One-step extraction kit, extraction method, and high-throughput sequencing method for vertebrate mitochondrial genomic DNA and use thereof

Abstract

Provided are a one-step extraction kit, an extraction method, and a high-throughput sequencing method for vertebrate mitochondrial genomic DNA and the use thereof. The kit comprises: a reagent A comprising 3.25-4.75M guanidinium isothiocyanate, 0.65-0.85M sodium citrate, and 5-15v / v% sodium N-lauroyl sarcosinate as a cell lysis and equilibration reagent; a reagent B comprising sodium acetate; and a reagent C comprising phenol, chloroform and isoamyl alcohol at a volume ratio of (120-130) : (19-29) : 1. Reagents B and C are used for mixing with a sample of the total DNA from the lysed cells and undergoing phase separation so as to obtain the aqueous phase of the mitochondrial DNA.

Description

Spinal cord mitochondrial genome DNA one-step extraction kit, extraction method, high-throughput sequencing method and application thereof

[0001] Cross-reference

[0002] The present application claims priority to the Chinese patent application No. 202411030534.9, filed on July 30, 2024, entitled "Spinal cord mitochondrial genome DNA one-step extraction kit, extraction method, high-throughput sequencing method and application thereof", the whole content of which is incorporated herein by reference. TECHNICAL FIELD

[0003] The present application relates to the field of gene sequencing and biological detection, in particular to a spinal cord mitochondrial genome DNA one-step extraction kit, a spinal cord mitochondrial genome DNA one-step extraction method, a high-throughput sequencing method and their applications in preparing mitochondrial genome base and structural mutation analysis products, species identification products and biological diversity analysis products. BACKGROUND

[0004] Animal mitochondrial DNA is a cytoplasmic DNA that is different from the nuclear genome DNA and exists independently in the mitochondrial organelle. Mitochondrial DNA is not only one of the important mechanisms for regulating cell mitochondrial energy metabolism, but also a main genetic marker molecule for animal evolution analysis and species identification. Mitochondrial genome sequencing analysis is widely used in human energy metabolism genetic disease diagnosis and various animal species identification and biological diversity analysis. Mitochondrial genome is a small, circular and multiple copy DNA, and its quality accounts for only 0.05% or less of the total DNA of the nuclear genome of the vertebrate. If the total DNA of the animal tissue is directly sequenced (or called whole genome sequencing, WGS) to obtain mitochondrial genome data, a large amount of waste, high cost and low efficiency will be caused. Therefore, after the total DNA of the tissue cells is extracted, the mitochondrial DNA is usually prepared by using different methods for secondary enrichment, and then the enriched mitochondrial DNA is used for library preparation and high-throughput sequencing analysis.

[0005] At present, the commonly used mitochondrial genome enrichment methods include various forms of PCR amplification method and probe capture method, etc.

[0006] Firstly, the species-specific PCR amplification method is the most commonly used method for mitochondrial DNA enrichment preparation. According to the number of primer pairs or amplified fragments, it can be divided into 1 whole genome fragment, 2 or more PCR fragment methods. Unlike the traditional single gene PCR amplification and Sanger sequencing analysis, mitochondrial whole genome sequencing needs to amplify the whole mitochondrial genome by PCR, so as to carry out effective whole genome sequencing analysis.

[0007] Secondly, probe capture method is another method for enriching mitochondrial genome DNA. The principle is to capture small fragments of mitochondrial DNA from total DNA by a large number of species-specific mitochondrial DNA probes, and then used for library preparation and high-throughput sequencing analysis.

[0008] Thirdly, other commercial non-PCR methods for enriching mitochondrial DNA include total DNA extraction kit, alkaline preparation kit, mitochondrial extraction kit and selective lysis kit. Generally speaking, the content of mitochondrial DNA in different tissues is different, but the content is relatively low. For example, in total DNA extraction method, the content of mitochondrial DNA in human tumor tissue is very low, about 0.1% (i.e. about 0.1% mtDNA ratio). The highest mitochondrial DNA enrichment efficiency of the three commonly used enrichment kits is only 10 times higher, that is, the mitochondrial DNA can only be increased to 1% of the total DNA after enrichment (i.e. from 0.1% mtDNA ratio to 1% mtDNA ratio), which is still a very low proportion. Even if the highest efficiency product after enrichment is treated with a series of complex exonuclease, the mitochondrial DNA enrichment ratio can only be increased to 27%, which is still a low proportion, and still brings great difficulties to the subsequent mitochondrial DNA sequencing analysis. Moreover, these enrichment methods only use high-quality isolated cells and cultured cells, which have a narrow range of application and have been proved to be unable to achieve practical results (Scientific Reports, 2018, 8:2261).

