market demands of the prior art, the invention aims to provide an accurate, effective, simple and feasible method for detecting the single copy number and the single copy position of an extrachromosomal gene sequence in a cell. In order to achieve the object, the method provided by the invention comprises the following steps:
(1) On a metaphase of a chromosome of a target cell or a cell smear, three primary probes are hybridized and combined with a target gene sequence; each primary probe is a nucleic acid sequence which is coupled with part of the nucleic acid sequence of the target gene in a pairing way; the target gene sequence comprises DNA or RNA sequence fragments;
(2) Hybridization combination is carried out by utilizing the pairing sequence of the secondary probe and the primary probe obtained in the step (1); the secondary probe is a nucleic acid sequence capable of specifically combining with the pairing sequences of two immediately adjacent primary probes in the three primary probes;
(3) Adding an amplification probe to the product obtained in the step (2) to combine with the secondary probe; the amplification probe is a nucleic acid probe marked by biotin;
(4) Adding and binding labeled probes (such as avidin) that specifically bind to the amplification probes;
(5) Microscopic examination was performed under a fluorescence microscope.
In the step (1), the invention establishes four gene signal amplification points through the combination of three primary probes corresponding to the target gene fragments, so that the specific signals of the target fragments can be amplified, but the non-specific combined signals cannot be amplified.
The invention releases the binding site through the primary probe modification, establishes the bridging and amplifying point of the probe after adjacent hybridization, then carries out the signal amplifying treatment in the step (3) and the signal marking treatment in the step (4), and then can observe and detect through a microscope.
In the present invention, as shown in FIG. 1, one of three primary probes is a target probe (primary probe 1), and the other two are specific signaling probes (primary probes 2, 3). In general, a gene fragment or a circular gene fragment that is separated from a chromosome may cause a failure of stable binding of the probe due to a deletion of one of the specific signaling probes or due to a point mutation or deletion (for example, in the case where the primary probe 1 or the primary probe 2 cannot bind). Because the invention adopts the design of three primary probes, 2 bridging amplification (as shown in figures 1-5) and 4 gene signal amplification points (as shown in figure 6) can be formed, and target gene sequences, including specific point hot spot mutations, can be detected as usual. In the invention, a single primary probe cannot form a signal amplification part, and finally cannot be detected, and only specific gene sequences are amplified and can be detected; this nucleic acid-specific sequence signal amplifies only specific nucleic acid sequences and not background signals that are not specific.
In metaphase of chromosome, extrachromosomal circular DNA can be detected, while in cell smears, the number of related gene or fragment sequences, including DNA and RNA, can be detected. In addition, in cell smears, when a chromosomal control gene or fragment sequence is set, the amount of extrachromosomal DNA and the location in the cell (including whether it is in the nucleus or cytoplasm) can be determined.
In step (3), the amplification probe is a signal-labeled probe, such as a Biotin or a fluorescent-labeled probe, or a Biotin-labeled probe which can further bind to a fluorescent-labeled Avidin protein, on the basis of the binding to the secondary probe, and new binding is continuously generated.
From the above description, it will be understood that the target fragment of the present invention may be one or more, and the target fragment includes a sequence or target of ribonucleic acid or deoxyribonucleic acid.
The metaphase or cell smear of the target cells refers to a sample that can be used for in situ hybridization after washing or immobilization and protease treatment.
As one embodiment of the present invention, the immobilization is an immobilization of a biological sample using methanol or acetic acid; and/or, the protease treatment is a treatment before the hybridization binding using a protease including pepsin.
As a preferred embodiment of the present invention, in step (1), the hybridization is performed in situ by combining hybridization solutions comprising:
sodium citrate buffer at 6-fold dilution, 25% W/W formamide, 0.2% W/W lithium dodecyl sulfate and blocking solution;
the sodium citrate buffer comprises the following components: 0.15mol/L NaCl and 0.0015mol/L sodium citrate;
and/or the number of the groups of groups,
in step (2), the hybridization is combined into in situ hybridization, and the hybridization solution used consists of the following components:
2nmol/L of the pre-amplified fragment of the secondary probe, 20% of W/W formamide, 5 times of sodium citrate buffer, 0.3% of W/W lithium dodecyl sulfate, 10% of dextran sulfate and blocking solution;
the sodium citrate buffer comprises the following components: 0.15mol/L NaCl and 0.0015mol/L sodium citrate.
As a preferable technical scheme of the invention, in the step (1), the hybridization time is 3 hours and the temperature is 40 ℃; and/or, in the step (2), the hybridization time is 30 minutes and the temperature is 40 ℃.
