Method for detecting chromosome outer circular DNA (deoxyribonucleic acid) in few cells

Abstract

The invention relates to the technical field of nucleic acid detection, in particular to a method for detecting chromosome outer circular DNA in a small number of cells. The method for extracting the circular DNA outside the chromosome in the cell comprises the following steps: performing cell lysis and enzyme digestion treatment on a to-be-detected human cell sample; the number of cells in the human cell sample to be detected is at least 250; the enzyme digestion treatment comprises the following steps: co-treating by adopting exonuclease and restriction endonuclease; the exonuclease is RecBCD, the restriction endonuclease is PacI, and the enzyme activity ratio of the exonuclease to the restriction endonuclease is (0.2-0.4): (0.2-0.4). The invention provides a DNA-extraction-free eccDNA detection method suitable for a trace sample, and efficient detection of eccDNA can be realized by directly carrying out enzyme digestion treatment and rolling circle amplification on a cell lysis product and combining a real-time fluorescence detection technology.

Description

Technical Field

[0001] The present invention relates to the technical field of nucleic acid detection, and in particular to a method for detecting extrachromosomal circular DNA in a small number of cells. Background Art

[0002] Extrachromosomal circular DNA (eccDNA) is a closed circular DNA molecule that exists in eukaryotic cells and is independent of chromatin DNA. The formation mechanism of eccDNA is diverse. Among the specific mechanisms of eccDNA formation through the apoptotic pathway: DNase γ-mediated apoptotic DNA fragmentation is one of the conditions for eccDNA production, and oligonucleosomal DNA fragments randomly ligate through DNA ligase 3 and circularize themselves to form eccDNA. EccDNA plays a role in a variety of biological processes, especially playing a key role in the occurrence, development, heterogeneity, and drug resistance of tumors. For example, it affects gene expression by carrying and amplifying regulatory elements such as oncogenes and enhancers. Due to its appearance frequency in specific pathological states such as tumors being closely related to the disease process, eccDNA is considered a potential biomarker and can be used for disease diagnosis, prognosis judgment, and treatment monitoring.

[0003] However, the analysis of eccDNA faces challenges. Techniques that rely on physical separation such as density gradient centrifugation and gel electrophoresis are usually cumbersome and time-consuming to operate, and often require a large amount of starting samples, which limits their applicability in clinical application scenarios that require processing of trace samples and rapid detection. Although fluorescence in situ hybridization can observe larger eccDNA at the cellular level, its resolution is limited, it is difficult to effectively detect smaller eccDNA molecules, and it can only detect known sequences. Sequencing techniques represented by circle-seq can provide detailed eccDNA sequence information, but their processes involve complex library construction, require a large amount of starting DNA, are costly, and have a large amount of data analysis work; more critically, the sequencing method itself has inherent difficulties in distinguishing circular and linear DNA, and often requires relying on complex pre-purification steps or special bioinformatics algorithms to infer the circular structure. Existing methods generally face the problem of interference from a large amount of linear genomic DNA in the sample, which seriously affects the specificity and accuracy of detection. Summary of the Invention

[0004] In order to solve the problems existing in the prior art, the present invention provides a method for detecting extrachromosomal circular DNA in a small number of cells.

[0005] There are methods in the prior art to remove interference from linear genomic DNA and mtDNA in cell samples, but it is difficult to achieve efficient detection of eccDNA in trace samples for samples with more than tens of thousands of cells. The present invention eliminates the process of purifying eccDNA, directly incubates the cell lysate with nuclease exonucleases and restriction endonucleases, and then performs rolling circle amplification to achieve detection of eccDNAs. The entire process is simple, fast and highly sensitive, and can be applied to a small number of cell samples with only 250 cells.

