Single-cell mitochondrial whole genome efficient and specific amplification method

Abstract

The application discloses a single-cell mitochondrial whole-genome efficient and specific amplification method. The application provides a single-cell or multi-cell mitochondrial genome DNA high-fidelity amplification method, which comprises the following steps: taking total DNA obtained by lysing a test single cell as a template, using a chimeric primer composed of a 5' end non-template matching common sequence and a 3' end mitochondrial DNA (mtDNA) specific matching sequence, and a primer corresponding to the 5' end non-template matching common sequence of the chimeric primer, and using a high-fidelity DNA polymerase with strand displacement activity to simultaneously amplify several overlapping amplicons which completely cover the single-cell or multi-cell mitochondrial genome DNA. The application avoids various problems in the current single-cell mtDNA in-vitro amplification approach, and is a very reliable mtDNA whole-genome amplification method.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a method for efficient and specific amplification of the entire mitochondrial genome in a single cell. Background Technology

[0002] Mitochondria are not only the energy factories of eukaryotic cells, but also play a crucial role in numerous cellular functions, including apoptosis regulation, immune responses, signal transduction, biomass synthesis, and cytoplasmic calcium ion regulation. Mitochondria possess mitochondrial DNA (mtDNA), a genetic material independent of the nuclear genome. mtDNA is located in the mitochondrial matrix. Human mtDNA is approximately 16.5 kb in length, existing in a closed circular double-stranded form, encoding 37 genes, including 13 respiratory chain protein-coding genes, 22 tRNA genes required for transcription and translation of these protein-coding genes, and 2 rRNA genes. The coding regions account for approximately 95% of all genes, with replication initiating in the D-loop region; other regions regulate mtDNA replication and transcription. Due to the lack of an efficient recombination repair system in mitochondria, the low fidelity of DNA polymerase γ responsible for mtDNA replication, the proximity of mtDNA to the oxidative phosphorylation reaction site, the lack of histone protection, and the susceptibility to attack by reactive oxygen species (ROS) free radicals, a byproduct of the reaction, mtDNA exhibits a high mutation rate. Studies show that the mutation rate of mtDNA is 10-17 times higher than that of nuclear DNA. mtDNA exists in multiple copies within cells, and the coexistence of mutated and wild-type mtDNA creates mtDNA heterogeneity. Unlike the diploid nuclear genome, mtDNA heterogeneity can vary between 0% and 100%. Even among different cells of the same tissue in the same individual, mtDNA heterogeneity can differ. Different proportions of mtDNA heterogeneity can lead to completely different phenotypes. Current multicellular hybrid DNA sequencing methods can only detect the average mtDNA heterogeneity within a cell population, ignoring the unique mtDNA heterogeneity characteristics of individual cells and masking harmful mtDNA mutations in a few cells, resulting in the loss of much important information.

[0003] mtDNA accounts for less than 1% of total cellular DNA, making it difficult to deeply cover the mitochondrial genome and accurately detect mtDNA heterogeneity when directly sequencing the entire cellular genome. Enriching single-cell mtDNA for deep sequencing allows for the study of mtDNA heterogeneity at the single-cell level. Early single-cell mtDNA enrichment was primarily achieved through PCR amplification. These methods typically target the highly variable D-loop regions of mtDNA, covering approximately 1 kb. However, this enrichment method is limited to a small portion of the mitochondrial genome, failing to provide a complete picture of single-cell mtDNA mutations. Subsequent research has primarily utilized in vivo amplification and in vitro nucleic acid amplification to address the challenge of insufficient single-cell DNA sample size for direct sequencing analysis.

[0004] The in vivo amplification pathway involves culturing isolated single cells in vitro until a sufficient number of cells have proliferated, then extracting DNA, followed by enriching the mtDNA for high-throughput sequencing. The biggest problem with this method is that a single cell needs to be propagated in vitro for multiple generations to obtain enough DNA for sequencing, making it unsuitable for non-immortalized cells or cells that cannot be cultured in vitro. Furthermore, prolonged in vitro culture may lead to changes in mtDNA heterogeneity, and whether the results accurately reflect the characteristics of the original sample is controversial.

[0005] In vitro nucleic acid amplification directly amplifies and enriches the mitochondrial genome from single-cell lysates. Unlike earlier methods that targeted only specific regions of the mitochondria, these improved methods employ various amplification strategies. They utilize multiple overlapping replicons, long-range PCR, or strand displacement-based rolling circle amplification to specifically amplify the single-cell mitochondrial genome, achieving near-complete coverage of mtDNA. Morris et al. further achieved intramitochondrial mtDNA amplification. These methods have enhanced our understanding of mtDNA heterogeneity at the single-cell level, but they also have certain limitations. To avoid interference between primers used in the amplification reaction, these methods typically distribute single-cell lysates into different reaction systems, resulting in each system only amplifying a portion of the initial sample's mtDNA, leading to loss of original information. While rolling circle amplification can amplify the entire mtDNA in a single reaction, issues such as pre-amplification single-cell extraction and purification, and the presence of numerous reaction byproducts require further improvement. Furthermore, the amplification products obtained through amplification methods all need to be purified by nucleic acid before they can be used for subsequent sequencing library construction, which increases the probability of sample cross-contamination and is not conducive to high-throughput sample preparation. Summary of the Invention