As one embodiment of the present invention, the target fragment is an epidermal growth factor receptor (epidermaI growth factor receptor, abbreviated as EGFR). The sequences of the three primary probes are respectively shown as SEQ ID No.1, SEQ ID No.2 and SEQ ID No. 3; alternatively, the sequences of the three primary probes are respectively shown as SEQ ID No.4, SEQ ID No.5 and SEQ ID No. 6.
The sequences of the three primary probes are specifically:
primary probe 1:
primary probe 2:
primary probe 3:
alternatively, the sequences of the three primary probes are specifically:
primary probe 1:
primary probe 2:
primary probe 3:
as a preferred embodiment of the present invention, in the step (3), the treatment time is 15 minutes, and the concentration of the amplification factor is 2nmol/L.
In the step (4), the fluorescent-labeled probe is biotin, and the hybridization solution used in the hybridization is sodium citrate buffer solution with 5 times of dilution and 0.3% W/W of lithium dodecyl sulfate; the sodium citrate buffer comprises the following components: 0.15mol/L NaCl and 0.0015mol/L sodium citrate.
As a preferable technical scheme of the invention, the washing treatment is required to be carried out at room temperature for three times after the treatment of each step; the washing treatment was carried out using the following washing solutions: sodium citrate buffer at 0.1 fold dilution and 0.03% W/W lithium dodecyl sulfate; the sodium citrate buffer comprises the following components: 0.15mol/L NaCl and 0.0015mol/L sodium citrate.
The invention has the beneficial effects that:
according to the invention, four amplification points are obtained by designing three probes, so that not only is random nonspecific combination of a single probe and a sample prevented, but also the defect that two probes cannot be detected due to the deficiency of any part of the gene sequence before or after the detection is prevented. Meanwhile, the design of three probes is more beneficial to identifying the molecular structure of DNA and RNA and detecting targets comprising gene mutation and gene deletion, and is also beneficial to substituting antibodies and carrying out pathological analysis.
The method for detecting the single copy number and the single copy position of the extrachromosomal gene sequence in the cells is accurate, effective, simple and feasible.
Drawings
FIG. 1 is a schematic diagram of in situ hybridization detection of gene copies of the present invention;
FIG. 2 is a schematic representation of three primary probes of the present invention;
FIG. 3 is a schematic representation of in situ hybridization of a primary probe of the present invention, showing that 3 primary probes specifically bind to a DNA or RNA template;
FIG. 4 is a schematic diagram of a primary probe after enzyme modification binding after in situ hybridization according to the present invention;
FIG. 5 is a schematic illustration of the release of secondary probe binding sites after warming upon hybridization of the primary probe of the present invention;
FIG. 6 is a schematic representation of hybridization of a secondary probe of the present invention, pre-amplification bridge formation and creation of 4 amplification sites;
FIG. 7 is a schematic diagram of the detection principle of the invention, wherein 3 primary probes can form 2 bridging and amplifying bridges, and 4 gene signal amplifying points are used for detecting specific gene sequences;
FIGS. 8 to 12 are diagrams illustrating a method of failing to realize the detection effect of the present invention;
FIG. 13 is a graph showing the fluorescence detection results of example 2 of the present invention, wherein the left side of the graph shows the DNA copy number of 4 EGFR in tumor cells, and the right side of the graph shows the DNA copy number of 2 EGFR in blood cells;
FIG. 14 is a graph showing the results of fluorescence detection in example 3 of the present invention, in which the left and middle parts show that tumor cells have RNA gene expression of a plurality of EGFR, and the right part shows that blood cells have no expression of the gene.
Detailed Description
The present invention is described in detail below by way of examples, which are necessary to be pointed out herein for further illustration of the invention and are not to be construed as limiting the scope of the invention, since numerous insubstantial modifications and adaptations of the invention will occur to those skilled in the art in light of the foregoing disclosure.
The following examples refer to raw material information and abbreviations thereof:
sodium citrate buffer: abbreviated as SSC, the components are: 0.15mol/L NaCl and 0.0015mol/L sodium citrate;
double dilution of the above sodium citrate buffer: 2XSSC
Sodium citrate buffer as described above at 6 fold dilution: 6XSSC
Sodium citrate buffer as described above at 0.1 fold dilution: 0.1XSSC
Blocking solution: 1% BSA (bovine serum albumin solution)
Example 1
The detection object is single copy of single cell EGFR gene (epidermal growth factor receptor) DNA
1. The target cells were centrifuged onto a slide glass, and subjected to fixation treatment using methanol as a fixation solution, followed by air-drying.