[0009] Therefore, the above several most commonly used mitochondrial DNA enrichment methods in the prior art still have many limitations when used for mitochondrial whole genome sequencing analysis:

[0010] Firstly, the biggest common feature of the above several enrichment methods is that the sequence information of the mitochondrial genome of the species to be tested must be obtained in advance to design species-specific PCR primers or capture probes. Therefore, these methods can only be limited to a few common animals, such as humans, mice, etc. In the five main groups of vertebrates (mammals, birds, reptiles, amphibians, fish) in the natural environment, each group has thousands of different species, most of which do not have mitochondrial genome sequence information, so they cannot be used for sequencing analysis by PCR amplification method / probe capture method. At present, the existing mitochondrial whole genome sequencing method of vertebrates is mostly based on PCR or capture method, which covers a small number of species and is difficult to apply to other animal species.

[0011] Secondly, even for a few species with mitochondrial genome sequences, it is a technically demanding, cumbersome and costly method to design effective PCR primers and amplify the expected PCR fragments, which has a small application range and poor feasibility in environmental animal mitochondrial genome analysis.

[0012] Thirdly, the mitochondrial DNA enrichment based on PCR amplification is easily affected by amplification errors and biases introduced by in vitro amplification of PCR and mitochondrial pseudogenes (NUMTs) in nuclear DNA, and sequencing data is prone to bias and other problems.

[0013] Therefore, it is necessary to provide a rapid and efficient universal technology for mitochondrial whole genome sequencing analysis of all vertebrate taxa. SUMMARY

[0014] OBJECTIVE

[0015] The purpose of the present application is to provide a one-step vertebrate mitochondrial genome DNA extraction kit, a one-step vertebrate mitochondrial genome DNA extraction method, a high-throughput sequencing method, and their applications in preparing mitochondrial genome base and structural mutation analysis products, species identification products, and biodiversity analysis products. The kit and method of the present application do not rely on mitochondrial genome sequence information of species, can directly extract high-purity mitochondrial DNA from animal tissue samples, and can be used for DNA library preparation and high-throughput sequencing, providing a rapid and efficient universal technology for mitochondrial whole genome sequencing analysis of all vertebrate taxa.

[0016] SOLUTION

[0017] To achieve the above-mentioned purpose, the present application provides the following technical solutions:

[0018] In a first aspect, the present application provides a one-step vertebrate mitochondrial genome DNA extraction kit, comprising the following components:

[0019] Reagent A: including cell lysis and balancing reagent;

[0020] Reagent B: including 1.7-2.5M sodium acetate;

[0021] Reagent C: including phenol, chloroform and isoamyl alcohol in a volume ratio of (120-130):(19-29):1;

[0022] Among them, reagents B and C are used to mix with total DNA samples lysed by cells and obtain water phase containing mitochondrial DNA through layering.

[0023] Further, in the reagent A, the cell lysis and the balancing reagent comprises 3.25-4.75 M guanidine isothiocyanate, 0.65-0.85 M sodium citrate and 5-15 v / v% N-lauroylsarcosine sodium salt; optionally comprises 3.85-4.25 M guanidine isothiocyanate, 0.7-0.8 M sodium citrate and 5-10 v / v% N-lauroylsarcosine sodium salt; optionally comprises 3.85-4.1 M guanidine isothiocyanate, 0.7-0.74 M sodium citrate and 5-10 v / v% N-lauroylsarcosine sodium salt.

[0024] Further, in the reagent B, the concentration of sodium acetate is 1.8-2 M.

[0025] Further, in the reagent C, the volume ratio of phenol, chloroform and isoamyl alcohol is (120-127):(22-26):1.

[0026] In the second aspect, a one-step method for extracting vertebrate mitochondrial genome DNA is provided, which uses the kit of the first aspect and comprises the following steps:

[0027] 1) obtaining fresh or frozen sample;

[0028] 2) adding the sample into the reagent A, mixing thoroughly to lyse the cells and release free nucleic acid, obtaining the lysis solution;

[0029] 3) adding the reagent B and the reagent C into the lysis solution, mixing thoroughly, and waiting for the water phase and the organic phase to separate;

[0030] 4) taking the water phase containing mitochondrial DNA, and recovering the mitochondrial DNA by alcohol precipitation.

[0031] Further, in the step 1), the large sample is cut or crushed to obtain small sample; optionally, the large sample is cut or crushed to obtain small sample.

[0032] Further, in the step 1), the sample amount is 5-20 mg; optionally, the tissue sample is obtained by using a sampling needle.

[0033] Further, in the steps 2) and 3), the mixing method comprises oscillation mixing.

[0034] Further, in the step 3), the water phase and the organic phase are accelerated to separate by low-temperature high-speed centrifugation. Generally, the low-temperature high-speed centrifugation refers to centrifugation at 4-6℃ and 10000-14000 rpm / min.

[0035] Further, in the step 4), the alcohol precipitation uses anhydrous ethanol or isopropanol.

[0036] In a third aspect, there is provided a high-throughput sequencing method, which uses the mitochondrial DNA sample extracted by the kit of the first aspect or the mitochondrial DNA sample extracted by the extraction method of the second aspect to perform high-throughput sequencing.

[0037] Further, the method comprises the following steps: constructing a gene library by using the extracted mitochondrial DNA, performing PCR amplification (the primers can be sequencing universal primers such as P5 / p7) and magnetic bead purification on the mitochondrial DNA library, performing multiplex sequencing by mixing a plurality of mitochondrial DNA libraries, and performing analysis.