[0006] To overcome the problems in existing technologies, the purpose of this invention is to provide a highly efficient and complete amplification method for single-cell mtDNA. This method includes obtaining single cells using single-cell preparation technology and lysing them to fully release DNA. Then, using chimeric primers composed of a 5' end non-template-matching common sequence and a 3' end mtDNA-specific matching sequence, and primers corresponding to the aforementioned 5' end non-template-matching common sequence, a high-fidelity DNA polymerase with strand displacement activity is used to simultaneously amplify several overlapping amplicones covering the complete single-cell mitochondrial genome. This invention achieves highly efficient and specific amplification of the entire single-cell mitochondrial genome sequence in a single reaction system. The mtDNA enriched using this method can be used for single-cell mtDNA sequencing, accurately detecting mtDNA heterogeneity variations at the single-cell level.

[0007] In a first aspect, the present invention claims protection for a method for amplifying mitochondrial genomic DNA.

[0008] The mitochondrial genomic DNA amplification method claimed in this invention may include the following steps: using a nucleic acid sample containing the mitochondrial genome as a template, performing multiplex PCR with a DNA polymerase having strand displacement activity, wherein the primer combination used for the multiplex PCR contains N chimeric primer pairs; N is a positive integer of 2 or more.

[0009] Each of the chimeric primer pairs consists of the upstream and downstream primers from the 5' end to the 3' end, which are composed of the following (a1) and (a2):

[0010] (a1) A universal sequence that does not match the mitochondrial genome;

[0011] (a2) A specific sequence that perfectly matches the mitochondrial genome;

[0012] Furthermore, the universal sequences in the different chimeric primer pairs are the same, but the specific sequences are different;

[0013] Furthermore, the specific sequences in the different chimeric primer pairs are distributed (relatively uniformly) at their binding sites on the mitochondrial genome;

[0014] The N-segment amplicon (terminal overlap) that completely covers the mitochondrial genome was obtained through the multiplex PCR.

[0015] Furthermore, the primer combination used for the multiplex PCR consists of the N chimeric primer pairs and one universal primer;

[0016] The universal primers are as follows: (b1) or (b2) or (b3) or (b4):

[0017] (b1) The general sequence;

[0018] (b2) is inversely complementary to the general sequence;

[0019] (b3) A continuous sequence selected from the general sequence;

[0020] (b4) is inversely complementary to a continuous sequence selected from the general sequence.

[0021] Furthermore, the nucleic acid sample containing the mitochondrial genome can be derived from a single cell or from multiple cells.

[0022] When the nucleic acid sample containing the mitochondrial genome to be tested comes from a single cell, the present invention provides a method for amplifying mitochondrial genomic DNA in a single cell.

[0023] The single-cell mitochondrial genomic DNA amplification method provided by the present invention may include the following steps: single-cell sorting of test cells, lysing cells to release total cellular DNA after obtaining single cells, using the obtained single-cell total DNA as a template, performing multiplex PCR using a DNA polymerase with strand displacement activity, wherein the primer combination used for the multiplex PCR consists of N chimeric primer pairs and one universal primer; N is a positive integer of 2 or more.

[0024] Each of the chimeric primer pairs consists of the upstream and downstream primers from the 5' end to the 3' end, which are composed of the following (a1) and (a2):

[0025] (a1) A universal sequence that does not match the mitochondrial genome of the single cell;

[0026] (a2) A specific sequence that perfectly matches the mitochondrial genome of the single cell;

[0027] Furthermore, the universal sequences in the different chimeric primer pairs are the same, but the specific sequences are different;

[0028] Furthermore, the binding sites of the specific sequences in the different chimeric primer pairs on the mitochondrial genome of the single cell are dispersed (relatively uniform);

[0029] The universal primers are as follows: (b1) or (b2) or (b3) or (b4):

[0030] (b1) The general sequence;

[0031] (b2) is inversely complementary to the general sequence;

[0032] (b3) A continuous sequence selected from the general sequence;

[0033] (b4) is inversely complementary to a continuous sequence selected from the general sequence;

[0034] The N-segment amplicon (terminal overlap) that can completely cover the mitochondrial genome of the single cell was obtained through the multiplex PCR.

[0035] Furthermore, in each chimeric primer pair, the binding site of the specific sequence in the upstream primer on the mitochondrial genome of the single cell is located within the amplification target of the adjacent upstream chimeric primer pair, and the binding site of the specific sequence in the downstream primer on the mitochondrial genome of the single cell is located within the amplification target of the adjacent downstream chimeric primer pair. Figure 1 Primers A and D form a chimeric primer pair, and primers B and C form another chimeric primer pair.

[0036] Furthermore, when performing the multiplex PCR, the working concentration of the universal primers is 0.1-1 μM, and the working concentrations of the upstream and downstream primers in each chimeric primer pair are 1 / 50 to 1 / 100 of the working concentration of the universal primers used. The working concentration is the final concentration in the reaction system.

[0037] In a specific embodiment of the present invention, the working concentration of the upstream and downstream primers in each chimeric primer pair is 0.0125 μM, and the working concentration of the universal primer is 0.8 μM.

