阅读说明：本技术 一种检测和定量单拷贝染色体外dna或rna的方法 (Method for detecting and quantifying single-copy extrachromosomal DNA or RNA ) 是由 邓亚光 于 2020-03-31 设计创作，主要内容包括：本发明提供了一种准确、有效、稳定可行的检测细胞中染色体外基因序列和病毒基因的单拷贝数量和部位的方法,所述方法包括如下步骤：(1)在靶细胞的染色体中期相或细胞涂片上,利用三个一级探针与目标基因序列杂交结合；(2)利用二级探针进行杂交结合；所述二级探针为可与三个上述一级探针中的紧邻的两个一级探针序列特异性结合的核酸序列；(3)加入扩增探针,与二级探针相结合；(4)将与扩增探针相结合的标记探针进行杂交结合；(5)于荧光显微镜下进行镜检。(The invention provides a method for accurately, effectively, stably and feasible detecting the single copy quantity and the single copy position of an extrachromosomal gene sequence and a virus gene in a cell, which comprises the following steps: (1) hybridizing and combining three primary probes with a target gene sequence in the metaphase of chromosomes or cell smear of a target cell; (2) performing hybridization and combination by using a secondary probe; the secondary probe is a nucleic acid sequence which can be specifically combined with two adjacent primary probe sequences in three primary probes; (3) adding an amplification probe, and combining with the secondary probe; (4) carrying out hybridization combination on the labeled probe combined with the amplification probe; (5) microscopic examination was performed under a fluorescent microscope.)

1. A method for in situ detection and quantification of single copy extrachromosomal DNA or RNA, comprising the steps of:

(1) hybridizing and combining three primary probes with a target gene sequence in the metaphase of chromosomes or cell smear of a target cell; each primary probe is a nucleic acid sequence which is matched and combined with a partial nucleic acid sequence in a target gene; the target gene sequence comprises a DNA or RNA sequence segment;

(2) hybridizing and combining a secondary probe and a pairing sequence of the primary probe obtained in the step (1); the secondary probe is a nucleic acid sequence which can be specifically combined with the pairing sequences of two adjacent primary probes in the three primary probes;

(3) adding an amplification probe into the product obtained in the step (2) to be combined with a secondary probe; the amplification probe is a nucleic acid probe marked by biotin;

(4) adding a labeled probe specifically bound to the amplification probe and allowing it to bind;

(5) microscopic examination was performed under a fluorescent microscope.

2. The method of claim 1, wherein the metaphase or cell smear of the target cell is a sample that can be used for in situ hybridization after washing or fixation and protease treatment.

3. The method of claim 2, wherein the immobilization is of the biological sample using methanol or acetic acid; and/or the protease treatment is a treatment before the hybridization binding using a protease including pepsin.

4. The method according to claim 1, wherein in step (1), the hybridization is combined as in-situ hybridization, and a hybridization solution is used, which comprises the following components:

6 times of dilution times of sodium citrate buffer solution, 25% of formamide in a W/W mode, 0.2% of lithium dodecyl sulfate in a W/W mode and blocking solution;

the sodium citrate buffer solution comprises 0.15 mol/L NaCl and 0.0015 mol/L sodium citrate;

and/or the presence of a gas in the gas,

in the step (2), the hybridization is combined as in-situ hybridization, and the used hybridization solution consists of the following components:

2 nmol/L of pre-amplified fragment of secondary probe, 20% W/W formamide, 5 times of dilution times of sodium citrate buffer solution, 0.3% W/W lithium dodecyl sulfate, 10% dextran sulfate and blocking solution;

the sodium citrate buffer solution comprises 0.15 mol/L NaCl and 0.0015 mol/L sodium citrate.

5. The method according to claim 4, wherein in the step (1), the hybridization time is 3 hours and the temperature is 45 ℃; and/or, in the step (2), the hybridization time is 30 minutes and the temperature is 45 ℃.

6. The method according to any one of claims 1 to 5, wherein the fragment of interest is epidermal growth factor receptor.

7. The method according to claim 6, wherein the sequences of the three primary probes are shown in SEQ ID No.1, SEQ ID No.2 and SEQ ID No.3, respectively; or, the sequences of the three primary probes are respectively shown as SEQ ID No.4, SEQ ID No.5 and SEQ ID No. 6.

8. The method according to claim 1, wherein in the step (3), the treatment time is 15 minutes, and the concentration of the amplification factor is 2 nmol/L.

9. The method according to claim 1, wherein in the step (4), the fluorescently labeled probe is biotin when the hybridization binding is performed, and the hybridization solution is 5-fold dilution of sodium citrate buffer solution and 0.3% W/W of lithium dodecyl sulfate, wherein the sodium citrate buffer solution comprises 0.15 mol/L NaCl and 0.0015 mol/L sodium citrate.

