CN116200459A - Efficient and specific amplification method for whole genome of single cell mitochondria - Google Patents

Abstract

The invention discloses a high-efficiency and specific amplification method for the whole genome of single-cell mitochondria. The invention provides a method for high-fidelity amplification of mitochondrial genomic DNA in single or multiple cells, comprising: using the total DNA obtained by lysing a single cell for testing as a template, using the non-template matching common sequence at the 5' end and the 3' end Chimeric primers composed of mitochondrial DNA (mitochondrial DNA, mtDNA) specific matching sequences, and primers corresponding to the 5'-end non-template matching common sequences of the above primers, use a high-fidelity DNA polymerase with strand displacement activity to simultaneously amplify Several overlapping amplicons that completely cover the mitochondrial genomic DNA of single or multicellular cells are amplified. The invention avoids various problems in the current single-cell mtDNA amplification method in vitro, and is a very reliable mtDNA whole genome amplification method.

Description

A method for efficient and specific amplification of the whole genome of single-cell mitochondria

Technical Field

The invention relates to the field of biotechnology, and in particular to a method for highly efficient and specific amplification of a single-cell mitochondrial whole genome.

Background Art

Mitochondria are not only the energy factories of eukaryotic cells, but also play an important role in many cellular functions such as apoptosis regulation, immune response, signal transduction, biomass synthesis, and cytoplasmic calcium ion regulation. Mitochondria have genetic material independent of the nuclear genome - mitochondrial DNA (mtDNA). mtDNA is located in the matrix of mitochondria. The total length of human mtDNA is about 16.5kb, existing in the form of closed circular double strands, encoding a total of 37 genes, including 13 respiratory chain protein encoding genes and 22 tRNA genes and 2 rRNA genes required for transcription and translation of these protein encoding genes. Its total gene coding region accounts for about 95%, and replication starts in the D-loop region. Other regions are responsible for regulating the replication and transcription functions of mtDNA. Due to the lack of an efficient recombination repair system in mitochondria, the fidelity of DNA polymerase γ responsible for mtDNA replication is low. mtDNA is close to the oxidative phosphorylation reaction site, without the protection of histones, and is susceptible to the attack of reactive oxygen species (ROS) free radicals by reaction byproducts, resulting in high-frequency mutations in mtDNA. Studies have shown that the mutation rate of mtDNA is 10-17 times higher than that of nuclear DNA. mtDNA exists in the form of multiple copies in cells, and mutant mtDNA coexists with wild-type mtDNA to form mtDNA heteroplasmy. Unlike the diploid nuclear genome, mtDNA heteroplasmy can vary between 0-100%. Even between different cells of the same tissue in the same individual, there may be differences in mtDNA heteroplasmy. Different proportions of mtDNA heteroplasmy may lead to completely different phenotypes. The current multi-cell mixed DNA sequencing method can only detect the average value of mtDNA heteroplasmy in the cell population, which not only ignores the unique mtDNA heteroplasmy characteristics of a single cell, but also masks harmful mtDNA mutations in a few cells, resulting in the loss of many important information.

mtDNA accounts for less than 1% of the total cellular DNA. Direct whole-genome sequencing of cells makes it difficult to deeply cover the mitochondrial genome and cannot accurately detect mtDNA heteroplasmy. Deep sequencing of mtDNA enriched in single cells can study mtDNA heteroplasmy from a single-cell dimension. Early single-cell mtDNA enrichment was mainly achieved through PCR amplification. This type of method generally selects the mtDNA D-loop region with large variations as the amplification target, with a coverage length of about 1kb. The enrichment target of this type of method is limited to a small part of the mitochondrial genome, and it is impossible to obtain a complete picture of single-cell mtDNA mutations. Subsequent research mainly relies on two methods: in vivo cell amplification and in vitro nucleic acid amplification to solve the problem that the amount of single-cell DNA samples is insufficient for direct sequencing analysis.

In vivo cell amplification pathway, isolated single cells are cultured in vitro, DNA is extracted after the cells proliferate to a sufficient number, and then the mtDNA is enriched for high-throughput sequencing. The biggest problem with this type of method is that a single cell needs to be propagated in vitro for multiple generations before sufficient DNA can be obtained for sequencing, so it is not suitable for non-immortalized cells or cells that cannot be cultured in vitro. In addition, long-term in vitro culture may also lead to changes in mtDNA heterogeneity, and whether the results obtained can accurately reflect the characteristics of the original sample is also controversial.