[0038] In a fourth aspect, there is provided an application of the kit of the first aspect or the extraction method of the second aspect or the high-throughput sequencing method of the third aspect in preparing a mitochondrial genome base and structural mutation analysis product, or a method for performing mitochondrial genome base and structural mutation analysis by using the kit of the first aspect or the extraction method of the second aspect or the high-throughput sequencing method of the third aspect.

[0039] In a fifth aspect, there is provided an application of the kit of the first aspect or the extraction method of the second aspect or the high-throughput sequencing method of the third aspect in preparing a species identification product, or a method for performing species identification by using the kit of the first aspect or the extraction method of the second aspect or the high-throughput sequencing method of the third aspect.

[0040] In a sixth aspect, there is provided an application of the kit of the first aspect or the extraction method of the second aspect or the high-throughput sequencing method of the third aspect in preparing a biodiversity analysis product, or a method for performing biodiversity analysis by using the kit of the first aspect or the extraction method of the second aspect or the high-throughput sequencing method of the third aspect. Advantages

[0041] (1) The kit and method of the present application can directly extract high-purity mitochondrial DNA from any animal tissue sample, and the product does not need any secondary enrichment and can be directly used for mitochondrial genome sequencing independent of the species type, which can effectively improve the sequencing ratio of mitochondrial DNA in total DNA, effectively improving the mitochondrial DNA ratio extracted from the existing method from 0.05-1% to 50-99.9%, indicating that the enrichment efficiency of the kit of the present application is thousands of times higher than that of the prior art, and far exceeds the commonly used enrichment extraction kits on the market.

[0042] (2) Due to the great improvement in the sequencing efficiency of mitochondrial DNA, a sequencing data amount equivalent to a total DNA sample can be used to simultaneously determine dozens or more mitochondrial DNA samples, greatly reducing the sequencing cost of the mitochondrial genome of a single sample.

[0043] (3) The kit of the present application also provides a feasible solution independent of species for the identification of numerous different animal species in five major groups of vertebrates, biodiversity surveys, and the discovery of new species, filling a gap in this field.

[0044] (4) The mitochondrial genome extraction kit / method of the present application can effectively obtain high-purity mitochondrial DNA from solid sample tissues in a one-step method, without the need for designing PCR primer enrichment, without dependence on the species, and can effectively improve the efficiency of high-throughput sequencing and reduce the difficulty of data analysis of sequencing.

[0045] (5) The present application overcomes various limitations of existing mitochondrial genome DNA enrichment and sequencing methods. Since cell lysis solution (such as guanidine isothiocyanate reagent) can only release total DNA, i.e., a mixture of nuclear DNA and a small amount of mitochondrial DNA is obtained, the enrichment and extraction of mitochondrial DNA is difficult; the present application overcomes this situation, effectively separates high-purity circular mitochondrial genome DNA from the total DNA mixture of the sample, and can be directly used for sequencing analysis.

[0046] (6) The present application does not require prior mitochondrial genome information of the species to be tested for sequencing analysis, and is a species-independent method, which not only simplifies the analysis of common vertebrates, but also provides an effective mitochondrial DNA preparation and sequencing method for a large number of vertebrates lacking mitochondrial genome information.

[0047] (7) The present application is a one-step method for directly extracting high-purity mitochondrial DNA from solid tissues, without the need to first prepare total DNA of animal tissues, and then to secondarily enrich mitochondrial DNA through PCR amplification or probe capture. Therefore, the present application is not only simple and fast, but also avoids the cumbersome technical operations and high experimental costs of PCR amplification or probe capture.

[0048] (8) The mitochondrial genome sequencing data generated by the mitochondrial DNA extracted by the present application is a high-fidelity data, avoiding the errors, biases of PCR and the influence of pseudogenes in nuclear DNA brought by PCR amplification enrichment. BRIEF DESCRIPTION OF DRAWINGS

[0049] One or more embodiments are illustrated by way of example in the figures that form a part of this patent document. This illustration, together with the specification, is not to be used to construe the embodiments in a limiting sense. The word “exemplary” is used herein to mean “serving as an example, instance, or illustration.” Any implementation described herein as “exemplary” is not necessarily to be construed as preferred or advantageous over other implementations.

[0050] Figure 1 is the sequencing coverage of the bluefin sunfish mitochondrial genome (reference genome NC-015984.2) of Example 1 of the present application.

[0051] Figure 2 is the assembly map of the bluefin sunfish mitochondrial genome assembled de novo from the sequencing results of Example 1 of the present application.

[0052] Figure 3 is the sequencing coverage of the domestic pigeon mitochondrial genome (reference genome OR120383.1) of Example 2 of the present application.

[0053] Figure 4 is the assembly map of the domestic pigeon mitochondrial genome assembled de novo from the sequencing results of Example 2 of the present application.

