Method for determining multiple functional indexes and mutations of single-cell mitochondria

Abstract

The invention relates to the technical field of biology, in particular to a method for determining multiple functional indexes and mutations of single-cell mitochondria. At the single cell level, the scheme can realize simultaneous detection of a plurality of mitochondrial indexes; in the cell level, the scheme can comprehensively evaluate the mitochondrial function and reduce the requirement for the sample size, and the problems that the number of rare sample cells is low and data acquisition is difficult are effectively solved. The mitochondrial DNA sequence of the cell is subjected to phenotype-genotype correlation analysis. According to the scheme, the mutation site screening accuracy can be improved while high mitochondrial heterogeneity is achieved.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a method for determining multiple functional indicators and mutations in single-cell mitochondria. Background Technology

[0002] Mitochondria are vital organelles in eukaryotic cells, serving as the cell's energy factories. They provide 90% of the ATP required for cellular metabolism and participate in crucial cellular functions such as regulating intracellular calcium homeostasis, cell proliferation, substance synthesis, and reactive oxygen species (ROS) levels. The number and metabolic characteristics of mitochondria vary among different cell types and under different physiological states (tissues are aggregates of different cell types and normal / abnormal cells, exhibiting significant mitochondrial heterogeneity). Each mitochondrion contains 2-10 closed circular mitochondrial DNA molecules (mtDNA). Human mitochondrial DNA encodes 33 genes, including 13 subunits of the oxidative phosphorylation (OXPHOS) complex, 2 rRNA genes, and 22 tRNA genes. Due to its susceptibility to oxidative damage and the lack of histone protection and gene repair mechanisms, the mutation rate of mitochondrial DNA is 10-17 times higher than that of nuclear DNA. When mitochondrial genomic heterogeneity reaches a certain level, it can lead to impaired cellular oxidative respiration, subsequently causing disease.

[0003] Currently, dozens of diseases caused by mitochondrial mutations have been identified, including MELAS syndrome, Leigh syndrome, mitochondrial myopathy, and mitochondrial encephalopathy. Mitochondrial mutations have also been found to be associated with many human diseases such as aging, Alzheimer's disease, Parkinson's disease, and diabetes. Currently, mitochondrial diseases are incurable; existing treatments only alleviate specific symptoms. Early detection of mitochondrial markers and gene mutations could aid in early diagnosis, improving cure rates and quality of life for patients with mitochondrial diseases.

[0004] With the development of next-generation sequencing technology, gene sequencing has been widely used in the screening and mechanistic research of genetic diseases. However, mitochondrial gene sequencing still faces some challenges. The human genome consists of more than 3 billion base pairs, while mitochondrial DNA is only 16,569 bp in length. If whole-genome sequencing is performed first, and then the mitochondrial DNA sequence obtained by comparison with a reference genome is further analyzed, the large amount of nuclear DNA sequencing data and pseudogenes can interfere with mitochondrial DNA mutation screening. Isolating mitochondria from cells requires methods such as density gradient centrifugation, which is relatively complex and requires a large sample volume. Currently, some researchers are attempting to construct mitochondrial genome-specific libraries based on multiplex PCR technology, but problems such as excessively long amplicon lengths, heterogeneous products, and interference from pseudogenes in the nuclear genome exist. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the present invention aims to provide a rapid and efficient method for determining multiple functional indicators and mutations in single-cell mitochondria. This method allows for the free selection of multiple mitochondrial indicators for combined detection and single-cell mitochondrial sequencing as needed.

[0006] To address the aforementioned technical problems, the present invention provides the following technical solution: a method for determining multiple functional indicators and mutations in single-cell mitochondria, wherein the indicators include cell viability, mitochondrial copy number, mitochondrial membrane potential, and reactive oxygen species; DAPI dye is used for cell viability; and MitoTracker is used for mitochondrial copy number. TM Deep Red FM (ThermoFisher) dye; MitoTracker was used to target mitochondrial membrane potential. TM Red CMXRos (ThermoFisher) dye, Rhodamine123 dye, and CellROX Green dye; MitoSOX Red (ThermoFisher) dye and DCFH dye are used for reactive oxygen species.

[0007] Furthermore, a first dye combination is used, which is co-stained with a flow cytometry antibody that does not require permeabilization, including:

[0008] DAPI dyes are used to determine cell viability;

[0009] DCFH dyes are used to target reactive oxygen species.

[0010] MitoTracker was used to target mitochondrial membrane potential. TM Red CMX dye;

[0011] MitoTracker was used to target mitochondrial copy number. TM Deep Red FM dye.

[0012] Furthermore, the preparation and dyeing method of the first dye combination includes the following steps:

[0013] S1.1: Take a 1.5 mL or 2 mL centrifuge tube and keep it protected from light. Add 1 mL of PBS to the centrifuge tube.

[0014] S1.2: Add 1.5 μL DCFH and 0.15 μL LitoTracker to the centrifuge tube. TM Red CMX and 0.15μM MitoTracker TM Deep Red FM, mix well to prepare a mixed staining working solution;

[0015] S1.3: Take 100 μL of mixed staining working solution and add single-cell sample precipitate, then mix by pipetting and place it in a dark environment at 37°C for 30 min, and mix it once every preset time period during the process.