In vitro nucleic acid amplification pathway, mitochondrial genome is directly amplified and enriched from single-cell lysate. Different from the early amplification of partial mitochondrial regions, this improved method adopts multiple amplification strategies. Either multiple overlapping replicons, or long-range PCR, or rolling circle amplification based on chain displacement to specifically amplify the single-cell mitochondrial genome, basically can achieve complete coverage of mtDNA. Morris et al. further achieved mtDNA amplification in single mitochondria. The application of these methods has enabled people to further understand the heterogeneity of mtDNA at the single-cell level, but there are also certain defects. In order to avoid mutual interference between primers used in the amplification reaction, this type of amplification method usually distributes single-cell lysate into different reaction systems, resulting in each reaction system only being able to amplify part of the mtDNA of the initial sample, resulting in the loss of original information. Although rolling circle amplification can complete the amplification of the entire mtDNA in one reaction, the extraction and purification of single cells before amplification and the large number of reaction byproducts need to be further improved. In addition, the amplification products obtained by the amplification method 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

In order to overcome the problems in the prior art, the purpose of the present invention is to provide a method for efficient and complete amplification of single-cell mtDNA. It includes obtaining a single cell through a single-cell preparation technology and lysing it, and after fully releasing the DNA, using a chimeric primer composed of a 5'-end non-template matching common sequence and a 3'-end mtDNA specific matching sequence, and a primer corresponding to the 5'-end non-template matching common sequence of the above primers, using a high-fidelity DNA polymerase with strand displacement activity, and simultaneously amplifying several overlapping amplicons covering the complete single-cell mitochondrial genome. The present invention realizes efficient and specific amplification of the entire sequence of the single-cell mitochondrial genome in a single reaction system. The mtDNA enriched based on the method of the present invention can be used for single-cell mtDNA sequencing to accurately detect mtDNA heteroplasmic variation at the single-cell level.

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

The mitochondrial genome DNA amplification method claimed in the present invention may include the following steps: using a nucleic acid sample to be tested containing the mitochondrial genome as a template, using a DNA polymerase having strand displacement activity to perform multiple PCR, wherein the primer combination used for the multiple PCR contains N chimeric primer pairs; N is a positive integer of 2 or greater;

The upstream primer and the downstream primer in each chimeric primer pair are composed of the following (a1) and (a2) from the 5' end to the 3' end:

(a1) a universal sequence that does not match the mitochondrial genome;

(a2) a specific sequence that completely matches the mitochondrial genome;

Furthermore, the universal sequences in different chimeric primer pairs are the same, and the specific sequences are different;

Furthermore, the binding positions of the specific sequences in different chimeric primer pairs on the mitochondrial genome are dispersedly distributed (relatively uniformly);

The multiplex PCR is used to obtain N segments of amplicons (with overlapping ends) that can completely cover the mitochondrial genome.

Further, the primer combination used for performing the multiplex PCR consists of the N chimeric primer pairs and a universal primer;

The universal primer is as follows (b1) or (b2) or (b3) or (b4):

(b1) the universal sequence;

(b2) being reverse complementary to the universal sequence;

(b3) a continuous sequence selected from the universal sequence;

(b4) reverse complementing a continuous sequence selected from the universal sequence.

Furthermore, the nucleic acid sample to be tested containing the mitochondrial genome may come from a single cell or from multiple cells.

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

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

The upstream primer and the downstream primer in each chimeric primer pair are composed of the following (a1) and (a2) from the 5' end to the 3' end:

(a1) a universal sequence that does not match the mitochondrial genome of the single cell;

(a2) a specific sequence that completely matches the mitochondrial genome of the single cell;

Furthermore, the universal sequences in different chimeric primer pairs are the same, and the specific sequences are different;

Furthermore, the binding positions of the specific sequences in different chimeric primer pairs on the mitochondrial genome of the single cell are dispersedly distributed (relatively uniformly);

The universal primer is as follows (b1) or (b2) or (b3) or (b4):

(b1) the universal sequence;

(b2) being reverse complementary to the universal sequence;

(b3) a continuous sequence selected from the universal sequence;

(b4) reverse complementing a continuous sequence selected from the universal sequence;

The multiplex PCR is used to obtain N amplicons (with overlapping ends) that can completely cover the mitochondrial genome of the single cell.

Further, in each chimeric primer pair, the binding position of the specific sequence in the upstream primer on the mitochondrial genome of the single cell is located within the amplification target of the chimeric primer pair adjacent to the upstream, and the binding position of the specific sequence in the downstream primer on the mitochondrial genome of the single cell is located within the amplification target of the chimeric primer pair adjacent to the downstream. For example, primer A and primer D in Figure 1 are one chimeric primer pair, and primer B and primer C are another chimeric primer pair.

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

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

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 mainly plays a role in DNA polymerase activity, and the latter mainly plays a role in strand displacement activity. Of course, the DNA polymerase can also be other DNA polymerases with strand displacement activity.

In the present invention, the design of each primer only needs to comply with the general primer design principles, GC%, 40-60%; Tm is greater than the 3'-end specific primer to avoid the formation of secondary structures such as hairpins or dimers.

In the method, the method for extracting total DNA from a single cell may be a conventional method in the art.

The nucleic acid sample to be tested containing the mitochondrial genome can come from any cell whose mitochondrial genome sequence is known, such as a human cell.