2. After RNase enzyme treatment of the target cells, the cells were further treated with 0.005% W/W pepsin at 37℃for 10 minutes, and then washed once with cell buffer.
3. Carrying out DNA denaturation treatment by using 75% W/W formamide and 2XSSC for 5 minutes at 75 ℃, adding 3 adjacent primary probe mixed solutions of EGFR genes, heating for denaturation, and carrying out in-situ hybridization treatment on the treated cells by adopting an in-situ hybridization solution A for 3 hours;
the 3 adjacent primary probes are as follows:
primary probe 1:
primary probe 2:
primary probe 3:
the in situ hybridization solution A consists of the following components:
6XSSC, 25% W/W formamide, 0.2% W/W lithium dodecyl sulfate and blocking solution;
4. the in situ hybridization solution B containing the gene pre-amplification of the secondary probes (designated as secondary probe 1, secondary probe 2, and secondary probe 3) coupled to the above primary probe pair was treated for 30 minutes.
The in situ hybridization solution B comprises the following components:
2nmol/L of the pre-amplified fragment of the secondary probe, 20% W/W formamide, 5XSSC, 0.3% W/W lithium dodecyl sulfate, 10% dextran sulfate and blocking solution.
5. 2nmol/L of the amplification factor was added to the product obtained in step 4, and the mixture was treated at 40℃for 15 minutes.
6. Treating for 15 min with fluorescent labeling probe (Biotin labeling) capable of binding to the secondary probe amplification site, and then binding with fluorescent labeling protein of fluorescent labeling streptavidine and Biotin of the labeling probe to obtain fluorescent labeling signal of in-situ gene; the hybridization solution used comprises the following components:
2nmol/L of fluorescent labeled probe, 5XSSC, 0.3% W/W lithium dodecyl sulfate and blocking solution.
Note that, in carrying out the above hybridization step, the washing solution was used for washing three times at room temperature. The washing solutions used were: 0.1XSSC and 0.03% W/W lithium dodecyl sulfate.
7. And (3) microscopic examination, fluorescence imaging and software treatment are carried out, and the DNA copy number of EGFR is counted to obtain a detection report.
Example 2
DNA molecule of EGFR gene of tumor cell single cell as detection object
1. The target cells were centrifuged onto a slide glass, and subjected to fixation treatment using methanol as a fixation solution, followed by air-drying.
2. After RNase enzyme treatment of the target cells, the cells were further treated with 0.005% W/W pepsin at 37℃for 10 minutes, and then washed once with cell buffer.
3. Carrying out DNA denaturation treatment by using 75% W/W formamide and 2XSSC for 5 minutes at 75 ℃, adding 3 adjacent primary probe mixed solutions of EGFR genes, heating for denaturation, and carrying out in-situ hybridization treatment on the treated cells by adopting an in-situ hybridization solution A for 3 hours;
the 3 adjacent primary probes are as follows:
primary probe 1:
primary probe 2:
primary probe 3:
the in situ hybridization solution A consists of the following components:
6XSSC, 25% W/W formamide, 0.2% W/W lithium dodecyl sulfate and blocking solution;
4. the in situ hybridization solution B containing the gene pre-amplification of the secondary probes (designated as secondary probe 1, secondary probe 2, and secondary probe 3) coupled to the above primary probe pair was treated for 30 minutes.
The in situ hybridization solution B comprises the following components:
2nmol/L of the pre-amplified fragment of the secondary probe, 20% W/W formamide, 5XSSC, 0.3% W/W lithium dodecyl sulfate, 10% dextran sulfate and blocking solution.
5. 2nmol/L of the amplification factor was added to the product obtained in step 4, and the mixture was treated at 40℃for 15 minutes.
6. Treating for 15 min with fluorescent labeling probe (Biotin labeling) capable of binding to the secondary probe amplification site, and then binding with fluorescent labeling protein of fluorescent labeling streptavidine and Biotin of the labeling probe to obtain fluorescent labeling signal of in-situ gene; the hybridization solution used comprises the following components:
2nmol/L of fluorescent labeled probe, 5XSSC, 0.3% W/W lithium dodecyl sulfate and blocking solution.
Note that, in carrying out the above hybridization step, the washing solution was used for washing three times at room temperature. The washing solutions used were: 0.1XSSC and 0.03% W/W lithium dodecyl sulfate.
7. And (3) after microscopic examination and fluorescent imaging software treatment, counting the DNA copy number of EGFR to obtain a detection report.
In this example, tumor cells were the experimental group and blood cells were the control group. The results are shown in FIG. 13, where the left side shows that tumor cells have 4 EGFR DNA copies, and the right side shows that blood cells have only 2 EGFR DNA copies.