[0016] S1.4: Add 1 μL of DAPI to 10 μL of PBS and mix well to prepare DAPI dilution solution. Add 1 μL of DAPI dilution solution to a single cell sample and mix well. Stain the sample at 37°C for 3 min in the dark.

[0017] S1.5: After staining, centrifuge at 500g for 5min, remove the supernatant, then add 100μL PBS for rinsing and mix by pipetting. After mixing, centrifuge at 500g for 5min, remove the supernatant, and obtain the first dye combination.

[0018] Furthermore, a second dye combination is used, which is co-stained with a flow cytometry antibody that does not require permeabilization, including:

[0019] DAPI dyes are used to determine cell viability;

[0020] Rhodamine 123 dye was used to target mitochondrial membrane potential;

[0021] MitoSOX Red dye was used to target reactive oxygen species.

[0022] MitoTracker was used to target mitochondrial copy number. TM Deep Red FM dye.

[0023] Furthermore, the preparation and dyeing method of the second dye combination includes the following steps:

[0024] S2.1: Take a 1.5 mL centrifuge tube and keep it protected from light, then add 1 mL of PBS to the centrifuge tube;

[0025] S2.2: Add 2 μL L hodamine 123, 2 μL Mito SOX Red, and 0.15 μL Mito Tracker to the centrifuge tube. TM Deep Red FM, mix well to prepare a mixed staining working solution;

[0026] S2.3: Take 100 μL of mixed staining working solution and add single-cell sample precipitate, then mix by pipetting and place it in a dark environment at 37℃ for 30 min, and mix it once every preset time period during the process.

[0027] S2.4: Add 1 μL of DAPI to 10 μL of PBS and mix well to prepare DAPI dilution solution. Add 1 μL of DAPI dilution solution to a single cell sample and mix well. Stain the sample at 37°C for 3 min in the dark.

[0028] S2.5: After staining, centrifuge at 500g for 5 min, remove the supernatant, then add 100μL PBS for rinsing and mix by pipetting. After mixing, centrifuge at 500g for 5 min, remove the supernatant, and obtain the second dye combination.

[0029] Furthermore, a third dye combination is employed, which is co-stained with the flow cytometry antibody requiring permeation, including: DAPI dye for cell viability; CellROX Green dye for mitochondrial membrane potential; and MitoTracker dye for reactive oxygen species. TM Red CMX dye; MitoTracker was used to target mitochondrial copy number. TM Deep Red FM dye.

[0030] Furthermore, the preparation and dyeing method of the third dye combination includes the following steps:

[0031] S3.1: Take a 1.5 mL centrifuge tube and keep it protected from light. Add 1 mL of PBS to the centrifuge tube.

[0032] S3.2: Add 2 μL CellROX Green and 0.15 μL MitoTracker to the centrifuge tube. TM Red CMX and 0.15μM MitoTracker TM Deep Red FM, mix well to prepare a mixed staining working solution;

[0033] S3.3: Add 100 μL of mixed staining working solution to the single-cell sample precipitate, then mix by pipetting, and place it in a dark environment at 37°C for 30 min for staining, mixing once every preset time period during the process;

[0034] S3.4: Add 1 μL of DAPI to 10 μL of PBS and mix well to prepare DAPI dilution solution. Add 1 μL of DAPI dilution solution to a single cell sample and mix well. Stain the sample at 37°C for 3 min in the dark.

[0035] S3.5: After staining, centrifuge at 500g for 5 min, remove the supernatant, then add 100μL PBS for rinsing and mix by pipetting. After mixing, centrifuge at 500g for 5 min, remove the supernatant, and obtain the third dye combination.

[0036] Furthermore, after staining with the third dye combination, the single-cell samples are permeabilized, fixed, and labeled with intracellular antibodies. The sample fixation includes the following steps:

[0037] S4.1: Add 100 μL of 4% cell fixative to the stained sample precipitate and mix by pipetting. Fix at 37°C for 20 min in the dark.

[0038] S4.2: After fixation, centrifuge at 500g for 5 minutes, and discard the supernatant after centrifugation;

[0039] S4.3: Add 100 μL of PBS to the sample from which the supernatant was removed in step S4.2 and mix by pipetting. Let stand for 2 min and then rinse.

[0040] S4.4: After rinsing, centrifuge at 500g for 5 minutes, and discard the supernatant after centrifugation;

[0041] S4.5: Repeat steps S4.3 and S4.4 once;

[0042] The sample permeability process includes the following steps:

[0043] S5.1: Add 100 μL of 0.1% Triton X-100 to the fixed sample precipitate and mix by pipetting. Place it in the dark at 37°C for 20 min for permeation.

[0044] S5.2: After the permeation is complete, centrifuge at 500g for 5 minutes, and discard the supernatant after centrifugation;

[0045] S5.3: Add 100 μL of PBS to the sample from which the supernatant was removed in step S5.2 and mix by pipetting. Let stand for 2 minutes and then rinse.