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

In a specific embodiment of the present invention, the universal primer is primer E; the sequence of primer E is shown as SEQ ID No.5.

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

In a specific embodiment of the present invention, the chimeric primers are two pairs, respectively as follows:

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;

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

In another specific embodiment of the present invention, the chimeric primers are three pairs in total, which are as follows:

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;

A second pair of chimeric primers consisting 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;

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

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

In a second aspect, the present invention claims a method for amplifying covalently closed circular DNA.

The covalently closed circular DNA amplification method claimed for protection in the present invention is different from the steps of the method described in the first aspect above, except that the nucleic acid sample to be tested containing the mitochondrial genome is replaced by a nucleic acid sample to be tested containing covalently closed circular DNA, and the mitochondrial genome is replaced by the covalently closed circular DNA.

In a third aspect, the present invention claims application of the method described in the first aspect above in any of the following:

(A1) Single-cell mitochondrial genome sequencing;

(A2) Detection of mitochondrial genome heteroplasmic variation down to the single-cell level;

(A3) Detection of mtDNA heteroplasmy in multi-cell mixed samples.

In a fourth aspect, the present invention claims a set of products for amplifying the human mitochondrial genome.

The complete set of products for amplifying the human mitochondrial genome claimed in the present invention is complete set of product A or complete set of product B.

The complete set of product A is composed of the following (c1)-(c3):

(c1) the primer A, the primer B, the primer C, and the primer D in the first aspect above;

(c2) the primer E described in the first aspect above;

(c3) The DNA polymerase having strand displacement activity as described in the first aspect above.

The complete set of product B consists of the following (d1)-(d3):

(d1) the primer F, the primer G, the primer H, the primer I, the primer J, and the primer K in the first aspect above;

(d2) the primer L in the first aspect above;

(d3) The DNA polymerase having strand displacement activity as described in the first aspect above.

In a fifth aspect, the present invention claims protection for the use of the set of products described in the fourth aspect above in any of the following:

(A1) Single-cell mitochondrial genome sequencing;

(A2) Detection of mitochondrial genome heteroplasmic variation down to the single-cell level;

(A3) Detection of mtDNA heteroplasmy in multi-cell mixed samples.

Beneficial effects of the present invention:

1. The heterogeneity of mtDNA in single cells can be preserved completely and truly. The mtDNA extracted from multi-cell mixed samples can only obtain the average value of mtDNA heterogeneity in the cell group, and the unique mtDNA heterogeneity characteristics of a single cell are lost during the DNA extraction process. As a result, the mtDNA from multiple cells masks the mtDNA mutations in a few cells, resulting in the loss of many important mutation information. The present invention specifically enriches the whole genome of mtDNA in single-cell lysate. It is suitable for high-resolution single-cell mtDNA sequencing, which will help reveal the changing mechanism of single-cell mtDNA heterogeneity in different cell lineages.

2. It is applicable to non-immortalized cells and cells that cannot be cultured in vitro. Conventional mtDNA enrichment usually relies on in vivo cell amplification. This process involves culturing isolated single cells in vitro, extracting DNA after the cells have proliferated to a sufficient number, and then enriching the mtDNA therein. The biggest problem with this type of method is that single cells need to be propagated in vitro for multiple generations to obtain enough starting DNA for subsequent enrichment, and therefore cannot be applied to non-immortalized cells and cells that cannot be cultured in vitro. In addition, long-term in vitro culture may also lead to changes in mtDNA heterogeneity, and the enriched mtDNA is difficult to accurately reflect the original appearance of the original starting cell mtDNA. This method can be directly applied to isolated single cells, does not require enrichment of mtDNA through cell culture, and will not produce changes in mtDNA heterogeneity. Therefore, the present invention has extensiveness for the selection of samples.

3. The full picture of single-cell mtDNA mutations can be fully displayed. Different from PCR amplification of the mtDNA D-loop vicinity or other partial regions of the mitochondrial genome, the present invention uses a multiplex PCR method based on strand displacement activity to amplify, and the amplicons contained in the amplified products can completely cover the mtDNA genome.

4. The mitochondrial genome can be directly amplified and enriched from the single-cell lysate. In order to avoid mutual interference between the primers used in conventional multiplex PCR amplification, the single-cell lysate is usually distributed to different reaction systems, resulting in each reaction system being able to amplify only part of the mtDNA of the initial sample, and the loss of original information. Although rolling circle amplification can complete the amplification of the entire mtDNA in one reaction, problems such as the extraction and purification of single cells before amplification and the large number of reaction by-products need to be further improved. In addition, the amplification products obtained by the amplification method 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. The primers in the mitochondrial single-cell mtDNA whole genome amplification method provided by the present invention can complete cell lysis and mtDNA whole genome amplification in one reaction, avoiding the loss of original information; at the same time, it avoids the possibility of sample cross-contamination due to liquid separation, which is conducive to high-throughput sample preparation.