[0054] Figure 5 is the sequencing coverage of the human peripheral blood mitochondrial genome extracted by the method of Comparative Example 1 of the present application (reference genome NC-012920.1). DETAILED DESCRIPTION

[0055] In order to make the objects, technical solutions, and advantages of the embodiments of the present application clearer, the technical solutions of the embodiments of the present application will be described clearly and completely below. Obviously, the described embodiments are only some of the embodiments of the present application, rather than all the embodiments of the present application. Based on the embodiments of the present application, all other embodiments obtained by those of ordinary skill in the art without creative work fall within the scope of protection of the present application. Unless otherwise explicitly indicated, in the entire specification and claims, the term “comprise” or its variants such as “contain” or “include” and the like are understood to include the stated elements or components, without excluding other elements or components.

[0056] In addition, in order to better illustrate the present application, numerous specific details are given in the specific embodiments below. Those skilled in the art should understand that the present application can also be implemented without some specific details. In some embodiments, the raw materials, elements, methods, means, and the like that are well known to those skilled in the art are not described in detail, in order to highlight the main ideas of the present application.

[0057] The present application is described in detail below.

[0058] In order to overcome the limitations of the prior art vertebrate mitochondrial genome sequencing, which requires prior knowledge of the sequence information of the mitochondrial genome to be measured to achieve mitochondrial genome sequencing by PCR amplification, the present application provides a new method and kit, which can effectively separate a small amount of mitochondrial DNA from nuclear DNA from animal solid tissue samples, rapidly extract high-purity mitochondrial genome DNA in one step, and can be effectively used for library preparation and genome sequencing, reducing the cost of subsequent sequencing analysis. The main steps of the method are as follows:

[0059] 1. Sample collection and preservation of vertebrate solid tissues

[0060] (1) Sample collection and preservation by animal dissection method: Different types of animals are killed according to ethical methods, and a small amount of fresh animal solid tissues is collected by routine dissection and preserved by freezing until use or immediately used for extraction and preparation, wherein the animal solid tissues include muscle and various internal organs.

[0061] (2) Rapid sampling and preservation by animal needle biopsy method: A main and auxiliary double-peak sampling needle or other sampling needle is used to quickly needle 5-20 mg of subcutaneous muscle tissue of the animal in a dead or live state, and the tissue is transferred to a sterile 2 mL microtube for preservation until use or immediately used for extraction and preparation.

[0062] 2. One-step rapid extraction and preparation of mitochondrial genome DNA from animal solid tissues

[0063] (1) A small amount of fresh or frozen fresh animal tissue (5-20 mg) is cut with scissors and then mixed with 600 μL of reagent A in a 2 mL microtube to promote the complete lysis of tissue cells, forming a uniform tissue lysis solution containing various free nucleic acids.

[0064] Among them, reagent A is a tissue cell lysis and balancing reagent, which includes 3.25-4.75 M guanidine isothiocyanate, 0.65-0.85 M sodium citrate, and 5-15 v / v% N-lauroylsarcosine sodium salt.

[0065] (2) Add reagent B (the amount of reagent B added is 7-13% of the tissue lysis solution) and reagent C (the volume ratio of reagent C to (tissue lysis solution + reagent B) is 1:1) to the tissue lysis solution in step (1) above, then mix well to promote the separation of the aqueous phase and the organic phase, and the mitochondrial DNA can be effectively separated from the nuclear DNA by liquid phase separation.

[0066] Among them, reagent B is a buffer reagent for promoting nucleic acid separation, which includes 1.7-2.5 M sodium acetate; reagent C is an organic reagent for promoting liquid phase separation, which includes phenol, chloroform and isoamyl alcohol, and the volume ratio of phenol, chloroform and isoamyl alcohol is (120-130):(19-29):1.

[0067] (3) The microtube in step (2) above is centrifuged at low temperature and high speed to effectively separate the aqueous phase and the organic phase, and the aqueous phase containing mitochondrial DNA is transferred to a new sterile microtube, and then the mitochondrial DNA is precipitated and recovered by isopropanol or anhydrous ethanol.

[0068] (4) Quantification and quality control of mitochondrial DNA. NanoDrop instrument and Qubit instrument are used together to determine the concentration of nucleic acid and DNA.

[0069] 3. Library preparation and high-throughput sequencing of mitochondrial genomic DNA:

[0070] (1) Single-tube DNA library preparation: The 1 / 4 to 1 / 2 of mitochondrial DNA extract obtained from step 2 of the one-step method can be used to prepare mitochondrial DNA library using a commonly used DNA library preparation kit, such as QIAseq FX DNA Library Kit (Qiagen), to complete mitochondrial DNA fragmentation and enzymatic repair, and end-joining with illumine sequencing adapters (provided in the kit) in a single microtube. The mitochondrial DNA library product is purified and recovered by AMpure XP (Agencourt) magnetic beads.

[0071] (2) PCR amplification of mitochondrial DNA library (using sequencing universal primers p5 / p7 amplification) and magnetic bead purification: The original mitochondrial DNA library is amplified by 8-12 cycles of PCR and purified by magnetic beads to obtain high-quality library products.