[0046] Compared with existing technologies, this approach offers significant advantages: at the single-cell level, it enables simultaneous detection of multiple mitochondrial indicators; at the cellular level, it comprehensively evaluates mitochondrial function while reducing sample size requirements, effectively addressing the challenges of low cell counts and data acquisition difficulties in rare samples. The mitochondrial DNA sequence of these cells is used for phenotypic-genotypic association analysis. This approach improves the accuracy of mutation site screening while addressing high mitochondrial heterogeneity. It also provides a novel human mitochondrial DNA sequencing library preparation method, enabling high-throughput sequencing of the mitochondrial genome in human-derived samples. Compared to whole-genome sequencing, specific mitochondrial gold and silver genome sequencing allows for in-depth research on mitochondrial gene mutations and functions, eliminating nuclear gene interference and more accurately identifying mutation frequencies at mitochondrial genome mutation sites; increasing sequencing depth for more sensitive detection of low-level mutation sites; and reducing library preparation costs and improving efficiency, thereby meeting the needs of large-scale mitochondrial genome mutation analysis in population studies. Attached Figure Description

[0047] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used in conjunction with embodiments of the invention to explain the invention and do not constitute a limitation thereof. In the drawings:

[0048] Figure 1 This is the fluorescence spectrum of the dye;

[0049] Figure 2 The results are from the flow cytometry analysis of the first dye combination.

[0050] Figure 3 The results are from flow cytometry analysis of the second dye combination.

[0051] Figure 4 The results are from flow cytometry analysis of the third dye combination;

[0052] Figure 5 This is a flowchart of the database creation process in this embodiment;

[0053] Figure 6 This is the electrophoresis image of the 8k fragment in this embodiment. Lane 1 and 8: 1K bp marker, 3μL sample loaded; Lane 2 to 7: 8k primer amplification products, 4μL sample loaded.

[0054] Figure 7 This is a peak graph of nucleic acid test results from LabChip Touch quality inspection.

[0055] Figure 8 The images show the electrophoresis results of the amplified products and the purified library. Lane 1: 100bp marker, 3μL loaded; Lanes 2-13: products after amplification and sequencing tag ligation, 3μL loaded; Lane 14: purified library, 6μL loaded.

[0056] Figure 9 This is a sample sequencing quality distribution diagram for this embodiment;

[0057] Figure 10 This is a diagram showing the base distribution of the sequencing samples in this embodiment;

[0058] Figure 11 The average sequencing depth of each locus in the mitochondrial genome;

[0059] Figure 12 This is a heatmap of point mutations in mitochondrial DNA, with mutation frequencies expressed as percentages (%).

[0060] Figure 13 Sanger sequencing validation diagram (R-end sequencing results) for the S1 mutation site G3316A in the sample. Detailed Implementation

[0061] The preferred embodiments of the present invention will be described below with reference to the accompanying drawings. It should be understood that the preferred embodiments described herein are for illustration and explanation only and are not intended to limit the present invention.

[0062] The method for determining multiple functional indicators and mutations in single-cell mitochondria described in this invention allows for flexible modification of dye combinations according to specific experimental needs. For the same mitochondrial phenotype, this scheme provides a variety of fluorescent dyes with different excitation / emission wavelengths, which can be selected based on different experimental protocols.

[0063] Specifically, the indicators include cell viability, mitochondrial copy number, mitochondrial membrane potential, and reactive oxygen species, with DAPI dye used to assess cell viability.

[0064] MitoTracker was used to target mitochondrial copy number. TM Deep Red FM (ThermoFisher) dye;

[0065] MitoTracker was used to target mitochondrial membrane potential. TM Red CMXRos (ThermoFisher) dye and Rhodamine 123 dye;

[0066] MitoSOX Red (ThermoFisher) dye and DCFH dye were used to target reactive oxygen species.

[0067] Single-cell mitochondrial phenotypes are obtained by detecting dye fluorescence intensity using flow cytometry. The combination of indicators and dyes needs to be determined based on the dye fluorescence channel. This protocol can simultaneously detect up to four mitochondrial indicators and provides three optional dye combinations. The first and second combinations can be co-stained with flow cytometry antibodies that do not require permeation, while the third combination can be co-stained with flow cytometry antibodies that require permeation.

[0068] The first dye combination includes:

[0069] DAPI dyes are used to determine cell viability;

[0070] DCFH dyes are used to target reactive oxygen species.

[0071] MitoTracker was used to target mitochondrial membrane potential. TM Red CMX dye;

[0072] MitoTracker was used to target mitochondrial copy number. TM Deep Red FM dye.

[0073] The second dye combination includes:

[0074] DAPI dyes are used to determine cell viability;

[0075] Rhodamine 123 dye was used to target mitochondrial membrane potential;

[0076] MitoSOX Red dye was used to target reactive oxygen species.

[0077] MitoTracker was used to target mitochondrial copy number. TM Deep Red FM dye.

[0078] The third dye combination includes:

[0079] DAPI dyes are used to determine cell viability;

[0080] CellROX Green dye was used to target mitochondrial membrane potential.

[0081] MitoTracker is used to target reactive oxygen species. TM Red CMX dye;

[0082] MitoTracker was used to target mitochondrial copy number. TM Deep Red FM dye.

[0083] When the experiment requires detection of mitochondrial indicators in 2-3 of the indicators, the indicators to be detected can be freely combined with their dyes from the three four-indicator detection schemes provided above, and the single cells sorted by flow cytometry can be used as templates for amplification of the whole mitochondrial genome.

[0084] The specific experimental procedure includes the following steps:

[0085] (1) Preparation of mitochondrial multi-index mixed dyes:

[0086] All dyes were prepared into stock solutions according to the instructions and stored at appropriate temperatures. Working solutions were obtained by diluting the stock solutions with PBS at a specific ratio. Except for DAPI, all other dyes could be prepared as mixed dye working solutions during staining, with a total volume of 1 mL. The volumes of each dye added are shown in the table below.