The present invention avoids various problems in the current nucleic acid in vitro amplification pathway and is a very reliable method for amplifying the entire mtDNA genome.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a schematic diagram of a method for multiplex PCR amplification of closed circular DNA templates based on DNA polymerase strand displacement activity of the present invention.

Figure 2 shows the flow cytometry sorted single cells in Example 1. Zombie NIR ^TM -: Jurkat cell line not stained with Zombie NIR ^TM (left); Zombie NIR ^TM+ : Jurkat cell line stained with Zombie NIR ^TM (right), R1 represents Jurkat living cells not stained with Zombie NIR ^TM ; R2 represents Jurkat cells stained with Zombie NIR ^TM .

Figure 3 is the gel electrophoresis detection result of the amplified products of Jurkat single cell mitochondrial genome sorted by multiple PCR amplification based on DNA polymerase chain displacement activity in Example 1. Lanes 1-5 are mitochondrial genome DNAs of five different Jurkat single cells amplified by multiple PCR method based on DNA polymerase chain displacement activity, and the fragment size is about 8.3 kb; Lane 6 is a negative control without LongAmp DNA polymerase; Lane 7 is a DNA marker.

Figure 4 shows the relative yields of two amplicons in two Jurkat single cell mtDNA amplification products detected by fluorescence quantitative PCR in Example 1. The Jurkat single cell mtDNA whole genome amplification product was diluted 100 times and then amplicon-specific primers were used for fluorescence quantitative PCR amplification, as shown in the CT value of the experimental group. The higher the CT value, the lower the mtDNA content; the negative control product without LongAmp DNA polymerase but containing template mtDNA was diluted 100 times and then amplified by specific primers for fluorescence quantitative PCR amplification, as shown in the CT value of the negative control group; the PCR reaction system without template was amplified by specific primers for fluorescence quantitative PCR amplification, as shown in the CT value of the blank control group.

Figure 5 shows the multiplex PCR amplification of human peripheral blood mononucleated cell mitochondrial genome based on DNA polymerase chain displacement activity in Example 2. Lanes 1-7 are 7 human peripheral blood mononucleated cell mitochondrial genome DNAs amplified by multiplex PCR based on DNA polymerase chain displacement activity, respectively, with a fragment size of about 8.3 kb; Lane 8 is a negative control without LongAmp DNA polymerase; Lane 9 is a DNA marker.

Figure 6 shows the yield of two amplicons in the mtDNA amplification product of human peripheral blood nucleated monocytes detected by fluorescence quantitative PCR in Example 2. The whole genome amplification product of human peripheral blood nucleated monocyte mtDNA was diluted 100 times and then used for fluorescence quantitative PCR detection using amplicon specific primers, as shown in the CT value of the experimental group, the higher the CT value, the lower the mtDNA content; the negative control product without LongAmp DNA polymerase but containing template mtDNA was diluted 100 times and then used for fluorescence quantitative PCR amplification using specific primers, as shown in the CT value of the negative control group; the PCR reaction system without template was amplified using specific primers for fluorescence quantitative PCR amplification, as shown in the CT value of the blank control group.

Figure 7 shows the amplification of HEK293T single cell mitochondrial genome by 3173 DNA polymerase with strand displacement activity in Example 3. Lanes 1-8 are mitochondrial genome DNA of 8 sorted HEK293T single cells amplified by 3173 DNA polymerase with strand displacement activity using a multiplex PCR method based on strand displacement activity, and the fragment size is about 8.3 kb; Lane 9 is a negative control without adding 3173 DNA polymerase; Lane 10 is a DNA marker.

Figure 8 shows the yield of two amplicons in the mtDNA amplification product of HEK293T single cells detected by fluorescence quantitative PCR in Example 3. The HEK293T single cell mtDNA whole genome amplification product was diluted 100 times and then amplicon-specific primers were used for fluorescence quantitative PCR amplification, as shown in the CT value of the experimental group. The higher the CT value, the lower the mtDNA content; the negative control product without 3173DNA polymerase but containing template mtDNA was diluted 100 times and then amplicon-specific primers were used for fluorescence quantitative PCR detection, as shown in the CT value of the negative control group; the PCR reaction system without template was used for fluorescence quantitative PCR amplification using amplicon-specific primers, as shown in the CT value of the blank control group.

Figure 9 shows that three amplicons completely cover the HEK293T single cell mtDNA genome in Example 4. Lanes 1-8 are HEK293T single cell mitochondrial genomic DNA amplified by a multiplex PCR method based on strand displacement activity, and three amplicons cover the HEK293T single cell mtDNA genome, with a fragment size of about 5.5 kb; Lane 9 is a negative control without LongAmp DNA polymerase; Lane 10 is a DNA marker.

