Primer group for human mitochondrial whole genome amplification, kit, library construction method and application

Abstract

The invention belongs to the technical field of gene detection, and discloses a primer group for human mitochondrial whole genome amplification, a kit, a library construction method and application, the primer group comprises 128 pairs of primers, the nucleotide sequences of forward primers in the 128 pairs of primers are sequentially shown as SEQ ID NO.1-SEQ ID NO.128, and the nucleotide sequences of reverse primers in the 128 pairs of primers are sequentially shown as SEQ ID NO.129-SEQ ID NO.256. The kit comprises the primer group, an amplification buffer solution, two enhancers, a universal primer and a specific primer. The library construction method comprises the following steps: acquiring DNA of a sample to be detected, performing two rounds of PCR amplification to obtain a mitochondrial genome library, and performing sequencing to obtain sequencing data. According to the method, more than 99% of the full length of human mitochondria can be covered, higher sequencing depth can be obtained under the condition of extremely small data volume, and the sequencing cost is greatly reduced.

Description

Technical Field

[0001] The present invention belongs to the technical field of gene detection, and in particular relates to a primer set, a kit, a library construction method and an application for amplifying the whole genome of human mitochondria. Background Art

[0002] Mitochondria are the energy-producing organelles of human cells, playing a crucial role in providing energy. Mitochondrial DNA (mtDNA) is passed down from mother to offspring through maternal inheritance. mtDNA in the cytoplasm has distinct characteristics distinct from nuclear DNA. Lacking the necessary proteins to protect it, it is exposed to the mitochondrial matrix, making it more susceptible to mutations. The frequency of mutations in cytoplasmic mtDNA is 10-25 times higher than that in nuclear DNA. Furthermore, mtDNA lacks the ability to repair damage, which can lead to the accumulation of mutations. Over time, abnormal mitochondrial numbers and the accumulation of mutations in tissue cells can lead to cellular dysfunction, disrupted energy metabolism, and even apoptosis. In more severe cases, mtDNA can affect tissue and organ function and cause death.

[0003] Due to the maternal inheritance of mitochondrial genes, women who carry mitochondrial pathogenic mutations are at risk of passing the pathogenic mutations to their offspring. Mitochondrial DNA also exhibits heteroplasmy. When the mutated mtDNA reaches the disease threshold, patients can develop severe illnesses. Currently, there are no effective treatments or therapies for diseases caused by mitochondrial gene mutations. Families carrying pathogenic mutations who wish to have healthy offspring can primarily rely on prenatal diagnosis and preimplantation mitochondrial genetic testing to avoid having offspring carrying the pathogenic gene.

[0004] Preimplantation embryo testing (PET) typically begins with whole-genome amplification (WGA) of extra-embryonic blastocyst biopsies (non-embryonic tissue). The amplified products are then used for mitochondrial gene analysis and chromosomal aneuploidy testing. Currently, commonly used WGA methods fall into three categories: polymerase chain reaction (PCR)-based WGA, isothermal WGA, and hybrid WGA. These three techniques differ in the amplification principles they employ, resulting in varying lengths of amplified product fragments.

[0005] According to literature reports, current pre-implantation mitochondrial testing mainly involves direct amplification of the variant site, followed by Sanger sequencing of the amplified product, or ddPCR, or NGS. Among these, Sanger detection of mitochondrial variants has low sensitivity, and Sanger first-generation sequencing struggles to detect sites with a mutation ratio below 5%. ddPCR is expensive, and primers must be designed for the pathogenic site each time, making it difficult to establish a unified operating procedure. It is difficult to amplify full-length mitochondrial DNA using the longRange PCR method from samples that have undergone whole-genome amplification. Currently, there is a lack of mitochondrial whole-genome amplification testing products on the market that can simultaneously detect different samples and different whole-genome amplification products.