[0087]

[0088]

[0089] Table 1. Volume of each dye added

[0090] Each 100 μL of mixed staining working solution can be used for 1*10 6 Each cell was stained.

[0091] (2) Mitochondrial multi-index staining:

[0092] After preparing the sample into a single-cell suspension, add the mixed staining working solution, invert and mix well, and stain at 37°C for 30 min in the dark. The DAPI staining conditions are different from other dyes. Add 1 μL of the prepared DAPI dilution to every 100 μL of the mixed staining system, invert and mix well, and stain at 37°C in the dark for 3 min.

[0093] (3) Flow cytometry fluorescence channel selection:

[0094] The excitation and absorption wavelengths of each dye are shown in the table below:

[0095]

[0096] Table 2 Excitation wavelengths for each dye

[0097] This protocol is applicable to samples such as fresh tissues, frozen tissues, and cells.

[0098] (4) The system and procedure for single-cell mitochondrial library construction and amplification of mitochondrial genes are shown in the table below:

[0099]

[0100]

[0101] Table 3 Primer Mixing System

[0102]

[0103] Table 4. PCR Procedure for Amplifying Mitochondrial Genes

[0104] (5) Further specific amplification of the complete mitochondrial genome PCR product obtained in the previous round was performed. The PCR amplification system and reaction procedure for this round are as follows:

[0105]

[0106]

[0107] Table 5. Specific amplification PCR system

[0108]

[0109] Table 6 PCR reaction procedures

[0110] DNA purification magnetic beads were used to purify PCR products.

[0111] (6) Sequencing tag linking:

[0112] The purpose of this step is to ligate the PCR product from the previous step with the sequencing adapter and the barcode sequence.

[0113] The PCR amplification system for this round is as follows:

[0114]

[0115]

[0116] Table 7. PCR systems for ligation sequencing of street and barcode sequences.

[0117] The PCR reaction procedure is as follows:

[0118]

[0119] Table 8. PCR reaction procedure for connecting sequencing adapters and barcode sequences.

[0120] Since sequencing tags have been attached, PCR products from different sources can now be mixed. After purification using DNA purification magnetic beads, the constructed mitochondrial genome library is obtained, which can be directly sequenced.

[0121] The following text provides a variety of fluorescent dyes with different excitation / emission wavelengths, which can be selected according to different experimental schemes. The specific steps are based on the first and third dye combinations as examples. The only difference between the second dye combination and the first dye combination is the preparation of the mixed dyeing working solution.

[0122] The first step is the preparation of the mixed staining working solution and the staining process:

[0123] First dye combination:

[0124] S1.1: Take a 1.5mL centrifuge tube, wrap the outside of the tube with aluminum foil to protect it from light, and add 1mL of PBS;

[0125] S1.2: Add 1.5 μL DCFH and 0.15 μL LitoTracker to PBS. TM Red CMX and 0.15μM MitoTracker TM Deep Red FM, invert and mix thoroughly to prepare a mixed staining working solution;

[0126] S1.3: Add 100 μL of the mixed staining working solution to the single-cell sample pellet and mix thoroughly by pipetting. Incubate in a dark environment at 37°C for 30 min, inverting the container every 5 min to mix and prevent cell clumping or precipitation.

[0127] S1.4: Add 1 μL of DAPI to 10 μL of PBS, invert and mix well to prepare DAPI dilution buffer. Add 1 μL of DAPI dilution buffer to a single-cell sample, invert and mix well, and continue staining at 37°C for 3 min in the dark.

[0128] S1.5: After staining, centrifuge at 500g for 5min, remove the supernatant, then add 100μL PBS for rinsing and mix by pipetting. After mixing, centrifuge at 500g for 5min, remove the supernatant, and obtain the first dye combination.

[0129] Second dye combination:

[0130] S2.1: Take a 1.5mL centrifuge tube, wrap the outside of the tube with aluminum foil to protect it from light, and add 1mL of PBS;

[0131] S2.2: Add 2 μL L hodamine 123, 2 μL Mito SOX Red, and 0.15 μL Mito Tracker to PBS. TM Deep Red FM, invert and mix thoroughly to prepare a mixed staining working solution;

[0132] S2.3: Add 100 μL of the mixed staining working solution to the single-cell sample pellet and mix thoroughly by pipetting. Incubate at 37°C in the dark for 30 min, inverting the container every 5 min to mix and prevent cell clumping or precipitation.

[0133] S2.4: Add 1 μL of DAPI to 10 μL of PBS, invert and mix well to prepare DAPI dilution buffer. Add 1 μL of DAPI dilution buffer to a single-cell sample, invert and mix well, and continue staining at 37°C for 3 min in the dark.

[0134] S2.5: After staining, centrifuge at 500g for 5 min, remove the supernatant, then add 100μL PBS for rinsing and mix by pipetting. After mixing, centrifuge at 500g for 5 min, remove the supernatant, and obtain the second dye combination.