[0006] In a first aspect, the present invention provides a method for extracting extrachromosomal circular DNA in a cell, comprising: Perform cell lysis and enzyme digestion on the human cell samples to be tested; The number of cells in the human cell sample to be tested is at least 250; The enzyme digestion treatment includes: using a nuclease exonuclease and a restriction endonuclease for joint treatment; the nuclease exonuclease is RecBCD, the restriction endonuclease is PacI, and the enzyme activity ratio of the nuclease exonuclease to the restriction endonuclease is (0.2~0.4): (0.2~0.4).

[0007] For human cell samples, the restriction endonuclease PacI used in the present invention can recognize four specific enzyme cutting sites (TTAATTAA sequences) on human mtDNA, which can specifically cut mtDNA in cell lysate and usually does not cut or rarely cuts nuclear genomic DNA or eccDNA. The exonuclease RecBCD can further degrade the endogenous linear DNA in the sample and the linearized mtDNA fragments generated by the restriction endonuclease cutting mtDNA. The two enzymes can be compatible with each other in an enzyme cutting system to achieve efficient removal of linear DNA and mtDNA.

[0008] Furthermore, the enzyme digestion treatment includes: The cell lysate obtained by cell lysis, rCutSmart buffer, ATP, water, the exonuclease and restriction endonuclease were mixed, incubated at 35-40°C for 1.5-2.5 hours, and then heat-inactivated at 65-75°C for 25-40 minutes.

[0009] Furthermore, based on the entire enzyme digestion system, the enzymatic activity of the exonuclease is 2-5 U / μL; and / or the enzymatic activity of the restriction endonuclease is 2-5 U / μL.

[0010] Further, the cell lysis comprises: Centrifuging the human cell sample to be tested and collecting the precipitate; Mix the cell lysate and then heat it at 93 - 98°C for 3 - 5 minutes; After adding Proteinase K, heat it at 50 - 60°C for 25 - 40 minutes.

[0011] Further, the cell lysate includes: 15 - 25 mM Tris-HCl and 0.05 - 0.15 mM EDTA; and / or, Calculated based on the whole cell lysis system, the enzyme activity of the Proteinase K is 0.01 - 0.1 U / μL.

[0012] Further, the human cell sample includes: plasmid DNA, cellular DNA, and tumor tissue DNA.

[0013] In a second aspect, the present invention provides a method for detecting extrachromosomal circular DNA in cells, including: Extract the extrachromosomal circular DNA in the human cell sample by using the aforementioned method; then perform rolling circle amplification and detection.

[0014] Those skilled in the art know that detecting extrachromosomal circular DNA in cells can be used not only in the field of disease diagnosis or treatment but also for environmental pollution monitoring. The method for detecting extrachromosomal circular DNA in cells provided by the present invention is a method for detecting extrachromosomal circular DNA in cells for non-disease treatment or diagnosis purposes.

[0015] Further, the rolling circle amplification includes: Mix the digestion product obtained by the digestion treatment, Phi29 Buffer, dNTP, random primer, and fluorescent dye, and treat it at 93 - 98°C for 2 - 5 minutes to denature the DNA; Treat it at 23 - 30°C for 2 - 5 minutes for annealing to bind the random primer to the DNA; After adding Phi29 DNA polymerase, perform isothermal amplification at 35 - 40°C.

[0016] Further, the random primer is a thiol-modified random hexamer primer; and / or, calculated based on the whole rolling circle amplification system, the enzyme activity of the Phi29 DNA polymerase is 0.05 - 0.15 U / μL.

[0017] Further, the fluorescent dye includes one or more of FAM, TET, HEX, ROX, Cy3, Cy5, Alexa Fluor, SYBR Green, DAPI, FITC, or Texas Red. SYBR Green is preferably used.

[0018] Furthermore, the detection includes: quantitatively or qualitatively analyzing eccDNA based on the fluorescence detection results.

[0019] The present invention has the following beneficial effects: The present invention establishes a rapid analysis method for eccDNA based on direct cell lysis, which eliminates the DNA purification process, shortens the experimental time, and reduces the loss of trace samples.