Example 3
The detection object is single copy of single cell RNA of circulating tumor cells
1. The target cells were centrifuged onto a slide glass, and subjected to fixation treatment using methanol as a fixation solution, followed by air-drying.
2. Target cells were treated with 0.005% W/W pepsin at 37 ℃ for 10 min and then washed once with cell buffer.
3. Carrying out DNA denaturation treatment by using 75% W/W formamide and 2XSSC for 5 minutes at 75 ℃, adding 3 adjacent primary probe mixed solutions of EGFR genes, heating for denaturation, and carrying out in-situ hybridization treatment on the treated cells by adopting an in-situ hybridization solution A for 3 hours;
the 3 adjacent primary probes are as follows:
primary probe 1:
primary probe 2:
primary probe 3:
4. the in situ hybridization solution B containing the gene pre-amplification of the secondary probes (designated as secondary probe 1, secondary probe 2, and secondary probe 3) coupled to the above primary probe pair was treated for 30 minutes.
The in situ hybridization solution B comprises the following components:
2nmol/L of the pre-amplified fragment of the secondary probe, 20% W/W formamide, 5XSSC, 0.3% W/W lithium dodecyl sulfate, 10% dextran sulfate and blocking solution.
5. 2nmol/L of the amplification factor was added to the product obtained in step 4, and the mixture was treated at 40℃for 15 minutes.
6. Treating for 15 min with fluorescent labeling probe (Biotin labeling) capable of binding to the secondary probe amplification site, and then binding with fluorescent labeling protein of fluorescent labeling streptavidine and Biotin of the labeling probe to obtain fluorescent labeling signal of in-situ gene; the hybridization solution used comprises the following components:
2nmol/L of fluorescent labeled probe, 5XSSC, 0.3% W/W lithium dodecyl sulfate and blocking solution.
Note that, in carrying out the above hybridization step, the washing solution was used for washing three times at room temperature. The washing solutions used were: 0.1XSSC and 0.03% W/W lithium dodecyl sulfate.
8. And (3) microscopic examination, fluorescence imaging and software treatment are carried out, and the DNA copy number of EGFR is counted to obtain a detection report.
In this example, tumor cells were the experimental group and blood cells were the control group
As a result, as shown in FIG. 14, tumor cells had the RNA genes of a plurality of EGFR expressed (left and middle portions of the figure), whereas blood cells had no expression of the genes (right portion of the figure)
Sequence listing
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<120> a method for detecting and quantifying single copy of extrachromosomal DNA or RNA
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<213> Artificial sequence (Artificial Sequence)
<400> 1
gttggtgact ttgattttcc tacacaaata aaattggaga aaatctaagt ggagaaaggc 60
ctgggcagaa ttccacttga agtgtgttta tttttgctat 100
<210> 2
<211> 100
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 2
ttttctcagc ctccagagga tgttcaataa ctgtgaggtg gtccttggga atttggaaat 60
tacctatgtg cagaggaatt atgatctttc cttcttaaag 100
<210> 3
<211> 100
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 3
ggcaatgaca agtcttacag agctacaaac gagagtttta tgagaaagcc attttaccag 60
ctaatgtcaa gtaataacta gaaaaggata tcaaatagaa 100
<210> 4
<211> 100
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 4
atttggaaat tacctatgtg cagaggaatt atgatctttc cttcttaaag accatccagg 60
aggtggctgg ttatgtcctc attgccctca acacagtgga 100
<210> 5
<211> 100
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 5
tgccaaggca cgagtaacaa gctcacgcag ttgggcactt ttgaagatca ttttctcagc 60
ctccagagga tgttcaataa ctgtgaggtg gtccttggga 100
<210> 6
<211> 100
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 6
gcgaattcct ttggaaaacc tgcagatcat cagaggaaat atgtactacg aaaattccta 60
tgccttagca gtcttatcta actatgatgc aaataaaacc 100
Claims (7)
1. A method for in situ detection and quantification of single copy extrachromosomal DNA or RNA for non-diagnostic purposes, comprising the steps of:
(1) On a metaphase of a chromosome of a target cell or a cell smear, three primary probes are hybridized and combined with a target gene sequence; each primary probe is a nucleic acid sequence which is coupled with part of the nucleic acid sequence of the target gene in a pairing way; the target gene sequence comprises DNA or RNA sequence fragments;
(2) Hybridization combination is carried out by utilizing the pairing sequence of the secondary probe and the primary probe obtained in the step (1); the secondary probe is a nucleic acid sequence capable of specifically combining with the pairing sequences of two immediately adjacent primary probes in the three primary probes;
(3) Adding an amplification probe to the product obtained in the step (2) to combine with the secondary probe; the amplification probe is a nucleic acid probe marked by biotin;
(4) Adding a labeled probe which specifically binds to the amplification probe and binding the labeled probe;
(5) Performing microscopic examination under a fluorescence microscope;
wherein the target gene is an epidermal growth factor receptor gene;
the three primary probes comprise a primary probe 1 serving as a target probe, and a primary probe 2 and a primary probe 3 serving as specific signal probes;
the sequences of the three primary probes are respectively shown as SEQ ID No.1, SEQ ID No.2 and SEQ ID No. 3; alternatively, the sequences of the three primary probes are respectively shown as SEQ ID No.4, SEQ ID No.5 and SEQ ID No. 6.