[0135] Third dye combination:

[0136] S3.1: Take a 1.5mL centrifuge tube, wrap the outside of the tube with aluminum foil to protect it from light, and add 1mL of PBS;

[0137] S3.2: Add 2 μL CellROX Green and 0.15 μL Mito Tracker to PBS. TM Red CMX, with 0.15μM MitoTracker TM Deep Red FM, invert and mix thoroughly to prepare a mixed staining working solution;

[0138] S3.3: Add 100 μL of the mixed staining working solution to the single-cell sample pellet and mix thoroughly by pipetting. Incubate at 37°C in the dark for 30 min, inverting the container every 5 min to mix and prevent cell clumping or precipitation.

[0139] S3.4: Add 1 μL of DAPI to 10 μL of PBS, invert and mix well to prepare DAPI dilution buffer. Add 1 μL of DAPI dilution buffer to a single-cell sample, invert and mix well, and continue staining at 37°C for 3 min in the dark.

[0140] S3.5: After staining, centrifuge at 500g for 5min, remove the supernatant, add 100μL PBS for rinsing, mix well by pipetting, centrifuge at 500g for 5min, remove the supernatant, and obtain the third dye combination.

[0141] The sample was then permeabilized and fixed.

[0142] After staining with the third dye combination, the sample can be permeabilized, fixed, and labeled with intracellular antibodies. Specifically, the sample fixation steps are as follows:

[0143] S4.1: Add 100 μL of 4% cell fixative to the stained sample precipitate and mix well by pipetting. Fix at 37°C for 20 min in the dark.

[0144] S4.2: After fixation, centrifuge at 500g for 5 minutes, and discard the supernatant after centrifugation;

[0145] S4.3: Add 100 μL of PBS to the sample from which the supernatant was removed in step S4.2 and mix by pipetting. Let stand for 2 min and then rinse.

[0146] S4.4: After rinsing, centrifuge at 500g for 5 minutes, and discard the supernatant after centrifugation;

[0147] S4.5: Repeat steps S4.3 and S4.4 once.

[0148] The steps for sample permeation are as follows:

[0149] S5.1: Add 100 μL of 0.1% Triton X-100 to the fixed sample precipitate and mix well by pipetting. Place in the dark at 37°C for 20 min for permeation.

[0150] S5.2: After the permeation is complete, centrifuge at 500g for 5 minutes, and discard the supernatant after centrifugation;

[0151] S5.3: Add 100 μL of PBS to the sample from which the supernatant was removed in step S5.2 and mix by pipetting. Let stand for 2 minutes and then rinse.

[0152] Intracellular antibody labeling was then performed:

[0153] In this example, mouse anti-human IgG1 Anti-alpha smooth muscle Actin (α-SMA) antibody and goat anti-mouse IgG1 heavy chain (APC / Cy7) pre-adsorbed secondary antibody were used as examples. The experimental steps are as follows:

[0154] S6.1: Prepare IgG1α-SMA primary antibody working solution using 10% goat serum, diluted at a ratio of 1:200;

[0155] S6.2: Add 100 μL of primary antibody working solution to the sample precipitate, mix well by pipetting, and incubate at 22°C for 30 min in the dark.

[0156] S6.3: Centrifuge at 500g for 5 minutes, and remove the supernatant after centrifugation;

[0157] S6.4: Add 100 μL of PBS to the sample precipitate, mix well by pipetting, and let stand for 2 min to rinse;

[0158] S6.5: Centrifuge at 500g for 5 minutes, and remove the supernatant after centrifugation;

[0159] S6.6: Repeat steps (4) to (5) once;

[0160] S6.7: Prepare the IgG1 heavy chain (APC / Cy7) secondary antibody working solution using PBS, with a dilution ratio of 1:100.

[0161] S6.8: Add 100 μL of secondary antibody working solution to the sample precipitate, mix well by pipetting, and incubate at 37°C for 30 min in the dark.

[0162] S6.9: Centrifuge at 500g for 5 minutes, and remove the supernatant after centrifugation;

[0163] S6.10: Add 100 μL of PBS to the sample precipitate, mix well by pipetting, and let stand for 2 min to rinse;

[0164] S6.11: Centrifuge at 500g for 5 minutes, and remove the supernatant after centrifugation;

[0165] S6.12: Repeat steps (10) to (11) once;

[0166] S6.13: Add 300-500 μL of PBS to the sample precipitate and mix thoroughly by pipetting. Transfer the sample to a flow cytometer tube.

[0167] Next, flow cytometry fluorescence detection will be performed:

[0168] Samples should be analyzed by flow cytometry as soon as possible after staining. The excitation and absorption wavelengths and excitation channels for each dye combination are shown in the table below, and the fluorescence spectra are as follows. Figure 1 As shown (A represents the first dye combination; B represents the second dye combination; C represents the third dye combination):

[0169]

[0170] Table 9 First Dye Combinations

[0171]

[0172] Table 10 Second Dye Combinations

[0173]

[0174] Table 11 Third Dye Combination

[0175] This protocol provides 122 pairs of multiplex PCR primers covering the full length of the mitochondrial genome and, through optimized primer concentration, achieves "one-tube" library construction of the mitochondrial genome. The library construction primers contain sequencer adapter sequences; after two rounds of amplification, the constructed library is ready for direct sequencing. The library construction process is as follows: Figure 5 As shown.

[0176] (1) Whole genome DNA extraction:

[0177] Sample lysis:

[0178] For tissue samples: Take 10-50 mg of tissue sample and add 500 μL of DNA lysis buffer (Tris-Cl 10 mmol / L, EDTA-2Na 0.1 mol / L, SDS 0.5% (m / V); proteinase K, 50 μg / ml, RNase 20 μg / ml, pH=8.0), and lyse overnight at 56℃.