[0072] (3) Multiplexing of mitochondrial DNA libraries and high-throughput sequencing: Each mitochondrial DNA library has a unique identification code, and multiple DNA libraries can be mixed in equal amounts, then multiplexed and sequenced using any model of illumine high-throughput sequencer for analysis.

[0073] Some specific embodiments are as follows:

[0074] Example 1 One-step extraction and sequencing of mitochondrial genomic DNA of Lepomis macrochirus

[0075] 1. Subcutaneous muscle tissue needle biopsy of Lepomis macrochirus

[0076] (1) Knock the head of the fish to kill it.

[0077] (2) Use a sterile animal sampling needle to insert into the subcutaneous muscle tissue of the fish back and obtain a 5-20 mg needle biopsy muscle tissue sample, transfer it to a sterile 2 ml microtube, and store it in the cold until use or immediately use for extraction experiments.

[0078] 2. Extraction and preparation of mitochondrial genomic DNA from fish needle biopsy muscle tissue

[0079] (1) Transfer the needle biopsy muscle tissue sample to a 2 ml microtube containing 600 uL of reagent A (containing 3.85 M guanidine isothiocyanate, 0.7 M sodium citrate and 5% N-lauroylsarcosine sodium salt), vortex for 1-2 minutes to completely lyse the muscle tissue.

[0080] (2) Add 10% reagent B (2M sodium acetate) and 600uL reagent C (phenol-chloroform-isoamyl alcohol, volume ratio 123:22:1) to the tissue lysate, vortex for 1 minute, and then centrifuge at low temperature (4°C) and high speed after a short rest to separate the water phase containing mitochondrial DNA and the organic phase containing nuclear DNA.

[0081] (3) Carefully transfer the upper water phase containing mitochondrial DNA to another sterile 1.5ml microtube, add an equal volume of isopropanol, and rest for a short time.

[0082] (4) Centrifuge at low temperature (4°C) and high speed, collect the DNA precipitate at the bottom of the tube, and recover it in 20ul TE solution after 75% ethanol washing.

[0083] (5) Measure the concentration of extracted nucleic acids using NanoDrop and Qubit kits.

[0084] 3. DNA library preparation and high-throughput sequencing

[0085] (1) Single-tube DNA library preparation: Use the QIAseq FX DNA Library Kit (Qiagen) library preparation kit to complete mitochondrial DNA fragmentation, enzymatic repair, and end ligation with illumine sequencing adapters in a single microtube with 10ul mitochondrial DNA extract. Mitochondrial DNA library products are purified and recovered by AMpure XP (Agencourt) magnetic beads.

[0086] (2) PCR amplification of mitochondrial DNA library (using sequencing universal primers p5 / p7) and magnetic bead purification: The original mitochondrial DNA library is amplified by 8-12 cycles of PCR and purified by magnetic beads to obtain high-quality library products.

[0087] (3) Multiplexing of mitochondrial DNA library and high-throughput sequencing: Each mitochondrial DNA library has a unique identification code, and multiple DNA libraries can be mixed in equal amounts, then multiplexed and analyzed using any model of illumine high-throughput sequencer.

[0088] Some sequencing results are shown in Table 1, Table 2, Figure 1, and Figure 2:

[0089] Table 1, sequencing parameters of the mitochondrial genome of the bluefin sunfish muscle needle sample analyzed in this example

[0090] The results in Table 1 show that the extracted mtDNA percentage in this embodiment accounts for 99.56% of the total DNA, indicating that the kit of this application can extract mitochondrial DNA with high purity. The sequencing coverage reaches 100%, and the average sequencing depth reaches 29707×, indicating that the sequencing data can cover all sites in the genome and has high sequencing coverage. This application does not require designing PCR primers for enrichment from total DNA, reducing the difficulty of sequencing data analysis. The mitochondrial genome sequencing data generated from the extracted mitochondrial DNA in this application is high-fidelity data, avoiding PCR errors and biases introduced by PCR amplification and enrichment, as well as the influence of pseudogenes in nuclear DNA, greatly reducing the sequencing error rate.

[0091] The results of the variant sites and frequencies of the bluegill sunfish sequencing data compared with the reference genome NC-015984.2 are shown in Figure 1 and Table 2.

[0092] The results in Figure 1 and Table 2 show that this embodiment can generate high-quality sequencing data. This application can generate high-quality sequencing data and effectively detect various variant sites and mutation rates. It can not only detect low-frequency variant sites, but also effectively detect them when the mutation abundance is 0.4%. Moreover, it can efficiently detect SNP variant sites, which greatly improves the analysis efficiency.

[0093] The mitochondria extracted in this embodiment have high purity. When performing sequencing analysis using them, the complete mitochondrial genome sequence of the bluegill sunfish can be assembled de novo without any sequence information of the sample to be tested. The assembly diagram is shown in Figure 2.

[0094] Table 2. Variation sites and frequencies of the bluegill sunfish mitochondrial genome compared with the reference genome (NC-015984.2) in this embodiment.