[0038] In a specific embodiment of the present invention, the DNA polymerase with strand displacement activity is LongAmp Taq DNA polymerase or 3173 DNA polymerase. LongAmp Taq DNA polymerase is a mixture of Taq and Deep Vent enzymes; the former primarily functions as a DNA polymerase, while the latter primarily functions as a strand displacement enzyme. Of course, the DNA polymerase can also be other DNA polymerases with strand displacement activity.

[0039] In this invention, the design of each primer only needs to conform to the general primer design principles, GC% 40-60%; Tm greater than 3' end specific primers, to avoid the formation of secondary structures such as hairpins or dimers.

[0040] In the method described, the method for extracting total DNA from a single cell is a conventional method in the art.

[0041] The nucleic acid sample containing the mitochondrial genome can be derived from any cell with a known mitochondrial genome sequence, such as human cells.

[0042] In a specific embodiment of the present invention, the cells are specifically Jurkat cells, human peripheral blood nucleated cells, or HEK293T cells.

[0043] In one specific embodiment of the present invention, the universal primer is primer E; the sequence of primer E is shown in SEQ ID No. 5.

[0044] In another specific embodiment of the present invention, the universal primer is primer L; the sequence of primer L is shown in SEQ ID No. 12.

[0045] In one specific embodiment of the present invention, there are two pairs of chimeric primers, as follows:

[0046] The first pair of chimeric primers consists of primer A and primer D; the sequence of primer A is shown in SEQ ID No. 1; the sequence of primer D is shown in SEQ ID No. 4;

[0047] The second pair of chimeric primers consists of primer B and primer C; the sequence of primer B is shown in SEQ ID No. 2; the sequence of primer C is shown in SEQ ID No. 3.

[0048] In another specific embodiment of the present invention, there are three pairs of chimeric primers, as follows:

[0049] The first pair of chimeric primers consists of primer F and primer G; the sequence of primer F is shown in SEQ ID No. 6; the sequence of primer G is shown in SEQ ID No. 7;

[0050] The second pair of chimeric primers consists of primer H and primer I; the sequence of primer H is shown in SEQ ID No. 8; the sequence of primer I is shown in SEQ ID No. 9;

[0051] The third pair of chimeric primers consists of primer J and primer K; the sequence of primer J is shown in SEQ ID No. 10; the sequence of primer K is shown in SEQ ID No. 11.

[0052] The amplification method of the present invention is not only applicable to the amplification of mtDNA, but also applicable to the amplification of any covalently closed circular DNA with a known sequence.

[0053] Secondly, the present invention claims protection for a method for covalently closed circular DNA amplification.

[0054] The method for amplifying covalently closed circular DNA claimed in this invention differs from the method described in the first aspect above only in that: the nucleic acid sample containing the mitochondrial genome is replaced with a nucleic acid sample containing covalently closed circular DNA, and the mitochondrial genome is replaced with the covalently closed circular DNA.

[0055] Thirdly, the present invention claims protection for the application of the method described in the first aspect above in any of the following:

[0056] (A1) Single-cell mitochondrial genome sequencing;

[0057] (A2) Detect mitochondrial genomic heterogeneity variations down to the single-cell level;

[0058] (A3) Detect mtDNA heterogeneity in multi-cell mixed samples.

[0059] Fourthly, this invention claims protection for a complete product for amplifying human mitochondrial genomes.

[0060] The kit for amplifying the human mitochondrial genome claimed in this invention is kit A or kit B.

[0061] The complete set of products A consists of the following (c1)-(c3):

[0062] (c1) Primers A, B, C, and D mentioned in the first aspect above;

[0063] (c2) Primer E mentioned in the first aspect above;

[0064] (c3) DNA polymerases with strand displacement activity as described in the first aspect above.

[0065] The complete set of products B consists of the following (d1)-(d3):

[0066] (d1) Primers F, G, H, I, J, and K mentioned in the first aspect above;

[0067] (d2) Primer L mentioned in the first aspect above;

[0068] (d3) DNA polymerases with strand displacement activity as described in the first aspect above.

[0069] Fifthly, the present invention claims protection for the use of the complete set of products described in the fourth aspect above in any of the following:

[0070] (A1) Single-cell mitochondrial genome sequencing;

[0071] (A2) Detect mitochondrial genomic heterogeneity variations down to the single-cell level;

[0072] (A3) Detect mtDNA heterogeneity in multi-cell mixed samples.

[0073] The beneficial effects of this invention are:

[0074] 1. It can faithfully and completely preserve the full heterogeneity of mtDNA in single cells. mtDNA extracted from multi-cell mixed samples only yields the average value of mtDNA heterogeneity within the cell population; the unique mtDNA heterogeneity characteristics of individual cells are lost during DNA extraction. Therefore, mtDNA from multiple cell sources masks mtDNA mutations in a few cells, leading to the loss of much important mutational information. This invention specifically enriches the whole genome of mtDNA in single-cell lysates. Suitable for high-resolution single-cell mtDNA sequencing, it will help reveal the mechanisms underlying the variation of single-cell mtDNA heterogeneity across different cell lineages.