[0179] For cultured cells or white membrane layers: After obtaining dispersed cells, wash twice with PBS, centrifuge at 300g for 3-5 min, and remove the supernatant. Add 500 μL of the above DNA lysis buffer to each 12-well plate and digest at 56℃ for 1-2 h.

[0180] For EDTA-anticoagulated whole blood samples: Take 100-200 μL of whole blood and add 2 times the volume of PBS. Centrifuge at 3500g for 15 min at room temperature, discard the supernatant, and resuspend the precipitate in 500 μL of the above DNA lysis buffer. Lyse at 56℃ for 1-2 h.

[0181] The samples described in this protocol are six human leukocyte samples.

[0182] DNA precipitation:

[0183] Add an equal volume of 25:24:1 phenol-chloroform-isoamyl alcohol solution to the sample lysis buffer and centrifuge at 12000 rpm for 10 min. Take the supernatant and add 2 volumes of anhydrous ethanol, mix thoroughly by inverting several times, and centrifuge at 10000 rpm for 10 min. Discard the supernatant, add 1 mL of 75% ethanol, and wash the precipitate 1–2 times. Discard the supernatant, air-dry the DNA precipitate, and add 30–50 μL of enzyme-free water or TE buffer for later use.

[0184] (2) Amplification of the 8k fragment of the mitochondrial genome:

[0185] The two pairs of primers used in this step are shown in the table below. They can amplify fragments of approximately 8000 bp in length on the mitochondrial genome. The two fragments overlap and can cover the full length of the mitochondrial genome.

[0186]

[0187] Table 12 Primers for amplifying the 9k fragment of the mitochondrial genome

[0188] Prepare a PCR reaction system with 8k primers on ice:

[0189]

[0190] Table 13 Amplification 8kPCR Reaction System

[0191] The PCR reaction procedure is as follows:

[0192]

[0193] Table 14 Amplification 8kPCR Reaction Procedure

[0194] The two sets of reaction products were purified using DNA purification magnetic beads for the next round of specific amplification.

[0195] (3) Mitochondrial DNA-specific amplification:

[0196] The complete mitochondrial genome PCR product obtained in the previous round was further amplified specifically. The amplification primers used were 122 pairs of multiplex PCR primers for the mitochondrial genome, which were mixed according to the concentrations in Table 2. The mixed primers can be used for "one-tube" mitochondrial genome specific amplification.

[0197]

[0198]

[0199]

[0200]

[0201]

[0202]

[0203]

[0204]

[0205]

[0206]

[0207]

[0208] Table 15. 122 pairs of multiplex PCR primer sequences and final concentrations of library construction systems

[0209] The PCR amplification system for this round is as follows:

[0210]

[0211] Table 16 Specific amplification system of 122 primer pairs

[0212] The PCR reaction procedure is as follows:

[0213]

[0214] Table 17 122 Primer-Specific Amplification Procedure

[0215] DNA purification magnetic beads were used to purify PCR products.

[0216] IV sequencing tag linking:

[0217] The PCR product from the previous step is ligated with a sequencing adapter and a barcode sequence.

[0218] The adapter sequence may vary depending on the sequencer. The table below shows the adapter and barcode sequences that are compatible with the Illumina Novaseq PE150 next-generation sequencing platform.

[0219]

[0220] Table 18 Sequencing adapter sequences and barcode sequences

[0221] The PCR amplification system for this round is as follows:

[0222]

[0223] Table 19. Linking Sequencing Adapters and Barcode PCR Systems

[0224] The PCR reaction procedure is as follows:

[0225]

[0226]

[0227] Table 20 Linking Sequencing Adapters and Barcode PCR Procedures

[0228] Because of the pre-linked barcode sequences, PCR products from different sources can now be mixed. After purification using DNA purification magnetic beads, a completed mitochondrial genome library can be obtained, which can be directly sequenced.

[0229] Experimental results:

[0230] Following the experimental protocol, mitochondrial staining of vascular smooth muscle cells was performed using a combination of three dyes in BDInflux. TM After detection by the cell sorter, fluorescence intensity dot plots and peak plots are obtained. Figures 2-4 The results of flow cytometry analysis using three dye combinations were presented, with the samples including unstained negative single-cell samples and stained positive samples. Figure 2 In the diagram, A is a two-dimensional dot plot of DAPI and DCFH fluorescence intensity; B is a two-dimensional dot plot of MitoTracker CMX Red and MitoTracker Deep Red fluorescence intensity; C is a peak plot of DAPI fluorescence intensity; D is a peak plot of DCFH fluorescence intensity; E is a peak plot of MitoTracker CMX Red fluorescence intensity; and F is a peak plot of MitoTracker Deep Red fluorescence intensity. Figure 3 In the diagram, A is a two-dimensional dot plot of fluorescence intensity for DAPI and Rhodamine 123; B is a two-dimensional dot plot of fluorescence intensity for MitoSOX Red and MitoTracker Deep Red; C is a peak plot of fluorescence intensity for DAPI; D is a peak plot of fluorescence intensity for Rhodamine 123; E is a peak plot of fluorescence intensity for MitoSOX Red; and F is a peak plot of fluorescence intensity for MitoTracker Deep Red. Figure 4In the diagram, A is a two-dimensional dot plot of fluorescence intensity for DAPI and CellROXGreen; B is a two-dimensional dot plot of fluorescence intensity for MitoTracker CMXRed and MitoTracker Deep Red; C is a peak plot of fluorescence intensity for CellROXGreen; D is a peak plot of fluorescence intensity for Rhodamine123; E is a peak plot of fluorescence intensity for MitoTracker CMX Red; and F is a peak plot of fluorescence intensity for MitoTracker Deep Red.