Figure 10 shows the yield of three amplicons in the mtDNA amplification product of HEK293T single cells detected by fluorescence quantitative PCR in Example 4. The HEK293T single cell mtDNA whole genome amplification product was diluted 100 times and then amplicon-specific primers were used for fluorescence quantitative PCR amplification, as shown in the CT value of the experimental group. The higher the CT value, the lower the mtDNA content; the negative control product without LongAmpDNA polymerase but containing template mtDNA was diluted 100 times and then amplified by specific primers for fluorescence quantitative PCR amplification, as shown in the CT value of the negative control group; the PCR reaction system without template was amplified by specific primers for fluorescence quantitative PCR amplification, as shown in the CT value of the blank control group.

DETAILED DESCRIPTION

The present invention adopts the following technical solution:

(1) Preparation of template for mtDNA amplification by single cell sorting and lysis:

a. Preparation of cell lysis solution and sorting plate. In a sterile environment, use pure water to prepare single-cell mitochondrial lysis solution: 200mM NaOH, 50mM DTT; prepare a sterile, enzyme-free 96-well plate or eight-row PCR tube; add the prepared lysis solution to the sorting well plate, 2μL lysis solution per well. Care should be taken to avoid contamination from other unrelated mtDNA during the operation;

b. Single cell preparation. If it is a suspension cell line, the cells should be collected, centrifuged at 350G and the supernatant discarded, and the cells should be washed twice with 1×PBS and resuspended in 100μL staining buffer; if it is an adherent cell line, the cells should be fully digested with trypsin, centrifuged at 350G and the supernatant discarded, and the cells should be washed twice with 1×PBS and resuspended in 100μL staining buffer; if it is a tissue sample, the single cell suspension should be prepared according to the method of tissue digestion. After the tissue is digested into single cells, the supernatant should be discarded after centrifugation at 350G, and the cells should be washed twice with 1×PBS and resuspended in 100μL staining buffer. Add Zombie NIR ^TM dye at a volume ratio of 1:100, incubate in the dark for 20 minutes (or use other methods to distinguish between dead and live cells for staining); wash twice with 1mL 1×PBS, and finally resuspend the cells with 500μL 1×PBS; sort single living cells (or use other single cell separation methods) by flow cytometry into a well plate or tube pre-added with lysis buffer;

c. Cell lysis and preparation of template DNA. Cells can be lysed using alkaline lysis combined with high temperature cell lysis (high temperature cell lysis program: 65°C, 10 minutes; 4°C, insulation) or other cell lysis methods to fully release the DNA in the cells into the lysis solution. The prepared template DNA should be used for PCR as soon as possible or frozen at -80°C.

(2) Multiplex PCR amplification of single-cell mitochondrial genome based on strand displacement activity:

a. Design of multiple PCR primers based on strand displacement activity. Use chimeric primers consisting of a non-template matching common sequence at the 5' end and a mtDNA-specific matching sequence at the 3' end, and primers corresponding to the non-template matching common sequence at the 5' end of the above primers (referred to as universal primers), wherein the chimeric primers can be two pairs or more pairs; the universal primer can be any sequence with efficient amplification effect. The amplification process is (taking the primer combination corresponding to two pairs of chimeric primers plus the non-template matching common sequence as an example) chimeric primer A extends to chimeric primer C, the DNA polymerase with strand displacement activity exerts strand displacement activity, and chimeric primer A continues to extend to the 5' end of primer D and beyond, providing a binding site for primer D for the next round of PCR reaction. Therefore, primer D should be designed downstream of the binding position of primer C on the template DNA to ensure the complete amplification of the template DNA; but the distance should not be too large to prevent the strand displacement activity from being difficult to displace too long a fragment of DNA. The design of other chimeric primers is similar. At this point, a preliminary amplification product is formed (as shown in Figure 1). After the initial amplification product is formed, the initial amplification product is further amplified more efficiently by a universal primer (ie, primer E in FIG. 1 ), forming a plurality of amplicons with overlapping ends (as shown in FIG. 1 ).

b. The components of the multiplex PCR based on strand displacement activity are mainly composed of 5×LongAmp Taq reaction buffer, 10mM dNTPs, chimeric primers, universal primers, template DNA, Tricine (200mM), LongAmp Taq DNA polymerase, and ddH ₂ O (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 plays the role of DNA polymerase, and the latter mainly plays the role of strand displacement. Other high-temperature resistant DNA polymerases with strand displacement activity, such as 3173 DNA polymerase, can also be used.

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

c. Multiplex PCR program based on strand displacement activity. Prepare the reaction solution on ice and place the reaction system in a PCR instrument as soon as possible to perform the PCR program. The PCR program is shown in Table 2, where 65°C in the amplification step is the extension temperature of LongAmp Taq DNA polymerase, the rate is 1kb/50s, and the extension time needs to be set according to the extension length; the subsequent 72°C and 30 seconds are the strand displacement reaction, because the length of the strand displacement only needs to be displaced to the 5' end of the next chimeric primer and beyond, providing a primer binding site for the next round of PCR reaction, so it does not take a long time. If other DNA polymerases are used, the PCR program should be set according to the instructions.