[0006] In addition, Chinese patent CN114317712A discloses a kit, library construction method, and sequencing method for pre-implantation mitochondrial gene detection. The kit includes a pre-amplification reagent and an exponential amplification reagent. The pre-amplification reagent contains a first specific primer pair for pre-amplifying the mitochondrial target gene, and the exponential amplification reagent contains a second specific primer pair for exponentially amplifying the pre-amplified product of the mitochondrial target gene. The second specific primer pair is connected to a sequencing adapter sequence. The kit of this invention pre-amplifies the mitochondrial target gene with the pre-amplification reagent, and then exponentially amplifies the pre-amplified product with the exponential amplification reagent. Compared with the original embryo sample, the proportion of the mitochondrial target gene in the whole genome DNA is amplified, and low-frequency mitochondrial gene mutations can also be accurately detected, thereby improving the sensitivity and accuracy of pre-implantation mitochondrial gene detection.

[0007] The invention of Chinese patent CN111172157B provides a method for constructing a human single-cell mitochondrial high-throughput sequencing library and a kit for library construction. The kit comprises primers for full-circle amplification of the mitochondrial genome, single-cell lysate, end repair, phosphorylation, and 3' adenylate addition components for high-throughput library construction, adapters for high-throughput library construction, and forward and reverse library amplification primers. This method realizes the construction of high-throughput sequencing libraries of mitochondrial genomic DNA of a single or several cells in a system based on complete full-circle amplification of the mitochondrial genome, while avoiding the high off-target rate and high proportion of false positive site detection rate brought by conventional single-cell amplification systems, thereby eliminating a large number of drawbacks in mitochondrial mutation detection at the single-cell level and achieving high sensitivity and accuracy detection; at the same time, because this method adopts the method of full-circle full-length amplification of mitochondria, it can detect large fragment duplications and deletions of mitochondrial DNA while detecting mutation sites.

[0008] However, the above-mentioned mitochondrial detection methods currently lack primers and methods for whole-genome amplification of mitochondrial DNA in different whole-genome amplification products, and the methods shown therein have problems such as high cost, multiple steps, low sensitivity and long cycle. Therefore, providing a primer for amplifying the whole mitochondrial genome and its application, which can be used for whole-genome amplification of mitochondria in different whole-genome amplification product samples, and combining with the technical means of second-generation sequencing to obtain human mitochondrial whole genome information has become one of the urgent problems to be solved in the current biotechnology field. Summary of the Invention

[0009] In order to solve the technical problems of the technical defects in the background technology, the present invention discloses a primer set, a kit, a library construction method and application for human mitochondrial whole genome amplification. The primer combination and kit can amplify complete human mitochondrial gene products from different sample DNAs, and obtain human mitochondrial whole genome information by combining the technical means of second-generation sequencing.

[0010] The technical solutions adopted by the present invention to solve the above technical problems are as follows: In a first aspect, the present invention provides a primer set for amplifying the whole human mitochondrial genome, comprising 128 pairs of primers, wherein the nucleotide sequences of the forward primers in the 128 pairs of primers are sequentially shown as SEQ ID NO.1 to SEQ ID NO.128, and the nucleotide sequences of the reverse primers are sequentially shown as SEQ ID NO.129 to SEQ ID NO.256.

[0011] Furthermore, the primer set is amplified using a short-segment PCR amplification method to obtain an amplification product covering the entire length of human mitochondria, wherein the length of the short-segment PCR amplification is 180-310 bp.

[0012] The present invention has designed a primer set for amplifying the entire human mitochondrial genome. Using multiplex PCR amplification, the 128 primer pairs in the primer set can achieve coverage of over 99% of the entire length of the human mitochondrial genome. This allows for higher sequencing depth with minimal data volume, significantly reducing sequencing costs. Furthermore, the primer set utilizes short-segment amplification, enabling efficient amplification of low-quality, trace samples or whole-genome amplification products, enabling the acquisition of complete human mitochondrial genome information.