[0020] The method for detecting extrachromosomal circular DNA in a small number of cells provided by the present invention can rapidly and ultra-sensitively detect eccDNA in as few as 250 cells, and is suitable for high-throughput analysis of trace samples. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0022] Figure 1 It is a schematic diagram of selective real-time fluorescence amplification provided in Embodiment 1 of the present invention.

[0023] Figure 2 It is a flowchart of the rapid analysis of eccDNA provided in Embodiment 1 of the present invention.

[0024] Figure 3 It is a graph showing the rapid analysis results of eccDNA in trace apoptotic cells provided in Embodiment 1 of the present invention; the left graph is the fluorescence amplification result, and the right graph is the statistical result of the eccDNA copy number.

[0025] Figure 4 It is the analysis result of the simulated mixed sample provided in Embodiment 2 of the present invention; the left graph is the grouping, the middle graph is the fluorescence amplification curve, and the right graph is the linear fitting result. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0026] To make the objectives, technical solutions, and advantages of the present invention clearer, the following will clearly and completely describe the technical solutions in the present invention with reference to the drawings in the present invention. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art without creative efforts based on the embodiments in the present invention belong to the scope of protection of the present invention.

[0027] The experimental methods involved in the following embodiments, unless otherwise specified, are all conventional methods in the art. For example, they can be referred to the experimental manuals in the art or carried out according to the conditions recommended in the manufacturer's instructions.

[0028] In the following examples, the experimental materials and reagents involved, unless otherwise specified, can be obtained from commercial sources.

[0029] In the following examples, the restriction enzyme PacI and exonuclease RecBCD were both purchased from New England Biolabs.

[0030] Example 1 The present invention provides a method for detecting extrachromosomal circular DNA in a small number of cells (as shown in Figure 1 and Figure 2 ), and the process is as follows: Step 1: 293T cells treated with an apoptosis inducer (staurosporine, Stau.) were centrifuged at 2000 rpm for 5 min at room temperature for collection. 15 μL of lysis buffer was added to 1000, 500, 250, 125, and 60 cells respectively. The components of the lysis buffer were: Tris-HCl (pH 8.0) at a final concentration of 20 mM and 0.1 mM EDTA. After heating at 95°C for 3 min, 0.8 U of Proteinase K was added, and the mixture was incubated at 55°C for 30 min and then heated at 95°C for 5 min to inactivate Proteinase K.

[0031] Step 2: Using the above 15 μL of cell lysate as a sample, 2 μL of 10×rCutSmart buffer, 0.5 μL of 10 U / μL restriction enzyme PacI, 0.5 μL of 10 U / μL exonuclease RecBCD, and 2 μL of 10 mM ATP were added and incubated at 37°C for 1 h in a PCR instrument, followed by heat inactivation at 70°C for 30 min.

[0032] Step 3: Prepare a sample denaturation and annealing system. In 10 μL of template, 1×phi29 Buffer, 0.6 mM dNTP, 2 μL of random primers, and 1×SYBR Green Ⅱ were added, and the mixture was denatured at 95°C for 3 min and annealed at 25°C for 3 min in a PCR instrument.

[0033] The random primers used in this example were thiol-modified random hexamer primers (primers composed of six random nucleotides).

[0034] Step 4: Add 1.5 U Equi-phi29 DNA Polymerase, adjust the volume to 40 μL with pure water, and mix well. Add 9 μL of the sample to each well of a 384-well plate and place it in a real-time fluorescence amplification PCR instrument for real-time rolling circle amplification. The amplification program is set as follows: 37°C for 5.25 min per cycle. Repeat the cycle 40 times. After the cycle ends, inactivate at 65°C for 10 min.

[0035] The results are as Figure 3 shown. As can be seen from the results Figure 3 , a normal amplification curve can still be obtained with a minimum of 250 cells, that is, the rolling circle amplification of eccDNA can be carried out normally. And the amplification results of 1000 cells, 500 cells, and 250 cells can be calculated through the Ct value. 398 eccDNAs were obtained from 250 cells by the aforementioned method for subsequent analysis (the calculation of eccDNA can be achieved according to the fitting curve of Example 2). This shows that the method provided by the present invention is applicable to small or even trace amounts of cell samples.