Table 2. PCR reaction conditions

d. Electrophoresis analysis: After the reaction is completed, take 3-5 μL for agarose gel electrophoresis analysis.

(3) Fluorescence quantitative PCR was used to detect the amplification efficiency of each amplicon. For each amplicon, amplicon-specific RT-PCR primers were designed. The experimental group and negative control (without LongAmp Taq DNA polymerase) were diluted 100 times respectively and used as fluorescence quantitative PCR templates. The reaction solution was prepared on ice and the reaction system was placed in a fluorescence quantitative PCR instrument as soon as possible for PCR reaction. The reaction procedure is shown in Table 3.

Table 3. RT-PCR reaction conditions

The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions, such as the conditions described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial sources.

1. The materials and reagents used in the examples of the present invention are shown in Table 4.

Table 4. Materials and reagents

2. The primers used in the examples of the present invention are shown in Table 5.

Table 5. Primers used in the examples of the present invention

Example 1: Amplification of the entire mtDNA genome of Jurkat cells

1. Preparation of cell lysis solution and sorting plate. Prepare sterile and enzyme-free 8-row PCR tubes; prepare lysis solution with pure water, the solute and concentration are as follows: 200mM NaOH; 50mM DTT. Add the prepared lysis solution to the sorting plate, 2μL lysis solution per well.

2. Single cell sorting. Jurkat cells were collected and centrifuged at 500G. The supernatant was discarded and washed twice with 1×PBS. The cells were resuspended in 100μL staining buffer and Zombie NIR ^TM dye was added at a volume ratio of 1:100. The cells were incubated in the dark for 20 minutes. The cells were washed twice with 1mL 1×PBS and resuspended with 500μL 1×PBS. Single live cells (Zombie NIR ^TM negative) were sorted by flow cytometry into a well plate containing cell lysate (as shown in Figure 2).

3. Cell lysis: Use alkaline lysis combined with high temperature cell lysis (65°C, 10 minutes) to fully release the DNA in the cells into the lysis buffer.

4. Multiplex PCR amplification of Jurkat single-cell genome 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 reaction.

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

Table 6. PCR reaction system

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 _ddH2O Make up to 20 μL

Table 7. PCR reaction program

The electrophoresis analysis results showed that an amplified product of about 8 kb appeared, as shown in Figure 3.

5. Fluorescence quantitative PCR analysis of multiple PCR results

The experimental group and negative control (without LongAmp Taq DNA polymerase) were diluted 100 times respectively and used as templates for fluorescence quantitative PCR. The reaction solution was prepared on ice and the reaction system was placed in ABI 7500 fluorescence quantitative PCR instrument as soon as possible for PCR reaction.

The fluorescence quantitative PCR reaction system and reaction procedure are shown in Table 8 and Table 3, respectively.

Table 8. PCR reaction system

Note: Primer 1 and Primer 2 in the table are respectively Primer 8k-1F and Primer 8k-1R in Table 5 (used to amplify Amplicon 1 in FIG. 4 ), or Primer 8k-2F and Primer 8k-2R (used to amplify Amplicon 2 in FIG. 4 ).

The results showed that the two amplicons covering the entire mtDNA genome of Jurkat cells were amplified evenly and efficiently (Figure 4).

Example 2: Preparation of peripheral blood nucleated cell single cells by limiting dilution method and amplification of mtDNA whole genome

1. Collect peripheral blood in an anticoagulant tube, lyse the red blood cells with red blood cell lysis buffer, and wash them twice with 1× PBS to prepare a peripheral blood nucleated cell suspension;

2. Count the peripheral blood nucleated cells, dilute the cells to about 50 cells per 100 μL, blow and beat evenly, use a 2 μL pipette to absorb the cell suspension and first spot a few wells, 2 μL per well, the droplet should be as big as sesame seeds, then observe under an inverted microscope, if there are 1 to 2 cells per well, it means the cell concentration is appropriate, and the 96-well plate can be spotted at one time. If not, the droplet can be enlarged or reduced, or more cells can be added, or more culture medium can be added for dilution.

3. Cell lysis: Use high temperature method to lyse cells (99°C, 10 minutes, hot cover 110°C) to fully release the DNA in the cells into the lysis solution.

4. Multiplex PCR amplification of single-cell genome 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 reaction.

The PCR reaction system and PCR reaction program are shown in Table 6 and Table 7, respectively.

The electrophoresis analysis results showed that an amplified product of about 8 kb appeared, as shown in Figure 5.

5. Fluorescence quantitative PCR analysis of multiple PCR results

The experimental group and negative control (without LongAmp Taq DNA polymerase) were diluted 100 times respectively and used as templates for fluorescence quantitative PCR. The reaction solution was prepared on ice and the reaction system was placed in ABI 7500 fluorescence quantitative PCR instrument as soon as possible for PCR reaction.