10. The method of claim 1, wherein the washing treatment is carried out three times at room temperature after each step, and the washing treatment is carried out by using 0.1-fold dilution of sodium citrate buffer solution and 0.03% W/W of lithium dodecyl sulfate, wherein the sodium citrate buffer solution comprises 0.15 mol/L NaCl and 0.0015 mol/L sodium citrate.

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 proto-oncogenes or by loss of function of oncogenes. Recent studies have found that a large number of cancer-related genes are not on the chromosome but are shed from the chromosome, and become a small circular DNA with a size varying from several hundred Kb to Mb, and is called extrachromosomal DNA (ecDNA). Extrachromosomal DNA is widely present in tumor cells, their copy number is often relatively high, RNA copy number of the corresponding gene may also be relatively high, and extrachromosomal DNA is expected to become an important marker of tumor heterogeneity, and also may become an important marker of tumor gene therapy and drug resistance, as cell replication or drug therapy dynamically changes.

Extrachromosomal DNA is not only present in blood, but is also found in other body fluids, including pleural effusions. The extrachromosomal DNA is not only related to tumors, but also possibly serves as a gene amplification mechanism in eukaryotic cells for coping with crisis, so that the extrachromosomal DNA also possibly exists in other cells for coping with crisis, and the importance and the application range of the extrachromosomal DNA are possibly very wide.

Traditional methods of analyzing chromosomal karyotypes plus extrachromosomal DNA are essentially undetectable in cells that do not hold metaphase, and are much more improbable in small numbers of circulating tumor cells. In the DNA microscope technology, the detection signal of single copy DNA/RNA is not strong enough, so that the precision requirements of the quantity and the position of DNA cannot be met. The same problem is encountered with the in situ detection of viral RNA or viral DNA in virus-infected cells.

Therefore, there is a need in the art for an accurate, efficient, simple and feasible method for detecting the amount and location of single copies of extrachromosomal DNA and RNA in cells.

Disclosure of Invention

In view of the shortcomings of the prior art and the market demand, the present invention aims to provide an accurate, effective, simple and feasible method for detecting the single copy number and the position of the extrachromosomal gene sequence in cells. In order to achieve the object, the invention provides a method comprising the steps of:

(1) hybridizing and combining three primary probes with a target gene sequence in the metaphase of chromosomes or cell smear of a target cell; each primary probe is a nucleic acid sequence which is matched and combined with a partial nucleic acid sequence in a target gene; the target gene sequence comprises a DNA or RNA sequence segment;

(2) hybridizing and combining a secondary probe and a pairing sequence of the primary probe obtained in the step (1); the secondary probe is a nucleic acid sequence which can be specifically combined with the pairing sequences of two adjacent primary probes in the three primary probes;

(3) adding an amplification probe into the product obtained in the step (2) to be combined with a secondary probe; the amplification probe is a nucleic acid probe marked by biotin;

(4) adding a labeled probe (e.g., avidin) that specifically binds to the amplification probe and allowing it to bind;

(5) microscopic examination was performed under a fluorescent microscope.

In the step (1), the invention establishes four gene signal amplification points by combining with three primary probes corresponding to the target gene fragment, so that the specific signal of the target fragment is amplified, and the non-specific binding signal cannot be amplified.

The invention releases the binding sites through the modification of the primary probe, establishes bridging and amplification points of adjacent hybridized probes, and then can carry out observation and detection through the signal amplification treatment in the step (3) and the signal labeling treatment in the step (4) and then a microscope.

In the present invention, as shown in FIG. 1, one of the three primary probes is used as a target probe (primary probe 1), and the other two are used as specific signaling probes (primary probes 2 and 3). In general, a gene fragment or circular gene fragment that is out of the chromosome may not be stably bound to a probe (for example, in the case where the primary probe 1 or the primary probe 2 is not bound to the probe) due to deletion of one of the specific signaling probes or due to point mutation or deletion. Because the invention adopts the design of three primary probes, 2 amplification bridges (shown in figures 1-5) and 4 gene signal amplification points (shown in figure 6) can be formed, so that the target gene sequence including the specific point hot spot mutation can be detected as usual. In the invention, a single primary probe cannot form a signal amplification part and cannot be detected finally, and only a specific gene sequence is subjected to signal amplification and can be detected; this nucleic acid specific sequence signal is amplified by amplifying only the specific nucleic acid sequence, but not the non-specific background signal.

During metaphase, 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, if 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 fluorescent labeled probe, or a Biotin label that further binds to a fluorescently labeled Avidin protein, based on the binding of the amplification probe to the secondary probe, and new binding is continuously generated.