[0075] 2. Applicable to non-immortalized cells and cells that cannot be cultured in vitro. Conventional mtDNA enrichment usually relies on in vivo amplification. This process involves culturing isolated single cells in vitro until a sufficient number of cells have proliferated, then extracting DNA and subsequently enriching the mtDNA. The biggest problem with this method is that a single cell needs to be propagated in vitro for multiple generations to obtain enough starting DNA for subsequent enrichment, thus making it unsuitable for non-immortalized cells and cells that cannot be cultured in vitro. In addition, prolonged in vitro culture may lead to changes in mtDNA heterogeneity, making it difficult for the enriched mtDNA to accurately reflect the original appearance of the mtDNA from the original starting cell. This method can be directly applied to isolated single cells without the need for cell culture to enrich mtDNA, and it does not cause changes in mtDNA heterogeneity. Therefore, this invention has broad applicability to sample selection.

[0076] 3. It can fully display the mutation profile of single-cell mtDNA. Unlike PCR amplification of the mtDNA D-loop region or other partial regions of the mitochondrial genome, this invention uses a multiplex PCR method based on strand displacement activity, and the amplicon contained in the amplification product can completely cover the mtDNA genome.

[0077] 4. Mitochondrial genome can be directly amplified and enriched from single-cell lysates. Conventional multiplex PCR amplification typically involves distributing single-cell lysates into different reaction systems to avoid interference between primers. This results in each reaction system only amplifying a portion of the initial sample's mtDNA, leading to a loss of original information. While rolling circle amplification can amplify the entire mtDNA genome in a single reaction, issues such as pre-amplification single-cell extraction and purification, and numerous reaction byproducts require further improvement. Furthermore, amplification products obtained through these methods require nucleic acid purification before use in subsequent sequencing library construction, increasing the probability of sample cross-contamination and hindering high-throughput sample preparation. The primers in the mitochondrial single-cell mtDNA whole-genome amplification method provided in this invention can complete cell lysis and mtDNA whole-genome amplification in a single reaction, avoiding the loss of original information. Simultaneously, it avoids the possibility of sample cross-contamination due to dispensing, which is beneficial for high-throughput sample preparation.

[0078] This invention avoids the various problems in current in vitro nucleic acid amplification methods and is a highly reliable method for amplifying the entire mtDNA genome. Attached Figure Description

[0079] Figure 1 This is a schematic diagram of the multiplex PCR amplification method for closed circular DNA templates based on DNA polymerase chain displacement activity according to the present invention.

[0080] Figure 2 This refers to the flow cytometry sorting of single cells in Example 1. (Zombie NIR) TM -: Jurkat cell lines have not undergone Zombie NIR testing. TM Staining (left); Zombie NIR TM+ Zombie NIR using Jurkat cell lines TM Staining (right), R1 indicates not stained by Zombie NIR TM Jurkat live cells stained; R2 indicates cells stained with Zombie NIR. TM Stained Jurkat cells.

[0081] Figure 3 The images show the gel electrophoresis results of the Jurkat single-cell mitochondrial genome amplification products sorted by multiplex PCR based on DNA polymerase chain displacement activity in Example 1. Lanes 1-5 contain five different Jurkat single-cell mitochondrial genome DNA fragments amplified using multiplex PCR based on DNA polymerase chain displacement activity, with a fragment size of approximately 8.3 kb. Lane 6 is a negative control without LongAmp DNA polymerase. Lane 7 is the DNA marker.

[0082] Figure 4In Example 1, the relative yields of two amplicones in two Jurkat single-cell mtDNA amplification products were detected by real-time PCR. The whole-genome Jurkat single-cell mtDNA amplification products were diluted 100-fold and amplicon-specific primers were used for real-time PCR amplification. The CT values ​​for the experimental group are shown below; a higher CT value indicates a lower mtDNA content. The negative control product, which contained template mtDNA but did not contain LongAmp DNA polymerase, was diluted 100-fold and amplified using specific primers. The CT values ​​for the negative control group are shown below. The PCR reaction system without template was amplified using specific primers. The CT values ​​for the blank control group are shown below.

[0083] Figure 5 This is an example of multiplex PCR amplification of the mitochondrial genome of a single nucleated cell from human peripheral blood based on DNA polymerase chain displacement activity, as described in Example 2. Lanes 1-7 contain mitochondrial genomic DNA amplified from seven single nucleated cells from human peripheral blood using multiplex PCR based on DNA polymerase chain displacement activity, with fragment sizes of approximately 8.3 kb. Lane 8 is a negative control without LongAmp DNA polymerase. Lane 9 contains a DNA marker.

[0084] Figure 6 In Example 2, the yields of two amplicones in the human peripheral blood nucleated single-cell mtDNA amplification products were detected by real-time PCR. The whole-genome mtDNA amplification products from human peripheral blood nucleated single-cell cells were diluted 100-fold and then subjected to real-time PCR using amplicon-specific primers. The CT values ​​for the experimental group are shown below; a higher CT value indicates a lower mtDNA content. The negative control product, which contained template mtDNA but did not contain LongAmp DNA polymerase, was diluted 100-fold and then amplified by real-time PCR using specific primers. The CT values ​​for the negative control group are shown below. The PCR reaction system without template was amplified by real-time PCR using specific primers. The CT values ​​for the blank control group are shown below.