The fluorescence quantitative PCR reaction system and reaction procedure are shown in Table 8 (Note: Primer 1 and Primer 2 in the table are primer 8k-1F and primer 8k-1R in Table 5, respectively, used to amplify amplicon 1 in Figure 6; or primer 8k-2F and primer 8k-2R, respectively, used to amplify amplicon 2 in Figure 6) and Table 3.

The results showed that the two amplicons covering the entire mtDNA genome of peripheral blood nucleated cells were amplified evenly and efficiently (as shown in Figure 6).

Example 3: Amplification of the whole genome of HEK293T cell mtDNA using 3173 DNA polymerase

1. Preparation of cell lysis solution and sorting plate. Prepare eight sterile PCR tubes in a row; prepare lysis solution with pure water, the solute and concentration are as follows: 200mM NaOH; 50mM DTT. Add the prepared lysis solution to the sorting plate, 1μL lysis solution per well;

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, add Zombie NIR ^TM dye at a volume ratio of 1:100, incubate in the dark for 20 minutes; wash twice with 1mL 1×PBS, and finally resuspend the cells with 500μL 1×PBS; sort single live cells (Zombie NIR ^TM negative) by flow cytometry into the well plate with lysis buffer.

3. Cell lysis: Use alkaline lysis combined with high temperature cell lysis (65°C, 10 minutes) to fully release the DNA in the cells into the lysis buffer.

4. Multiplex PCR amplification of single-cell genome 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 reaction.

The PCR reaction system and PCR reaction program are shown in Table 9 and Table 10, respectively.

Table 9, PCR reaction system:

Table 10. PCR reaction conditions

The electrophoresis analysis results showed that an amplification product of about 8K appeared, as shown in Figure 7.

5. Fluorescence quantitative PCR analysis of multiple PCR results

The experimental group and negative control (without 3173 DNA polymerase) were diluted 100 times respectively and used as templates for fluorescence quantitative PCR. The reaction solution was prepared on ice and the reaction system was placed in ABI 7500 fluorescence quantitative PCR instrument as soon as possible for PCR reaction.

The fluorescence quantitative PCR reaction system and reaction procedure are shown in Table 8 (Note: Primer 1 and Primer 2 in the table are primer 8k-1F and primer 8k-1R in Table 5, respectively, used to amplify amplicon 1 in Figure 8; or primer 8k-2F and primer 8k-2R, respectively, used to amplify amplicon 2 in Figure 8) and Table 11.

Table 11. PCR reaction conditions

The results showed that the two amplicons covering the entire HEK293T mtDNA genome were amplified evenly and efficiently (as shown in Figure 8).

Example 4: Multiplex PCR amplification of single cell genome based on strand displacement activity, three amplicons covering the HEK293T mtDNA genome

1. Preparation of cell lysis solution and sorting plate. Prepare eight sterile PCR tubes and prepare lysis solution with pure water. The solute and concentration are as follows: 200mM NaOH; 50mM DTT. Add the prepared lysis solution to the sorting plate, 1μL lysis solution per well;

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, add Zombie NIR ^TM dye at a volume ratio of 1:100, incubate in the dark for 20 minutes; wash twice with 1mL 1×PBS, and finally resuspend the cells with 500μL 1×PBS; sort single live cells (Zombie NIR ^TM negative) by flow cytometry into the well plate with lysis buffer.

3. Cell lysis: Use alkaline lysis combined with high temperature cell lysis (65°C, 10 minutes) to fully release the DNA in the cells into the lysis buffer.

4. Multiplex PCR amplification of single-cell genomes based on strand displacement activity. 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 as soon as possible for PCR reaction.

The PCR reaction system and PCR reaction program are shown in Table 12 and Table 13, respectively.

Table 12. PCR reaction system

Table 13. PCR reaction conditions

The electrophoresis analysis results showed that an amplified product of about 5.5 kb appeared, as shown in FIG9 .

5. Fluorescence quantitative PCR analysis of multiple PCR results

The experimental group and negative control (without LongAmp Taq DNA polymerase) were diluted 100 times respectively and used as templates for fluorescence quantitative PCR. The reaction solution was prepared on ice and the reaction system was placed in ABI 7500 fluorescence quantitative PCR instrument as soon as possible for PCR reaction.

The fluorescence quantitative PCR reaction system and reaction procedure are respectively shown in Table 8 (Note: Primer 1 and Primer 2 in the table are primer 5.5k-1F and primer 5.5k-1R in Table 5, respectively, used to amplify amplicon 1 in Figure 10; or primer 5.5k-2F and primer 5.5k-2R, respectively, used to amplify amplicon 2 in Figure 10; or primer 5.5k-3FF and primer 5.5k-3R, respectively, used to amplify amplicon 3 in Figure 10) and Table 11.

The results showed that the three amplicons covering the entire HEK293T mtDNA genome were amplified evenly and efficiently, as shown in Figure 10.