As can be understood from the above description, the target fragment of the present invention may be one or more, and the target fragment includes a sequence of ribonucleic acid or deoxyribonucleic acid or a target.

The metaphase or cell smear of the target cell refers to a sample which can be used for in situ hybridization after washing or fixing and protease treatment.

As an embodiment of the present invention, the immobilization is 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 technical scheme of the invention, in the step (1), the hybridization binding is in situ hybridization, and a hybridization solution is composed of the following components:

6 times of dilution times of sodium citrate buffer solution, 25% of formamide in a W/W mode, 0.2% of lithium dodecyl sulfate in a W/W mode and blocking solution;

the sodium citrate buffer solution comprises 0.15 mol/L NaCl and 0.0015 mol/L sodium citrate;

and/or the presence of a gas in the gas,

in the step (2), the hybridization is combined as in-situ hybridization, and the used hybridization solution consists of the following components:

2 nmol/L of pre-amplified fragment of secondary probe, 20% W/W formamide, 5 times of dilution times of sodium citrate buffer solution, 0.3% W/W lithium dodecyl sulfate, 10% dextran sulfate and blocking solution;

the sodium citrate buffer solution comprises 0.15 mol/L NaCl and 0.0015 mol/L sodium citrate.

In a preferred embodiment of the present invention, in 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 epidermal growth factor receptor (EGFR for short). The sequences of the three primary probes are respectively shown as SEQ ID No.1, SEQ ID No.2 and SEQ ID No. 3; or, 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 respectively as follows:

a primary probe 1:

a primary probe 2:

a primary probe 3:

or, the sequences of the three primary probes are respectively:

a primary probe 1:

a primary probe 2:

a primary probe 3:

in 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 2 nmol/L.

As a preferable technical scheme of the invention, in the step (4), when the hybridization binding is carried out, the fluorescence labeling probe is biotin, the used hybridization solution is a sodium citrate buffer solution with 5-fold dilution and 0.3% W/W of lithium dodecyl sulfate, and the sodium citrate buffer solution comprises 0.15 mol/L NaCl and 0.0015 mol/L sodium citrate.

As a preferred technical scheme of the invention, after each step of treatment, washing treatment needs to be carried out at room temperature for three times, and when the washing treatment is carried out, the used washing solutions comprise 0.1-time dilution multiple sodium citrate buffer solution and 0.03% W/W lithium dodecyl sulfate, and the sodium citrate buffer solution comprises 0.15 mol/L NaCl and 0.0015 mol/L sodium citrate.

The invention has the beneficial effects that:

the invention obtains four amplification points by designing three probes, thereby not only preventing random non-specific combination of a single probe and a sample, but also preventing the defect that two probes cannot be detected due to deletion of any part of gene sequences before or after the two probes. Meanwhile, the design of the three probes is more favorable for identifying molecular structures of DNA and RNA and detecting target spots including gene mutation and gene deletion, and is also favorable for replacing antibodies to perform pathological analysis.

The invention is a method for detecting the single copy quantity and the single copy position of the extrachromosomal gene sequence in the cell accurately, effectively, simply and feasibly.

Drawings

FIG. 1 is a schematic diagram of the detection of gene copy in situ hybridization according to 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 the primary probes 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 diagram of the release of the binding sites of the secondary probe after temperature rise during hybridization of the primary probe according to the present invention;

FIG. 6 is a schematic diagram of the hybridization of the secondary probe, the formation of pre-amplification bridge and the establishment of 4 amplification sites according to the present invention;

FIG. 7 is a schematic diagram of the detection principle of the present invention, in which 3 primary probes can form 2 bridges with amplification and 4 amplification sites for gene signal detection of specific gene sequences;

FIGS. 8-12 are diagrams illustrating the method of detecting the effect of the present invention;

FIG. 13 is a graph showing the results of fluorescence detection in example 2 of the present invention, in which the left side shows that tumor cells have 4 DNA copy numbers of EGFR, and the right side shows that blood cells have only 2 DNA copy numbers of EGFR;

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 multiple RNA gene expressions of EGFR, and the right part shows that blood cells do not have the expression of the gene.

Detailed Description

The present invention is described in detail below by way of examples, and it should be noted that the following examples are only for illustrating the present invention and should not be construed as limiting the scope of the present invention.

The following examples relate to the information on the raw materials and their abbreviations:

sodium citrate buffer solution (SSC for short) containing 0.15 mol/L NaCl and 0.0015 mol/L sodium citrate;

two-fold dilution of the sodium citrate buffer described above: 2XSSC

6-fold dilution of the sodium citrate buffer: 6XSSC

0.1-fold dilution of the sodium citrate buffer: 0.1XSSC

Blocking liquid: 1% BSA (bovine serum albumin solution)