[0013] A second aspect of the present invention provides a kit for amplifying the whole human mitochondrial genome, comprising the primer set described in the first aspect, an amplification buffer, a first enhancer PEG6000 DMSO, a second enhancer dNTP, a universal primer, and a specific primer. Both the universal primer and the specific primer comprise a sequencer common sequence, which can be used for library construction and subsequent sequencing.

[0014] Furthermore, when the DNA sample to be tested is a mixed sample, ie, includes multiple samples, the specific primer further includes a sample distinguishing sequence, and the sample distinguishing sequence is used to distinguish different samples when performing data analysis after sequencing.

[0015] A third aspect of the present invention provides a method for constructing a human mitochondrial whole genome library, the method comprising: Step 1: Obtain the DNA of the sample to be tested; Step 2: using the primer set described in the first aspect or the kit described in the second aspect, perform two rounds of PCR amplification on the DNA of the sample to be tested to obtain a sequencing library of the human mitochondrial whole genome; Step 3: Use a high-throughput sequencing method to directly perform whole-genome sequencing on the sequencing library.

[0016] Furthermore, the sample DNA to be tested is derived from one or more of embryonic biopsy cell full gene amplification products, blood, amniotic fluid, hair, muscle tissue, and semen.

[0017] Furthermore, when the test sample DNA is derived from whole-genome amplification products of embryonic biopsy cells, MDA amplification or picoplex amplification is performed on non-embryonic extra-embryonic blastocyst biopsy cells to obtain the whole-genome amplification products of the embryonic biopsy cells. When the test sample DNA is derived from any of blood, amniotic fluid, hair, muscle tissue, and semen, no further processing is required and the DNA can be directly used for library construction.

[0018] Furthermore, when the test sample DNA is subjected to two rounds of PCR amplification, the reaction system for the first round of PCR amplification includes: 40-50 ng of the test sample DNA, 5 ul of the primer set, 10 ul of the amplification buffer, 3.5 ul of the first enhancer PEG6000 DMSO, 22.5 ul of the second enhancer dNTP, and the system volume is supplemented to 30 ul with nuclease-free water; the reaction system for the second round of PCR amplification includes: 13.5 ul of the first round PCR amplification product, 2.5 ul of the second enhancer dNTP, 2 ul of nuclease-free water, 2 ul of the universal adapter, and 10 ul of the amplification reaction solution.

[0019] Furthermore, the first round amplification program was as follows: initial denaturation at 95°C for 3–4 min, 10–16 cycles, extension at 72°C for 5–10 min, and holding at 16°C, with denaturation at 98°C for 20 s and annealing at 60°C for 2–5 min during each cycle; The second round of PCR amplification program is as follows: initial denaturation at 95°C for 3-4 min, 6-15 cycles, extension at 72°C for 30s-2min, and holding at 16°C, with denaturation at 98°C for 20s and annealing at 58°C for 1-3min in each cycle.

[0020] The fourth aspect of the present invention provides a use of the primer set described in the first aspect in constructing the kit described in the second aspect or the library construction method described in the third aspect.

[0021] Compared with existing technologies, the present invention offers the following advantages: the primer set designed for amplifying the entire human mitochondrial genome utilizes a short-segment amplification approach, achieving coverage of over 99% of the entire human mitochondrial genome using 128 primer pairs. Furthermore, the amplified products can be directly used for high-throughput sequencing, simplifying the process. Furthermore, the primer set can effectively amplify sample DNA or whole-genome amplification products from various sources to obtain complete human mitochondrial genome information. Bioinformatics analysis of the amplified products can then reveal the mutation sites and ratios of human mitochondrial genes, providing effective guidance for pre-implantation embryo screening. DETAILED DESCRIPTION

[0022] In order to better understand the content of the present invention, the present invention is described in detail below in conjunction with Examples 1 to 3. The following describes the embodiments of the present application by means of specific specific examples, and those skilled in the art can easily understand other advantages and effects of the present application from the content disclosed in this specification. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. The present application can also be implemented or applied through other different specific embodiments, and the various details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present application. It should be noted that, in the absence of conflict, the features of the following embodiments and embodiments can be combined with each other. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without making creative work are within the scope of protection of the present application.