[0085] Figure 7 The HEK293T single-cell mitochondrial genome was amplified using the strand displacement-active 3173 DNA polymerase in Example 3. Lanes 1-8 show the mitochondrial genomic DNA of eight sorted HEK293T single cells amplified using a strand displacement-based multiplex PCR method with the strand displacement-active 3173 DNA polymerase, with fragment sizes of approximately 8.3 kb. Lane 9 is a negative control without 3173 DNA polymerase. Lane 10 is a DNA marker.

[0086] Figure 8In Example 3, the yields of two amplicones in the HEK293T single-cell mtDNA amplification products were detected by real-time PCR. The HEK293T single-cell mtDNA whole-genome amplification products were diluted 100-fold and then amplified using amplicon-specific primers. The CT values ​​for the experimental group are shown below; a higher CT value indicates a lower mtDNA content. The negative control product, which contained template mtDNA but did not contain 3173 DNA polymerase, was diluted 100-fold and then detected by real-time PCR using amplicon-specific primers. The CT values ​​for the negative control group are shown below. The PCR reaction system without template was amplified using amplicon-specific primers. The CT values ​​for the blank control group are shown below.

[0087] Figure 9 In Example 4, three amplicons were used to completely cover the HEK293T single-cell mtDNA genome. Lanes 1-8 contain HEK293T single-cell mitochondrial genomic DNA amplified using a strand displacement-based multiplex PCR method, with three amplicons covering the HEK293T single-cell mtDNA genome, and the fragment size is approximately 5.5kb; lane 9 is a negative control without LongAmp DNA polymerase; lane 10 is a DNA marker.

[0088] Figure 10 In Example 4, the yields of three amplicones in the HEK293T single-cell mtDNA amplification products were detected by real-time PCR. The HEK293T single-cell mtDNA whole-genome amplification products were diluted 100-fold and then amplified using amplicon-specific primers using real-time PCR. The CT values ​​for the experimental group are shown below; a higher CT value indicates a lower mtDNA content. The negative control product, which contained template mtDNA but did not contain LongAmpDNA polymerase, was diluted 100-fold and then amplified using specific primers using real-time PCR. The CT values ​​for the negative control group are shown below. The PCR reaction system without template was amplified using specific primers using real-time PCR, as shown below the CT values ​​for the blank control group. Detailed Implementation

[0089] The present invention adopts the following technical solution:

[0090] (1) Templates for mtDNA amplification were prepared by single-cell sorting and lysis:

[0091] a. Preparation of cell lysis buffer and sorting plate. Under sterile conditions, prepare single-cell mitochondrial lysis buffer using pure water: 200mM NaOH, 50mM DTT; prepare sterile, enzyme-free 96-well plates or eight-tube PCR tubes; add 2 μL of the prepared lysis buffer to each well of the sorting plate. Care should be taken to avoid contamination by other unrelated mtDNA during the procedure.

[0092] b. Single-cell preparation. For suspension cell lines, collect cells, centrifuge at 350G, discard the supernatant, wash twice with 1×PBS, and resuspend the cells in 100μL staining buffer. For adherent cell lines, digest thoroughly with trypsin, centrifuge at 350G, discard the supernatant, wash twice with 1×PBS, and resuspend the cells in 100μL staining buffer. For tissue samples, follow the method for preparing single-cell suspensions by tissue digestion: digest the tissue into single cells, centrifuge at 350G, discard the supernatant, wash twice with 1×PBS, and resuspend the cells in 100μL staining buffer. Add Zombie NIR at a volume ratio of 1:100. TM Apply dye and incubate in the dark for 20 minutes (or use other methods to distinguish between live and dead cells); wash twice with 1 mL of 1×PBS, and finally resuspend the cells in 500 μL of 1×PBS; use a flow cytometer to sort single live cells (or use other single-cell isolation methods) into well plates or tubes pre-filled with lysis buffer;

[0093] c. Cell lysis and template DNA preparation. Cells can be lysed using alkaline lysis combined with high-temperature lysis (high-temperature cell lysis procedure: 65℃, 10 minutes; 4℃, incubation) or other cell lysis methods to fully release the DNA from the cells into the lysis buffer. The prepared template DNA should be used for PCR as soon as possible or frozen at -80℃.

[0094] (2) Multiplex PCR amplification of the whole mitochondrial genome in single cells based on strand displacement activity:

[0095] a. Design of multiplex PCR primers based on strand displacement activity. Chimeric primers consisting of a common 5' end non-template-matching sequence and a 3' end mtDNA-specific matching sequence are used, along with primers corresponding to the common 5' end non-template-matching sequence of the aforementioned primers (denoted as universal primers). There can be two or more pairs of chimeric primers; universal primers can be any sequence with high amplification efficiency. The amplification process (taking two pairs of chimeric primers plus primers corresponding to the common non-template-matching sequence as an example) is as follows: chimeric primer A extends to chimeric primer C, where the DNA polymerase with strand displacement activity performs strand displacement activity. Chimeric primer A continues to extend to the 5' end and beyond of primer D, providing a binding site for primer D in the next round of PCR. Therefore, primer D should be designed downstream of the binding site of primer C on the template DNA to ensure complete amplification of the template DNA; however, the distance should not be too large to prevent the strand displacement activity from being unable to displace excessively long DNA fragments. The design of other chimeric primers follows the same principle. This forms the initial amplification product (e.g., Figure 1 As shown). After the initial amplification product is formed, it is amplified using universal primers (i.e., Figure 1 Primer E in the middle further amplifies the initial amplification product more efficiently, forming numerous amplicons with overlapping ends (such as...). Figure 1 (As shown).