The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be implemented in a wide range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the present invention and without unnecessary experimentation. Although the present invention provides specific embodiments, it should be understood that further improvements may be made to the present invention. In short, according to the principles of the present invention, this application is intended to include any changes, uses or improvements to the present invention, including changes made by conventional techniques known in the art that depart from the scope disclosed in this application. Applications of some of the basic features may be made within the scope of the following appended claims.

Claims (10)

1. A method for amplifying mitochondrial genomic DNA, comprising the steps of: taking the nucleic acid sample to be tested containing the mitochondrial genome as a template, adopting a DNA polymerase with strand displacement activity to carry out multiple PCR, and carrying out the primer combination used in the multiple PCR containing N chimeric primer pairs; N is a positive integer of 2 or more; Each upstream primer and downstream primer in the chimeric primer pair is composed of the following (a1) and (a2) from the 5' end to the 3' end: (a1) a universal sequence that does not match said mitochondrial genome; (a2) a specific sequence that fully matches the mitochondrial genome; Moreover, the general sequences in different pairs of chimeric primers are the same, and the specific sequences are different; And, the binding positions of the specific sequences in the different chimeric primer pairs on the mitochondrial genome are dispersedly distributed; The N-segment amplicon that can completely cover the mitochondrial genome is obtained through the multiplex PCR. 2. The method according to claim 1, characterized in that: the primer combination used for the multiplex PCR is composed of the N chimeric primer pairs and a universal primer; The universal primer is as follows (b1) or (b2) or (b3) or (b4): (b1) said universal sequence; (b2) is reverse complementary to said universal sequence; (b3) a continuous sequence selected from the universal sequence; (b4) is reverse complementary to a continuous sequence selected from the general sequence. 3. The method according to claim 1 or 2, characterized in that: the nucleic acid sample to be tested containing the mitochondrial genome comes from a single or multiple cells. 4. The method according to any one of claims 1-3, characterized in that: in each of the chimeric primer pairs, the binding position of the specific sequence in the upstream primer on the mitochondrial genome Located in the amplification target of the upstream adjacent chimeric primer pair, the binding position of the specific sequence in the downstream primer on the mitochondrial genome is located in the downstream adjacent amplification target of the chimeric primer pair . 5. according to the method described in any one in claim 1-4, it is characterized in that: when carrying out described multiplex PCR, the working concentration of described universal primer is 0.1-1 μ M, the upstream in each described chimeric primer pair The working concentration of primers and downstream primers is 1/50 to 100/100 of the working concentration of the universal primer used; and / or The DNA polymerase with strand displacement activity is LongAmp Taq DNA polymerase or 3173 DNA polymerase; and / or The nucleic acid sample to be tested containing the mitochondrial genome comes from human cells. 6. The method according to any one of claims 1-5, characterized in that: said universal primer is primer E or primer L; the sequence of said primer E is as shown in SEQ ID No.5; said primer L The sequence of is shown in SEQ ID No.12. 7. according to the method described in any one in claim 1-6, it is characterized in that: described chimeric primer pair has two pairs, respectively as follows: The first pair of chimeric primers consisting 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; The second pair of chimeric primers consisting 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; or There are three pairs of chimeric primer pairs, which are as follows: The first pair of chimeric primers consisting 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; The second pair of chimeric primers consisting of primer H and primer I; the sequence of the primer H is shown in SEQ ID No.8; the sequence of the primer I is shown in SEQ ID No.9; The third pair of chimeric primers is composed 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. 8. A method for amplifying covalently closed circular DNA, compared with the steps of any one of claims 1-6, the only difference is that the nucleic acid sample to be tested containing the mitochondrial genome is replaced by The nucleic acid sample to be tested contains covalently closed circular DNA, and the mitochondrial genomic DNA therein is replaced by the covalently closed circular DNA. 9. The application of the method of any one of claims 1-7 in any of the following: (A1) Single-cell mitochondrial genome sequencing; (A2) Detection of mitochondrial genomic heterogeneity down to the single-cell level; (A3) Detection of mtDNA heterogeneity in multicellular mixed samples. 10. A set of products for amplifying the human mitochondrial genome, consisting of the following (c1)-(c3): (c1) said primer A, said primer B, said primer C, said primer D in claim 7; (c2) said primer E in claim 6; (c3) the DNA polymerase with strand displacement activity described in claim 1 or 4; or A set of products for amplifying the human mitochondrial genome, consisting of the following (d1)-(d3): (d1) said primer F, said primer G, said primer H, said primer I, said primer J, said primer K in claim 7; (d2) said primer L in claim 6; (d3) the DNA polymerase with strand displacement activity described in claim 1 or 4; or The application of the complete set of products in any of the following: (A1) Single-cell mitochondrial genome sequencing; (A2) Detection of mitochondrial genomic heterogeneity down to the single-cell level; (A3) Detection of mtDNA heterogeneity in multicellular mixed samples.

Images