[0023] In an embodiment of the present invention, a primer set for amplifying the whole mitochondrial genome is designed based on the whole human mitochondrial genome sequence. The primer set includes 128 pairs of primers. The nucleotide sequences of the forward primers in the 128 pairs of primers are shown in SEQ ID NO.1 to SEQ ID NO.128, and the nucleotide sequences of the reverse primers are shown in SEQ ID NO.129 to SEQ ID NO.256.

[0024] Furthermore, the primer set is amplified using short-segment PCR amplification to obtain an amplification product covering the entire length of human mitochondria, wherein the length of the short-segment PCR amplification is 180-310 bp, preferably, the length of the short-segment PCR amplification is 250±10 bp.

[0025] The present invention conducts feasibility verification on the above primer set through this Example 1, which specifically includes the following process: First, whole-genome amplification of GM02800 cells (3-5 cells can be selected) was performed using both picoplex amplification and MDA amplification methods, and the amplified products were set aside. GM02800 cells are a human skin fibroblast cell line obtained from the NIH (National Institutes of Health) Human Cell Bank and are generally considered representative of normal human cells. GM02800 cells have a normal genetic background and are commonly used to study gene function, genetics, cell biology, and disease modeling. Fibroblasts are commonly involved in tissue repair and collagen synthesis in the body.

[0026] Secondly, the above-mentioned primer set or a kit prepared using the primer set is used to construct a library of the above-mentioned amplified products, wherein the kit includes the above-mentioned primer set, an amplification buffer, a first enhancer PEG6000 DMSO, a second enhancer dNTP, a universal primer and a specific primer, and the universal primer and the specific primer both include a sequencer public sequence, and the sequencer public sequence can be used for library construction and sequencing.

[0027] Preferably, the forward sequence of the sequencer public sequence (as shown in SEQ ID NO. 257) is: 5'-ATGATACGGCGACCACCGAGATCTACACNNNNNNNNACACTCTTTCCCTACACGACGCCTTCCGATCT-3'; The reverse sequence of the sequencer public sequence (as shown in SEQ ID NO. 258) is: 5'-GTTCGTCTTCTGCCGTATGCTCTANNNNNNNNCACTGACCTCAAGGAACCGTGGGCTCTTAAGGT-3').

[0028] Preferably, when the sample DNA to be tested is a mixed sample, that is, includes multiple samples, the specific primer also includes a sample distinguishing sequence, and the sample distinguishing sequence is used to distinguish different samples when performing data analysis after sequencing, wherein the sample distinguishing sequence is NNNNNNNN.

[0029] The steps for library construction are as follows: (1) Establish the first round of PCR amplification system according to Table 1 below Table 1: First round PCR amplification system

[0030] In Table 1 above, gDNA can be derived from one or more of human blood, amniotic fluid, hair, muscle tissue, and semen. When using gDNA for library construction, DNA extraction is sufficient, requiring no additional processing. Whole-genome amplification products (i.e., whole-genome amplification products from embryonic biopsy cells) are derived from extraembryonic blastocyst biopsy cells from non-embryonic tissue. These products are obtained by whole-genome amplification of extraembryonic blastocyst biopsy cells from non-embryonic tissue using MDA amplification or picoplex amplification techniques.

[0031] (2) Perform the first round of PCR amplification on the amplified product according to Table 2 below to obtain the first round of PCR amplification product Table 2: First round PCR amplification program

[0032] (3) Purify the first-round PCR amplification products of (2) by magnetic beads.

[0033] (4) Using the purified product after the first round of PCR amplification in (3), a second round of PCR amplification is performed using the second round PCR amplification system in Table 3 and the second round PCR amplification program in Table 4 to obtain the second round PCR amplification product.