[0096] b. The components of multiplex PCR based on strand displacement activity mainly consist of 5×LongAmp Taq reaction buffer, 10mM dNTPs, chimeric primers, universal primers, template DNA, Tricine (200mM), LongAmp Taq DNA polymerase, and ddH2O (see Table 1 for details). The working concentration of each chimeric primer is 0.0125μM, and the working concentration of the universal primer is 0.8μM. The LongAmp Taq DNA polymerase in the LongAmp Kit mainly contains two enzymes: Taq and Deep vent. The former mainly functions as a DNA polymerase, while the latter mainly functions as a strand displacement enzyme. Other thermostable DNA polymerases with strand displacement activity, such as 3173 DNA polymerase, can also be used.

[0097] Table 1. Components of multiplex PCR based on strand displacement activity:

[0098]

[0099]

[0100] c. Procedure for multiplex PCR based on strand displacement activity. Prepare the reaction solution on ice and place the reaction system in a PCR instrument as soon as possible for PCR. The PCR procedure is shown in Table 2. In the amplification step, 65°C is the extension temperature for LongAmp Taq DNA polymerase, with a rate of 1kb / 50s. The extension time needs to be set according to the extension length. The subsequent 72°C for 30 seconds is the strand displacement reaction. Since the strand displacement length only needs to extend to the 5' end and beyond of the next chimeric primer to provide primer binding sites for the next round of PCR, a long time is not required. If using other DNA polymerases, follow the instructions for PCR program settings.

[0101] Table 2. PCR reaction conditions

[0102]

[0103] d. Electrophoretic analysis. After the reaction, take 3-5 μL for agarose gel electrophoresis analysis.

[0104] (3) Quantitative real-time PCR (qPCR) was used to detect the amplification efficiency of each amplicon. Amplicon-specific RT-PCR primers were designed for each amplicon. The experimental group and the negative control (without LongAmp Taq DNA polymerase) were diluted 100-fold to serve as templates for qPCR. The reaction solution was prepared on ice, and the reaction system was placed in a qPCR instrument for PCR reaction as soon as possible. The reaction procedure is shown in Table 3.

[0105] Table 3. RT-PCR reaction conditions

[0106]

[0107]

[0108] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0109] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. For example, the conditions described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or the conditions recommended by the manufacturer. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0110] 1. The materials and reagents used in the embodiments of the present invention are shown in Table 4.

[0111] Table 4. Materials and Reagents

[0112]

[0113] 2. The primers used in the embodiments of the present invention are shown in Table 5.

[0114] Table 5. Primers used in the embodiments of this invention

[0115]

[0116]

[0117] Example 1: Amplification of the entire mtDNA genome in Jurkat cells

[0118] 1. Preparation of cell lysis buffer and sorting plate. Prepare sterile, enzyme-free eight-tube PCR apparatus; prepare lysis buffer using pure water, with the following solute concentrations: 200mM NaOH; 50mM DTT. Add 2μL of the prepared lysis buffer to each well of the sorting plate.

[0119] 2. Single-cell sorting. Collect Jurkat cells, centrifuge at 500G, discard the supernatant after centrifugation, and wash twice with 1×PBS; resuspend the cells in 100μL staining buffer, and add Zombie NIR at a volume ratio of 1:100. TM Dye, incubate in the dark for 20 minutes; wash twice with 1 mL 1×PBS, and finally resuspend the cells in 500 μL 1×PBS; sort single live cells by flow cytometry (Zombie NIR). TM (Negative) to wells containing cell lysis buffer (e.g.) Figure 2 ).

[0120] 3. Cell lysis. Cells are lysed using an alkaline lysis method combined with high-temperature lysis (65°C, 10 minutes) to fully release the DNA from the cells into the lysis buffer.

[0121] 4. Amplify the Jurkat single-cell genome using multiplex PCR based on strand displacement activity. Prepare the reaction solution on ice and place the reaction system in a PCR instrument for PCR reaction as soon as possible.

[0122] The PCR reaction system and reaction procedure are shown in Tables 6 and 7.

[0123] Table 6. PCR Reaction System

[0124] Element volume 5×LongAmp Taq Reaction Buffer 4μL 10mM dNTPs 0.6μL Primer A (10 μM) 0.025μL Primer B (10 μM) 0.025μL Primer C (10 μM) 0.025μL Primer D (10 μM) 0.025μL Primer E (10 μM) 1.6μL DNA template (lysis buffer) 2μL Tricine (200mM) 2μL LongAmp Taq DNA polymerase 0.8μL <![CDATA[ddH2O]]> Make up to 20 μL

[0125] Table 7. PCR Reaction Procedure

[0126]

[0127]

[0128] Electrophoresis analysis showed the presence of approximately 8kb of amplified products, such as... Figure 3 .