[0231] The whole genome of human white membrane cell samples was extracted according to the experimental procedure. The DNA concentration and purity were measured using Nanodrop, and the results are shown in the table. The sample concentration and purity were qualified, and the next step of amplification can be carried out.

[0232] Mitochondrial genome 8k amplification in six samples is as follows: Figure 6 As shown, distinct bands of the amplification products can be observed. The 8k fragment of each sample is diluted to 10-50 ng / μL before proceeding to the next amplification step.

[0233] After mitochondrial DNA-specific amplification and sequencing tag ligation, the electrophoresis results of the PCR products are as follows: Figure 7 As shown, after DNA magnetic bead fragment selection, a uniform library with a fragment size of approximately 350 bp was obtained. Figure 7 -Lane 14).

[0234] Using LabChip Touch to perform quality checks on the constructed library, this library construction method can obtain a single-peak library free from dimer contamination and non-target fragment contamination. Figure 8 After passing quality control, the library can be directly sequenced using the Illumina Novaseq sequencing platform at a high-throughput read length of PE150.

[0235] The initial results of Illumina high-throughput sequencing are image data files. After sequence base identification using bcl-convert software, they are converted into raw sequencing sequences, i.e., Raw Data or Raw Reads, which are stored in FASTQ format. A sequencing quality distribution map can be obtained by plotting the base positions of the filtered sequences on the x-axis and the average quality value at each position on the y-axis. Generally speaking, the higher the proportion of Q30 sequences, the better the sequencing quality. Figure 9 As shown, the sequencing quality of the library constructed by this method is satisfactory.

[0236] Sequencing sample base distribution map ( Figure 10The graph is plotted with the base positions of the filtered sequence on the x-axis and the proportions of A, T, G, and C bases at each position on the y-axis. The results show that the proportions of A and T bases, and G and C bases at each sequencing position in the library are relatively uniform.

[0237] The raw sequencing reads obtained need to undergo data filtering to remove sequencing adapter sequences and low-quality sequences, resulting in high-quality clean reads for further analysis. The data filtering results are shown in Table 24. The sequencing quality Q30rate > 90%, meeting sequencing standards.

[0238]

[0239]

[0240] Table 21 Data Filtering Results

[0241] After the data quality control is passed, short sequence alignment is performed on the FASTQ data obtained from sequencing. Following alignment with the mitochondrial reference genome (NC_012920.1), the aligned position of each read on the genome can be obtained. Sequencing depth is the ratio of sequencing depth to the size of the target gene region; the sequencing depth of each amplicon is an important indicator for evaluating sequencing depth and the reliability of the sequencing method. Figure 7 The sequencing depth of each locus in the mitochondrial genome is shown, with an average sequencing depth of 100×, covering the entire mitochondrial genome sequence. This method demonstrates good genome coverage and uniformity. The single nucleotide variations (SNVs) identified in the sequencing samples after alignment with the reference genome are as follows: Figure 8 As shown, 96 heterogeneous sites with a mutation frequency >1% were identified in the 6 samples.

[0242] To verify the sequencing accuracy of this invention, Sanger sequencing was used to sequence 3316 sites in sample 6. The results are as follows: Figure 9 As shown, the sequencing results are accurate.

[0243] Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for determining multiple functional indicators and mutations in single-cell mitochondria, said indicators including cell viability, mitochondrial copy number, mitochondrial membrane potential, and reactive oxygen species, characterized in that, DAPI dyes are used to determine cell viability; MitoTracker was used to target mitochondrial copy number. TM Deep Red FM (ThermoFisher) dye; MitoTracker was used to target mitochondrial membrane potential. TM Red CMXRos (ThermoFisher) dye, Rhodamine 123, and CellRox Green dye; MitoSOX Red (ThermoFisher) dye and DCFH dye were used to target reactive oxygen species.

2. The method for determining multiple functional indicators and mutations of single-cell mitochondria according to claim 1, characterized in that, The first dye combination, which is co-stained with a flow cytometry antibody that does not require permeabilization, includes: DAPI dyes are used to determine cell viability; DCFH dyes are used to target reactive oxygen species. MitoTracker was used to target mitochondrial membrane potential. TM Red CMXRos (ThermoFisher) dye; MitoTracker was used to target mitochondrial copy number. TM Deep Red FM (ThermoFisher) dye.