[0034] Table 3: Second round PCR amplification system

[0035] Table 4: First round PCR amplification program

[0036] (5) Use XP magnetic beads to purify the products after the second round of PCR amplification to obtain a sequencing library, and perform quantitative and quality control tests on the sequencing library. Specifically, the library can be quantitatively tested using Qubit and the library fragments can be measured using Q-sep technology.

[0037] Then, the obtained sequencing library is sequenced using a high-throughput sequencing method to obtain sequencing data. Specifically, the sequencing data can be aligned to the mitochondrial reference genome NC_012920, and the data can be analyzed using the GATK mitochondrial analysis mode to obtain the mitochondrial mutation sites and mutation ratios.

[0038] Finally, the sequencing data was analyzed, and the analysis results are shown in Table 5 below.

[0039] Table 5: Sequencing data analysis results

[0040] According to the results in Table 5 above, the primer set of the present invention can be used to construct mitochondrial whole genome libraries for different types of samples. The coverage of 1x can reach 100%, and the coverage of 30x can reach more than 99%. This result shows that the primer set of the present invention can meet the library construction requirements of different whole genome amplification samples. The constructed library can be directly sequenced, which is more time-saving.

[0041] The present invention also recruits patients and embryos clinically diagnosed with mitochondrial genetic diseases as sample DNA sources as Example 2, and performs tests on them to verify the feasibility of the primer set of the present invention, specifically comprising the following steps: First, peripheral blood was collected from the patient, and gDNA was obtained using a blood extraction kit. Extraembryonic blastocyst biopsy cells or aborted embryonic tissue, which were not considered embryonic tissue, were obtained as embryo samples and amplified using an MDA amplification kit to generate whole-genome amplification products. The theoretical mitochondrial variation ratios in the patient's gDNA and whole-genome amplification products are shown in Table 6 below.

[0042] Table 6: Mitochondrial theoretical variation results in patient gDNA and whole genome amplification products

[0043] Secondly, according to the sequencing library construction method shown in Example 1 of the present invention, two rounds of PCR amplification were performed on the above 7 samples to obtain a sequencing library, and the data after sequencing the sequencing library were subjected to bioinformatics analysis to obtain the results shown in Table 7 below.

[0044] Table 7: Mitochondrial variation results of 7 samples constructed and analyzed using the primer set

[0045] From the mitochondrial variation results in Tables 6 and 7 above, it can be seen that the present invention can construct mitochondrial whole genome libraries for different types of samples, with a 1x coverage of up to 100% and a 1000x coverage of over 99%. The primer set of the present invention can accurately detect the variation sites and variation ratios in the whole genome amplification products.

[0046] The present invention also uses clinical samples with known mutation sites and mutation ratios as Example 3 to verify the feasibility of the primer set provided by the present invention, specifically including the following process: First, four samples with variation ratios of 2%, 5%, 8%, and 10% were prepared, and the four samples were diluted to 18 pg respectively to simulate biopsy cells. The diluted samples were then amplified by MDA.

[0047] Next, the sequencing library construction method in the embodiment of the present invention was used to construct and sequence the four simulated samples obtained by amplification, and the sequencing results were analyzed to obtain the results shown in Table 8 below.

[0048] Table 8: Mitochondrial variation results in MDA amplification products of 4 samples

[0049] According to Table 8 above, the primer sets provided by the present invention were used to amplify, construct libraries, and perform sequencing analysis on the different variation ratios of the simulated samples, and the actual variation ratios detected were consistent with the theoretical variation ratios.

[0050] According to the results of Examples 1 to 3 above, the primer set provided by the present invention can amplify the whole mitochondrial genome of human mitochondrial DNA, achieving coverage of more than 99% of the full length of human mitochondria, and can obtain a higher sequencing depth with a very small amount of data, which greatly reduces the sequencing cost. In addition, the mitochondrial genes of DNA and whole-genome amplification products are directly library-built by two-round PCR amplification methods, without the need for further enzyme excision and modification to build the library. The operation is simple and convenient, and it saves more time. The relative cost is also relatively low. And from the above-mentioned various variation result tables, it can be seen that the primer set, kit and library construction method of the present invention can stably and accurately detect the mitochondrial variation sites and variation ratios in the whole-genome amplification products, and can stably detect sites with a variation ratio of less than 10%, which is close to the theoretical variation ratio, with high detection accuracy and high sensitivity.