[0129] 5. Quantitative real-time PCR analysis of multiplex PCR results

[0130] The experimental group and the negative control (without LongAmp Taq DNA polymerase) were diluted 100-fold to serve as templates for real-time PCR. The reaction solution was prepared on ice, and the reaction system was placed in an ABI 7500 real-time PCR instrument for PCR reaction as soon as possible.

[0131] The reaction system and reaction procedure for real-time PCR are shown in Table 8 and Table 3, respectively.

[0132] Table 8. PCR Reaction System

[0133]

[0134] Note: Primer 1 and Primer 2 in the table are primer 8k-1F and primer 8k-1R from Table 5, respectively (used for amplification). Figure 4 The amplicon 1 in the middle, or primers 8k-2F and 8k-2R respectively (used for amplification) Figure 4 Amplicon 2 in the middle.

[0135] The results showed that the two amplicones covering the entire mtDNA genome of Jurkat cells were amplified in a balanced and efficient manner (e.g., Figure 4 ).

[0136] Example 2: Preparation of peripheral blood nucleated single cells using the extreme dilution method and amplification of the entire mtDNA genome.

[0137] 1. Collect peripheral blood in anticoagulant tubes, lyse red blood cells with red blood cell lysis buffer, wash twice with 1×PBS to prepare a peripheral blood nucleated cell suspension;

[0138] 2. Count the nucleated cells in peripheral blood. Dilute the cells to approximately 50 cells per 100 μL, pipette them evenly, and use a 2 μL pipette to spot the cell suspension into several wells, 2 μL per well, with each droplet about the size of a sesame seed. Observe under an inverted microscope. If there are 1-2 cells per well, the cell concentration is appropriate, and the entire 96-well plate can be plated at once. If not, the droplet size can be adjusted, more cells can be added, or the culture medium can be added to dilute the solution further.

[0139] 3. Cell lysis: Use high temperature method to lyse cells (99℃, 10 minutes, hot cap 110℃) to fully release the DNA in the cells into the lysis buffer.

[0140] 4. Amplify single-cell genomes using multiplex PCR based on strand displacement activity. Prepare the reaction solution on ice and place the reaction system in a PCR instrument for PCR reaction as soon as possible.

[0141] The PCR reaction system and PCR reaction procedure are shown in Table 6 and Table 7, respectively.

[0142] Electrophoresis analysis showed the presence of approximately 8kb of amplified products, such as... Figure 5 .

[0143] 5. Quantitative real-time PCR analysis of multiplex PCR results

[0144] The experimental group and the negative control (without LongAmp Taq DNA polymerase) were diluted 100-fold to serve as templates for real-time PCR. The reaction solution was prepared on ice, and the reaction system was placed in an ABI 7500 real-time PCR instrument for PCR reaction as soon as possible.

[0145] The reaction system and procedure for real-time PCR are shown in Table 8. (Note: Primers 1 and 2 in the table are primers 8k-1F and 8k-1R from Table 5, respectively, used for amplification.) Figure 6 Amplicon 1; or primers 8k-2F and 8k-2R, respectively, are used for amplification. Figure 6 Amplicon 2) and Table 3.

[0146] The results showed that the two amplicones covering the entire genome of peripheral blood nucleated cell mtDNA were amplified in a balanced and efficient manner (e.g., Figure 6 ).

[0147] Example 3: Amplification of the complete mtDNA genome of HEK293T cells using 3173 DNA polymerase

[0148] 1. Preparation of cell lysis buffer and sorting plate. Prepare sterile eight-tube PCR tubes; prepare lysis buffer with pure water, solute and concentration as follows: 200mM NaOH; 50mM DTT. Add 1μL of the prepared lysis buffer to each well of the sorting plate;

[0149] 2. Single-cell sorting. Collect HEK293T cells, centrifuge at 500G, discard the supernatant after centrifugation, and wash twice with 1×PBS; resuspend the cells in 100μL staining buffer, and add Zombie NIR at a volume ratio of 1:100. TM Dye, incubate in the dark for 20 minutes; wash twice with 1 mL 1×PBS, and finally resuspend the cells in 500 μL 1×PBS; sort single live cells by flow cytometry (Zombie NIR). TM (Negative) to the well plate into which the lysis buffer was added.

[0150] 3. Cell lysis. Cells are lysed using an alkaline lysis method combined with high-temperature lysis (65°C, 10 minutes) to fully release the DNA from the cells into the lysis buffer.

[0151] 4. Amplify single-cell genomes using multiplex PCR based on strand displacement activity. Prepare the reaction solution on ice and place the reaction system in a PCR instrument for PCR reaction as soon as possible.

[0152] The PCR reaction system and PCR reaction procedure are shown in Table 9 and Table 10, respectively.

[0153] Table 9. PCR reaction system:

[0154]

[0155]

[0156] Table 10. PCR Reaction Conditions

[0157]

[0158] Electrophoresis analysis showed the presence of approximately 8K amplification products, such as... Figure 7 .