2. The method of claim 1, wherein the metaphase or cell smear of the target cells is a sample that is washed or immobilized and treated with a protease for in situ hybridization.
3. The method according to claim 2, wherein the immobilization is of biological samples with methanol or acetic acid; and/or, the protease treatment is a treatment before the hybridization binding using a protease including pepsin.
4. The method of claim 1, wherein in step (1), the hybridization is performed in situ by combining hybridization solutions comprising:
sodium citrate buffer at 6-fold dilution, 25% W/W formamide, 0.2% W/W lithium dodecyl sulfate and blocking solution;
the sodium citrate buffer comprises the following components: 0.15mol/L NaCl and 0.0015mol/L sodium citrate;
and/or the number of the groups of groups,
in step (2), the hybridization is combined into in situ hybridization, and the hybridization solution used consists of the following components:
2nmol/L of the pre-amplified fragment of the secondary probe, 20% of W/W formamide, 5 times of sodium citrate buffer, 0.3% of W/W lithium dodecyl sulfate, 10% of dextran sulfate and blocking solution;
the sodium citrate buffer comprises the following components: 0.15mol/L NaCl and 0.0015mol/L sodium citrate.
5. The method of claim 4, wherein in step (1), the hybridization is performed for 3 hours at 45 ℃; and/or, in the step (2), the hybridization time is 30 minutes and the temperature is 45 ℃.
6. The method according to claim 1, wherein in step (4), the fluorescent-labeled probe is biotin, and the hybridization solution used is composed of 5-fold dilution of sodium citrate buffer and 0.3% W/W of lithium dodecyl sulfate; the sodium citrate buffer comprises the following components: 0.15mol/L NaCl and 0.0015mol/L sodium citrate.
7. The method according to claim 1, wherein the treatment in each step is carried out by three washing treatments at room temperature; the washing treatment is carried out with the following components of the washing solution: sodium citrate buffer at 0.1 fold dilution and 0.03% W/W lithium dodecyl sulfate; the sodium citrate buffer comprises the following components: 0.15mol/L NaCl and 0.0015mol/L sodium citrate.
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Citations (3)
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|---|---|---|---|---|
| US6242184B1 (en) * | 1988-10-18 | 2001-06-05 | University Of Massachusetts | In-situ hybridization of single-copy and multiple-copy nucleic acid sequences |
| CN102653789A (en) * | 2012-03-14 | 2012-09-05 | 河北大学 | Quantitative biomolecule detection method |
| CN110730825A (en) * | 2017-05-23 | 2020-01-24 | 新泽西鲁特格斯州立大学 | Target-mediated in situ signal amplification with dual-phase interacting hairpin probes |
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| CA2940111C (en) * | 2014-02-21 | 2022-03-15 | Ventana Medical Systems, Inc. | Single-stranded oligonucleotide probes for chromosome or gene copy enumeration |
| WO2017189525A1 (en) * | 2016-04-25 | 2017-11-02 | President And Fellows Of Harvard College | Hybridization chain reaction methods for in situ molecular detection |
| US20190382838A1 (en) * | 2018-06-01 | 2019-12-19 | The Trustees Of The University Of Pennsylvania | Methods For Single-Molecule Fluorescence Amplification Of RNA |
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|---|---|---|---|---|
| US6242184B1 (en) * | 1988-10-18 | 2001-06-05 | University Of Massachusetts | In-situ hybridization of single-copy and multiple-copy nucleic acid sequences |
| CN102653789A (en) * | 2012-03-14 | 2012-09-05 | 河北大学 | Quantitative biomolecule detection method |
| CN110730825A (en) * | 2017-05-23 | 2020-01-24 | 新泽西鲁特格斯州立大学 | Target-mediated in situ signal amplification with dual-phase interacting hairpin probes |
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