[0095] Example 2: Subcutaneous muscle tissue sampling of domestic pigeons (Columba livia) using needle biopsy

[0096] 1. Subcutaneous muscle tissue sampling of domestic pigeons (Columba livia) using needle biopsy

[0097] (1) The pigeon was euthanized by bleeding it out of its neck.

[0098] (2) Use a sterile animal sampling needle to insert into the subcutaneous muscle tissue on the back of the pigeon and obtain 5-20 mg of needle-tested muscle tissue sample. Transfer the sample to a sterile 2 ml microtube and store it under cold storage until use or immediately for extraction experiments.

[0099] 2. Extraction and preparation of mitochondrial genomic DNA from muscle tissue of domestic pigeons (using needle extraction)

[0100] (1) The muscle tissue sample was transferred to a 2ml microtube containing 600uL Reagent A (containing 4.1M guanidinium isothiocyanate, 0.74M sodium citrate and 10% N-lauroylsarcosine sodium salt) and vortexed for 1 minute to fully lyse the muscle tissue.

[0101] (2) 10% Reagent B (2M sodium acetate) and 600uL Reagent D (phenol-chloroform-isoamyl alcohol with a volume ratio of 127:26:1) were added to the tissue lysate and vortexed for 1 minute to mix, and after a brief standing, low-temperature high-speed centrifugation was performed to separate the water phase and the organic phase, wherein the water phase contained mitochondrial DNA and the organic phase contained nuclear DNA, so that the two could be effectively separated.

[0102] (3) The upper water phase containing mitochondrial DNA was carefully transferred to another sterile 1.5ml microtube, and an equal volume of isopropanol was added, and the mixture was briefly stood.

[0103] (4) Low-temperature (4°C) high-speed centrifugation was performed, and the DNA precipitate at the bottom of the tube was collected and washed with 75% ethanol and recovered into 20ul TE solution.

[0104] (5) The concentration of the extracted nucleic acid was determined by NanoDrop and Qubit kit.

[0105] 3. DNA library preparation and high-throughput sequencing

[0106] (1) Single-tube DNA library preparation: QIAseq FX DNA Library Kit (Qiagen) library preparation kit was used, and 10ul mitochondrial DNA extract was used to complete mitochondrial DNA fragmentation, enzymatic repair, and end connection with illumine sequencing adapters in a single microtube. The mitochondrial DNA library product was purified and recovered by AMpure XP (Agencourt) magnetic beads.

[0107] (2) PCR amplification of mitochondrial DNA library (amplified using sequencing universal primers p5 / p7) and magnetic bead purification: the original mitochondrial DNA library was amplified by 8-12 cycles of PCR and magnetic bead purification to obtain a high-quality library product.

[0108] (3) Multiplexing of mitochondrial DNA library and high-throughput sequencing: each mitochondrial DNA library has a unique identification code, and multiple DNA libraries can be mixed in equal amounts, and then subjected to multiplex sequencing and analysis using any model of illumine high-throughput sequencer.

[0109] Part of the sequencing results are shown in Table 3, Table 4, Figure 3, and Figure 4:

[0110] Table 3, sequencing parameters of the mitochondrial genome of the muscle needle detection sample of the domestic pigeon analyzed in this example

[0111] The results of Table 3 show that the ratio of mtDNA% of total DNA extracted in this example is 99.89%, indicating that the kit of the present application can extract mitochondrial DNA with very high purity. The sequencing coverage reaches 100%, and the average sequencing depth reaches 9156x, indicating that the sequencing data can cover all sites of the genome, with very high sequencing coverage. The present application does not need to design PCR primers from total DNA for enrichment, reducing the difficulty of data analysis of sequencing. The mitochondrial DNA extracted by the present application produces mitochondrial genome sequencing data which is a high-fidelity data, avoiding the errors, biases of PCR and the influence of pseudo genes in nuclear DNA brought by PCR amplification enrichment. Greatly reducing the sequencing error rate.

[0112] The results of the comparison of the variation sites and frequencies of the mitochondrial genome of the domestic pigeon with the reference genome (OR120383.1) are shown in Figure 3 and Table 4.

[0113] The results of Figure 3 and Table 4 show that this example can produce high-quality sequencing data and effectively detect various variation sites and variation rates. Not only can it detect low-frequency variation sites, but it can also effectively detect mutation abundance of 1%, and it can efficiently detect SNP variation sites of the domestic pigeon, greatly improving the analysis efficiency.

[0114] Table 4, this example analyzes the variation sites and frequencies of the mitochondrial genome of the domestic pigeon compared with the reference genome (OR120383.1)

[0115] The mitochondrial purity extracted in this example is high. When sequencing analysis is performed, the complete mitochondrial genome sequence of the domestic pigeon can be de novo assembled without any sequence information of the sample to be tested, and the assembly map is shown in Figure 4.

[0116] Comparative Example 1

[0117] 1. Extract DNA from human peripheral blood according to the method of Example 1 disclosed in the reference patent application CN101250522A

[0118] NanoDrop and Qubit were used to determine the concentration of the nucleic acid extracted in this comparative example.