[0051] Obviously, the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. A primer set for amplifying the whole human mitochondrial genome, characterized in that: The primer set includes 128 pairs of primers, the nucleotide sequences of the forward primers in the 128 pairs of primers are shown in SEQ ID NO.1 to SEQ ID NO.128, and the nucleotide sequences of the reverse primers are shown in SEQ ID NO.129 to SEQ ID NO.

256.

2. The primer set for human mitochondrial whole genome amplification according to claim 1, characterized in that The primer set is amplified by short-fragment PCR amplification to obtain an amplification product covering the entire length of human mitochondria, wherein the length of the short-fragment PCR amplification is 180-310 bp.

3. A kit for amplifying the whole genome of human mitochondria, characterized in that: The kit comprises the primer set according to claim 1 or 2, an amplification buffer, a first enhancer PEG6000 DMSO, a second enhancer dNTP, a universal primer and a specific primer, wherein both the universal primer and the specific primer comprise a sequencer public sequence.

4. The kit for human mitochondrial whole genome amplification according to claim 3, characterized in that The specific primer further includes a sample distinguishing sequence, and the sample distinguishing sequence is used to distinguish different samples.

5. A method for constructing a human mitochondrial whole genome library, characterized in that: The library construction method comprises: Obtaining DNA from the sample to be tested; Using the primer set of claim 1 or 2 or the kit of claim 3 or 4, performing two rounds of PCR amplification on the DNA of the sample to be tested to obtain a sequencing library of the human mitochondrial whole genome; The whole genome sequencing of the sequencing library is directly performed using a high-throughput sequencing method.

6. The method for constructing a human mitochondrial whole genome library according to claim 5, characterized in that: The DNA sample to be tested is derived from one or more of embryonic biopsy cell full gene amplification products, blood, amniotic fluid, hair, muscle tissue, and semen.

7. The method for constructing a human mitochondrial whole genome library according to claim 6, characterized in that: The embryonic biopsy cell whole-genome amplification product is obtained by performing whole-genome amplification on the extra-blastocyst biopsy cells of non-embryonic tissue using MDA amplification technology or picoplex amplification technology.

8. The method for constructing a human mitochondrial whole genome library according to claim 5, characterized in that: When the test sample DNA is subjected to two rounds of PCR amplification, the reaction system for the first round of PCR amplification includes: 40-50 ng of the test sample DNA, 5 μl of the primer set, 10 μl of amplification buffer, 3.5 μl of the first enhancer PEG6000 DMSO, 22.5 μl of the second enhancer dNTP, and the system volume is supplemented to 30 μl with nuclease-free water; the reaction system for the second round of PCR amplification includes: 13.5 μl of the first round PCR amplification product, 2.5 μl of the second enhancer dNTP, 2 μl of nuclease-free water, 2 μl of the universal adapter, and 10 μl of the amplification reaction solution.

9. The method for constructing a human mitochondrial whole genome library according to claim 8, characterized in that: The first round of amplification was performed as follows: initial denaturation at 95°C for 3–4 min, 10–16 cycles, extension at 72°C for 5–10 min, and holding at 16°C, with denaturation at 98°C for 20 s and annealing at 60°C for 2–5 min during each cycle. The second round of PCR amplification program was as follows: initial denaturation at 95°C for 3-4 min, 6-15 cycles, extension at 72°C for 30s-2min, and holding at 16°C, with denaturation at 98°C for 20s and annealing at 58°C for 1-3 min during each cycle.

10. Use of the primer set according to claim 1 or 2 in constructing the kit according to claim 3 or 4 or the library construction method according to any one of claims 5 to 9.

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