[0036] Example 2 In the present invention, 50 ng of total DNA was respectively mixed with 0 pg, 2 pg, 20 pg, 200 pg, and 2000 pg of plasmid (circular DNA) as simulated mixed samples, and the methods of Step 3 and Step 4 of Example 1 were repeated for analysis, replacing the cell samples.

[0037] The results showed that 50 ng of total DNA was hardly amplified (Ct value was 35). While the simulated samples of 50 ng of total DNA mixed with plasmids were efficiently amplified (Ct value ≤ 29). Among them, 2 pg of plasmid began to be efficiently amplified when the Ct value was 29 ( Figure 4 ). It shows that the detection method can specifically detect plasmids as low as 2 pg in 50 ng of total DNA, that is, when the circular DNA accounts for 0.004% of the total sample amount, it can be detected, and linear DNA will not be amplified, having high specificity and high sensitivity. The present invention further performed linear fitting on the Ct value and the template amount and found that there was a good linear relationship (R 2 = 0.948).

[0038] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements on some of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. A method for extracting extrachromosomal circular DNA in cells, characterized in that, Including: Performing cell lysis and enzymatic digestion on a human cell sample to be tested; The minimum number of cells in the human cell sample to be tested is 250; The enzymatic digestion includes: co-treating with an exonuclease and a restriction endonuclease; The exonuclease is RecBCD, the restriction endonuclease is PacI, and the enzyme activity ratio of the exonuclease to the restriction endonuclease is (0.2 - 0.4):(0.2 - 0.4).

2. The method according to claim 1, wherein The enzymatic digestion includes: Mixing the cell lysate obtained by cell lysis, rCutSmart buffer, ATP, water with the exonuclease and the restriction endonuclease, incubating at 35 - 40 °C for 0.5 - 2.5 hours, and then heat-inactivating at 65 - 75 °C for 25 - 40 minutes.

3. The method according to claim 2, wherein Based on the entire system of the enzymatic digestion, the enzyme activity of the exonuclease is 0.2 - 0.4 U / μL; and / or, the enzyme activity of the restriction endonuclease is 0.2 - 0.4 U / μL.

4. The method according to any one of claims 1-3, characterized in that The cell lysis includes: Centrifuging the human cell sample to be tested and collecting the precipitate; Mixing the cell lysate and then heating at 93 - 98 °C for 3 - 5 minutes; Adding Proteinase K and then heating at 50 - 60 °C for 25 - 40 minutes.

5. The method according to claim 4, wherein The cell lysate includes: 15 - 25 mM Tris-HCl and 0.05 - 0.15 mM EDTA; and / or, Based on the entire cell lysis system, the enzyme activity of Proteinase K is 0.01 - 0.1 U / μL.

6. A method for detecting extrachromosomal circular DNA in cells, characterized in that, Including: Treating a human cell sample by the method according to any one of claims 1 - 5, and then performing rolling circle amplification and detection.

7. The method according to claim 6, characterized in that The rolling circle amplification includes: Mixing the enzymatic digestion product obtained by the enzymatic digestion, Phi29 Buffer, dNTP, random primer and fluorescent dye, and treating at 93 - 98 °C for 2 - 5 minutes to denature the DNA; Treating at 23 - 30 °C for 2 - 5 minutes for annealing to bind the random primer to the DNA; Adding Phi29 DNA polymerase and then performing isothermal amplification at 35 - 40 °C.

8. The method according to claim 7, characterized in that, The random primer is a thiol-modified random hexamer primer; and / or, based on the entire rolling circle amplification system, the enzyme activity of Phi29 DNA polymerase is 0.05 - 0.15 U / μL.

9. The method according to any one of claims 6 - 8, characterized in that, The detection includes: quantitatively or qualitatively analyzing eccDNA through the fluorescence detection result.

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