[0119] The DNA library prepared in reference to step 3 of example 1 was used to detect the extraction efficiency of mitochondrial DNA by high-throughput sequencing method, and the results are shown in Table 5 and Figure 5. The results show that the ratio of mtDNA% in total DNA extracted by the comparative example is 0.05%, and no high-purity mitochondrial DNA is obtained. The inventors further analyzed that the claimed mitochondrial DNA obtained is not actually mitochondrial DNA, but may be genomic DNA. Specifically, in the effect part (page 3 / 7 of the specification), it is claimed that the length of mitochondrial DNA is 30 kb to 50 kb, but those skilled in the art know that the length of human mitochondrial DNA is generally not more than 20 kb, so the inventors cannot effectively obtain mitochondrial DNA after referring to this method.

[0120] Comparative Example 2

[0121] Mitochondrial DNA was extracted from frozen tumor tissue using Qproteome mitochondria isolation kit (purchased from Qiagen) kit. The ratio of mtDNA% in total DNA extracted by Qproteome mitochondria isolation kit (purchased from Qiagen) kit is 0.2% (this data is from the literature Weerts MJA, et al. Sensitive detection of mitochondrial DNA variants for analysis of mitochondrial DNA-enriched extracts from frozen tumor tissue. Sci Rep. 2018 Feb 2;8(1):2261.). Although Qproteome mitochondria isolation kit (purchased from Qiagen) kit has obvious enrichment effect, it still cannot meet the requirements.

[0122] Comparative Example 3

[0123] The mitochondrial DNA was extracted from the frozen tumor tissue by using QIAamp Circulating Nucleic Acid kit (purchased from Qiagen), and the ratio of mtDNA% in total DNA extracted by QIAamp Circulating Nucleic Acid kit (purchased from Qiagen) was 1% (this data is from the literature Weerts MJA, et al. Sensitive detection of mitochondrial DNA variants for analysis of mitochondrial DNA-enriched extracts from frozen tumor tissue. Sci Rep. 2018 Feb 2;8(1):2261.), although QIAamp Circulating Nucleic Acid kit (purchased from Qiagen) has obvious enrichment effect, but still not meet the requirements.

[0124] The ratio of mtDNA% in total DNA and sequencing coverage of Comparative Examples 1, 2 and 3 are shown in Table 5.

[0125] Table 5, the ratio of mitochondrial genome extracted from the sample of Comparative Examples 1, 2 and 3 and the sequencing coverage

[0126] The above results show that the existing mitochondrial DNA ratio is only about 0.05-1%, and the enrichment efficiency is low, resulting in high cost and difficulty of subsequent sequencing analysis.

[0127] And the ratio of mitochondrial DNA extracted from human peripheral blood and tumor tissue by the mitochondrial genome extraction kit of the present application accounts for more than 75% of the total DNA sequencing, that is, the one-step rapid extraction method of mitochondrial genome of the present application can effectively improve the sequencing ratio of mitochondrial DNA in total DNA, which is effectively improved from the existing mitochondrial DNA ratio of 0.05-1% to 50-99.9% (for example, the mitochondrial DNA ratio of mammals can be more than 75%, even more than 90%), which shows that the enrichment efficiency of the kit of the present application is thousands of times higher than that of the prior art, and far exceeds the commonly used enrichment extraction kit on the market.

[0128] Due to the great improvement of the sequencing efficiency of mitochondrial DNA, a sequencing data amount equivalent to a total DNA sample can be used to determine dozens or more mitochondrial DNA samples at the same time, greatly reducing the sequencing cost of mitochondrial genome of a single sample.

[0129] Moreover, the kit of the present application also provides a feasible solution independent of species for the identification of numerous different animal species in five major groups of vertebrates, biodiversity surveys, and the discovery of new species, filling a gap in this field.

[0130] The mitochondrial genome extraction kit / method of the present application can effectively obtain high-purity mitochondrial DNA from solid sample tissues in one step, without the need for designing PCR primer enrichment, without dependence on species, and can effectively improve the efficiency of high-throughput sequencing and reduce the difficulty of data analysis.

[0131] The present application overcomes various limitations of existing mitochondrial genome DNA enrichment and sequencing methods. Since cell lysates (such as guanidinium isothiocyanate and other reagents) can only release total DNA, i.e., a mixture of nuclear DNA and a small amount of mitochondrial DNA is obtained, the enrichment and extraction of mitochondrial DNA is difficult. The present application overcomes this situation, effectively separates high-purity circular mitochondrial genome DNA from the total DNA mixture of the sample, and can be directly used for sequencing analysis.

[0132] The present application does not require prior mitochondrial genome information of the species to be tested for sequencing analysis, and is a species-independent method. It not only simplifies the analysis of common vertebrates, but also provides an effective mitochondrial DNA preparation and sequencing method for a large number of vertebrates lacking mitochondrial genome information.

[0133] The present application is a one-step method for directly extracting high-purity mitochondrial DNA from solid tissues, without the need to first prepare total DNA of animal tissues, and then secondarily enrich mitochondrial DNA through PCR amplification or probe capture. Therefore, the present application is not only simple and fast, but also avoids the cumbersome technical operations and high experimental costs of PCR amplification or probe capture.