[0159] 5. Quantitative real-time PCR analysis of multiplex PCR results

[0160] The experimental group and the negative control (without 3173 DNA polymerase) were diluted 100-fold to serve as templates for real-time PCR. The reaction solution was prepared on ice and the reaction system was placed in an ABI 7500 real-time PCR instrument as soon as possible for PCR reaction.

[0161] The reaction system and procedure for real-time PCR are shown in Table 8. (Note: Primers 1 and 2 in the table are primers 8k-1F and 8k-1R from Table 5, respectively, used for amplification.) Figure 8 Amplicon 1; or primers 8k-2F and 8k-2R, respectively, are used for amplification. Figure 8 Amplicon 2) and Table 11.

[0162] Table 11. PCR Reaction Conditions

[0163]

[0164]

[0165] The results showed that the two amplicones covering the entire HEK293T mtDNA genome were amplified in a balanced and efficient manner (e.g., Figure 8 ).

[0166] Example 4: Multiplex PCR amplification of single-cell genome based on strand displacement activity, with three amplicones covering the HEK293TmtDNA genome.

[0167] 1. Preparation of cell lysis buffer and sorting plate. Prepare sterile eight-tube PCR tubing. Prepare lysis buffer using pure water with the following solute concentrations: 200mM NaOH; 50mM DTT. Add 1 μL of the prepared lysis buffer to each well of the sorting plate.

[0168] 2. Single-cell sorting. Collect HEK293T cells, centrifuge at 500G, discard the supernatant after centrifugation, and wash twice with 1×PBS; resuspend the cells in 100μL staining buffer, and add Zombie NIR at a volume ratio of 1:100. TM Dye, incubate in the dark for 20 minutes; wash twice with 1 mL 1×PBS, and finally resuspend the cells in 500 μL 1×PBS; sort single live cells by flow cytometry (Zombie NIR). TM (Negative) to the well plate into which the lysis buffer was added.

[0169] 3. Cell lysis. Cells are lysed using an alkaline lysis method combined with high-temperature lysis (65°C, 10 minutes) to fully release the DNA from the cells into the lysis buffer.

[0170] 4. Multiplex PCR amplification of single-cell genomes based on strand displacement activity. For multiplex PCR amplification of single-cell genomes based on strand displacement activity, prepare the reaction solution on ice and place the reaction system in a PCR instrument for PCR reaction as soon as possible.

[0171] The PCR reaction system and PCR reaction procedure are shown in Table 12 and Table 13, respectively.

[0172] Table 12. PCR Reaction System

[0173]

[0174]

[0175] Table 13. PCR Reaction Conditions

[0176]

[0177] Electrophoresis analysis showed the presence of approximately 5.5 kb of amplification products, such as... Figure 9 As shown.

[0178] 5. Quantitative real-time PCR analysis of multiplex PCR results

[0179] The experimental group and the negative control (without LongAmp Taq DNA polymerase) were diluted 100-fold to serve as templates for real-time PCR. The reaction solution was prepared on ice, and the reaction system was placed in an ABI 7500 real-time PCR instrument for PCR reaction as soon as possible.

[0180] The real-time PCR reaction system and procedure are shown in Table 8. (Note: Primers 1 and 2 in the table are primers 5.5k-1F and 5.5k-1R from Table 5, respectively, used for amplification.) Figure 10 Amplicon 1; or primers 5.5k-2F and 5.5k-2R, respectively, are used for amplification. Figure 10 Amplicon 2; or primers 5.5k-3FF and 5.5k-3R, respectively, are used for amplification. Figure 10 Amplicon 3) and Table 11.

[0181] The results showed that the three amplicones covering the entire HEK293T mtDNA genome were amplified in a balanced and efficient manner, such as... Figure 10 .

[0182] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. A method for amplifying mitochondrial genome DNA for non-disease diagnosis and treatment purposes, comprising the following steps: using a nucleic acid sample containing mitochondrial genome as a template, and performing multiplex PCR with a DNA polymerase having strand displacement activity; the primer combination used in the multiplex PCR consists of a chimeric primer pair and a universal primer; the chimeric primer pair consists of three pairs of primers, and the nucleotide sequences of the primers are shown in SEQ ID No. 6-11; the nucleotide sequence of the universal primer is shown in SEQ ID No. 12; and the DNA polymerase having strand displacement activity is LongAmp Taq DNA polymerase or 3173 DNA polymerase. The nucleic acid sample containing mitochondrial genome is from a single cell or multiple cells. In the multiplex PCR, the working concentration of the universal primer is 0.1-1 µM, and the working concentrations of the upstream primer and the downstream primer in each chimeric primer pair are 1 / 100-1 / 50 of the working concentration of the universal primer. And / or 2. The method of claim 1, wherein: The nucleic acid sample containing mitochondrial genome is from a human cell.

3. The method according to claim 1 or 2, characterized in that: 4.The method according to any one of claims 1-3, for non-disease diagnosis and treatment purposes in any one of the following: (A1) single cell mitochondrial genome sequencing; (A2) detecting mitochondrial genome heterogeneity variation at a low level of single cell; (A3) detecting mtDNA heterogeneity in a multi-cell mixed sample. ​ ​ ​

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