3. The method for determining multiple functional indicators and mutations of single-cell mitochondria according to claim 2, characterized in that, The preparation and dyeing method of the first dye combination includes the following steps: S1.1: Take a 1.5 or 2 mL centrifuge tube and keep it protected from light. Add 1 mL of PBS to the centrifuge tube. S1.2: Add 1.5 μL DCFH and 0.15 μL LitoTracker to the centrifuge tube. TM Red CMX and 0.15μM MitoTracker TM Deep Red FM, mix well to prepare a mixed staining working solution; S1.3: Take 100 μL of mixed staining working solution and add single-cell sample precipitate, then mix by pipetting and place it in a dark environment at 37℃ for 30 min, and mix it once every preset time period during the process. S1.4: Add 1 μL of DAPI to 10 μL of PBS and mix well to prepare DAPI dilution solution. Add 1 μL of DAPI dilution solution to a single cell sample and mix well. Stain the sample at 37°C for 3 min in the dark. S1.5: After staining, centrifuge at 500g for 5min, remove the supernatant, then add 100μL PBS for rinsing and mix by pipetting. After mixing, centrifuge at 500g for 5min, remove the supernatant, and obtain the first dye combination.

4. The method for determining multiple functional indicators and mutations of single-cell mitochondria according to claim 1, characterized in that, A second dye combination is used, which is co-stained with flow cytometry antibodies that do not require permeabilization, including: DAPI dyes are used to determine cell viability; Rhodamine 123 dye was used to target mitochondrial membrane potential; MitoSOX Red dye was used to target reactive oxygen species. MitoTracker was used to target mitochondrial copy number. TM Deep Red FM dye.

5. The method for determining multiple functional indicators and mutations of single-cell mitochondria according to claim 4, characterized in that, The preparation and dyeing method of the second dye combination includes the following steps: S2.1: Take a 1.5 mL centrifuge tube and keep it protected from light, then add 1 mL of PBS to the centrifuge tube; S2.2: Add 2 μL L hodamine 123, 2 μL Mito SOX Red, and 0.15 μL Mito Tracker to the centrifuge tube. TM Deep Red FM, mix well to prepare a mixed staining working solution; S2.3: Take 100 μL of mixed staining working solution and add single-cell sample precipitate, then mix by pipetting and place it in a dark environment at 37℃ for 30 min, and mix it once every preset time period during the process. S2.4: Add 1 μL of DAPI to 10 μL of PBS and mix well to prepare DAPI dilution solution. Add 1 μL of DAPI dilution solution to a single cell sample and mix well. Stain the sample at 37°C for 3 min in the dark. S2.5: After staining, centrifuge at 500g for 5 min, remove the supernatant, then add 100μL PBS for rinsing and mix by pipetting. After mixing, centrifuge at 500g for 5 min, remove the supernatant, and obtain the second dye combination.

6. The method for determining multiple functional indicators and mutations of single-cell mitochondria according to claim 1, characterized in that, A third dye combination is used, which is co-stained with the flow cytometry antibody that requires permeation, including: DAPI dyes are used to determine cell viability; MitoTracker was used to target mitochondrial membrane potential. TM Red CMX dye; Dyes are used to target reactive oxygen species; CellROX Green MitoTracker was used to target mitochondrial copy number. TM Deep Red FM dye.

7. The method for determining multiple functional indicators and mutations of single-cell mitochondria according to claim 6, characterized in that, The preparation and dyeing method of the third dye combination includes the following steps: S3.1: Take a 1.5 mL centrifuge tube and keep it protected from light. Add 1 mL of PBS to the centrifuge tube. S3.2: Add 2 μL CellROX Green and 0.15 μL MitoTracker to the centrifuge tube. TM Red CMX and 0.15μM MitoTracker TM Deep Red FM, mix well to prepare a mixed staining working solution; S3.3: Add 100 μL of mixed staining working solution to the single-cell sample precipitate, then mix by pipetting, and place it in a dark environment at 37°C for 30 min for staining, mixing once every preset time period during the process; S3.4: Add 1 μL of DAPI to 10 μL of PBS and mix well to prepare DAPI dilution solution. Add 1 μL of DAPI dilution solution to a single cell sample and mix well. Stain the sample at 37°C for 3 min in the dark. S3.5: After staining, centrifuge at 500g for 5 min, remove the supernatant, then add 100μL PBS for rinsing and mix by pipetting. After mixing, centrifuge at 500g for 5 min, remove the supernatant, and obtain the third dye combination.

8. The method for determining multiple functional indicators and mutations of single-cell mitochondria according to any one of claims 6 or 7, characterized in that, After staining with the third dye combination, the single-cell samples are permeabilized, fixed, and labeled with intracellular antibodies. The sample fixation includes the following steps: S4.1: Add 100 μL of 4% cell fixative to the stained sample precipitate and mix by pipetting. Fix at 37°C for 20 min in the dark. S4.2: After fixation, centrifuge at 500g for 5 minutes, and discard the supernatant after centrifugation; S4.3: Add 100 μL of PBS to the sample from which the supernatant was removed in step S4.2 and mix by pipetting. Let stand for 2 min and then rinse. S4.4: After rinsing, centrifuge at 500g for 5 minutes, and discard the supernatant after centrifugation; S4.5: Repeat steps S4.3 and S4.4 once; The sample permeability process includes the following steps: S5.1: Add 100 μL of 0.1% Triton X-100 to the fixed sample precipitate and mix by pipetting. Place it in the dark at 37°C for 20 min for permeation. S5.2: After the permeation is complete, centrifuge at 500g for 5 minutes, and discard the supernatant after centrifugation; S5.3: Add 100 μL of PBS to the sample from which the supernatant was removed in step S5.2 and mix by pipetting. Let stand for 2 minutes and then rinse.

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