[0134] The mitochondrial genome sequencing data generated by the mitochondrial DNA extracted by the present application is a high-fidelity data, avoiding the errors, biases of PCR, and the influence of pseudogenes in nuclear DNA brought by PCR amplification enrichment.

[0135] Finally, it should be noted that the above examples are only used to illustrate the technical solutions of the present application, and not to limit them; although the present application has been described in detail with reference to the foregoing examples, those of ordinary skill in the art should understand that they can still modify the technical solutions recorded in the foregoing examples, or make equivalent substitutions for some technical features; and these modifications or substitutions do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present application. Industrial applicability

[0136] The application provides a vertebrate mitochondrial genome DNA one-step extraction kit, an extraction method, a high-throughput sequencing method and application thereof, wherein the kit comprises: reagent A, which comprises a cell lysis and balance reagent; reagent B, which comprises 1.7-2.5M sodium acetate; and reagent C, which comprises phenol, chloroform and isoamyl alcohol in a volume ratio of (120-130):(19-29):1; wherein the reagents B and C are used for mixing with a total DNA sample of cell lysis and obtaining a water phase containing mitochondrial DNA through layering. The kit and the method thereof do not depend on the sequence information of the mitochondrial genome of a species, can directly extract high-purity mitochondrial DNA from an animal tissue sample, can be used for DNA library preparation and high-throughput sequencing, and provide a fast and efficient universal technology for mitochondrial whole genome sequencing analysis of all groups of vertebrates.

Claims

1. A vertebrate mitochondrial genomic DNA one-step extraction kit, characterized in that, Comprising the following components: Reagent A: comprising cell lysis and equilibration reagent; Reagent B: comprising 1.7-2.5M sodium acetate; Reagent C: comprising phenol, chloroform and isoamyl alcohol in a volume ratio of (120-130):(19-29):1; wherein, reagent B and C are used to mix with the total DNA sample lysed from cells and obtain the aqueous phase containing mitochondrial DNA by layering.

2. The kit of claim 1, wherein In reagent A, the cell lysis and equilibration reagent comprises 3.25-4.75M guanidine isothiocyanate, 0.65-0.85M sodium citrate and 5-15v / v% N-lauroylsarcosine sodium salt; optionally comprising 3.85-4.25M guanidine isothiocyanate, 0.7-0.8M sodium citrate and 5-10v / v% N-lauroylsarcosine sodium salt; optionally comprising 3.85-4.1M guanidine isothiocyanate, 0.7-0.74M sodium citrate and 5-10v / v% N-lauroylsarcosine sodium salt; and / or, in reagent B, the concentration of sodium acetate is 1.8-2M; and / or, in reagent C, the volume ratio of phenol, chloroform and isoamyl alcohol is (120-127):(22-26):

1.

3. A method for one-step extraction of vertebrate mitochondrial genomic DNA, characterized in that, The kit of claim 1 or 2 comprises the following steps: 1) obtaining fresh or frozen sample; 2) adding the sample to reagent A, mixing thoroughly to lyse the cells and release free nucleic acids, obtaining a lysate; 3) adding reagent B and reagent C to the lysate, mixing thoroughly, and waiting for the aqueous phase and organic phase to layer; 4) taking the aqueous phase containing mitochondrial DNA, and recovering the mitochondrial DNA by alcohol precipitation.

4. The extraction method according to claim 3, characterized in that, In step 1), the large sample is cut or crushed to obtain a small sample; Alternatively, in step 1), the sample amount is 5-20mg; optionally, the tissue sample is obtained using a sampling needle.

5. The extraction method of claim 3 or 4, wherein, In steps 2) and 3), the thorough mixing includes shaking; and / or, in step 3), the aqueous phase and organic phase are accelerated to layer by low-temperature high-speed centrifugation; and / or, in step 4), the alcohol precipitation uses anhydrous ethanol or isopropanol.

6. A high-throughput sequencing method, characterized by, The mitochondrial DNA sample extracted using the kit of claim 1 or 2 or the mitochondrial DNA sample extracted using the extraction method of any one of claims 3 to 5 is subjected to high-throughput sequencing.

7. The high-throughput sequencing method of claim 6, wherein, The method comprises the following steps: constructing a gene library using the extracted mitochondrial DNA, PCR amplifying the mitochondrial DNA library and magnetic bead purification, and performing multiplex sequencing and analysis by mixing multiple mitochondrial DNA libraries.

8. Use of the kit of claim 1 or 2 or the extraction method of any one of claims 3 to 5 or the high-throughput sequencing method of claim 6 or 7 in the preparation of a product for analyzing base and structural mutations of mitochondrial genomes.

9. Use of the kit of claim 1 or 2 or the extraction method of any one of claims 3 to 5 or the high-throughput sequencing method of claim 6 or 7 in the preparation of a product for species identification.

10. Use of a kit according to claim 1 or 2 or an extraction method according to any of claims 3 to 5 or a high-throughput sequencing method according to claim 6 or 7 for the preparation of a product for the analysis of biodiversity.