A primer set, a kit and a detection method for detecting mutation and copy number of human mitochondrial DNA

Abstract

The present invention relates to a primer set, a kit and a detection method for detecting mutation and copy number of human mitochondrial DNA. The primer set includes primers Y1 to Y5; primer Y1 is a primer P1 with a UMI sequence and a sequence of primer Y5 added to the 5' end; primer Y2 is a primer P2 with a Barcode sequence added to the 5' end; primer Y3 is a primer P3 with a UMI sequence and a sequence of primer Y5 added to the 5' end; primer Y4 is a primer P4 with a Barcode sequence added to the 5' end; primer Y5 is a primer P5 with a Barcode sequence added to the 5' end; primers P1 and P2, P3 and P4 are human mitochondrial DNA and TBP gene amplification specific primer pairs, respectively, and primer P5 is a universal primer. The present invention can simultaneously detect different types of mutations in human mitochondrial DNA and mitochondrial DNA copy numbers associated with human mitochondrial DNA depletion.

Description

Technical Field

[0001] The present invention relates to the technical field of gene detection, and in particular to a primer set, a kit and a detection method for detecting mutation and copy number of human mitochondrial DNA. Background Art

[0002] Mitochondria exist in almost all human cells, participate in the cell respiratory chain and oxidative phosphorylation, and provide energy for cell activities. In addition, mitochondria are also involved in cell differentiation, signal transduction, apoptosis and other processes, and can regulate cell growth and cell cycle. Mitochondrial dysfunction will directly affect human health and aging. Mitochondrial DNA damage and mitochondrial DNA depletion in mitochondria are important factors affecting mitochondrial function.

[0003] Mitochondrial DNA is a double-stranded circular DNA with a total length of 16569bp, encoding 37 genes. Point mutations or fragment deletions in mitochondrial DNA can lead to disorders in the synthesis of enzymes or carriers required for mitochondrial oxidative phosphorylation, thereby leading to disorders in cellular energy metabolism. In addition, mutations in nuclear genes (TK2, SUCLA2, SUCLG1, RRM2B, DGUOK, MPV17, POLG, C10orf2, TYMP) involved in nucleotide synthesis or mitochondrial DNA replication in the human cell nucleus can cause a decrease in the amount of mitochondrial DNA synthesis, that is, mitochondrial depletion, leading to energy production disorders in affected tissues and organs. Both of the above mitochondrial abnormalities can cause diseases of multiple organs and systems in the human body, including nervous system, cardiovascular, endocrine, tumor and other systemic diseases.

[0004] Methods for detecting mitochondrial DNA point mutations include Sanger sequencing, second-generation sequencing, and third-generation sequencing. Methods for detecting mitochondrial DNA fragment deletions include Southern hybridization, fluorescence quantitative PCR, long-fragment PCR, multiplex ligation probe amplification technology, second-generation sequencing, and third-generation sequencing. Sanger sequencing can only detect hotspot point mutations in mitochondrial DNA, but cannot detect other point mutations in mitochondrial DNA. Southern hybridization, fluorescence quantitative PCR, and multiplex ligation probe amplification technology can only detect fragment deletions in mitochondrial DNA, but cannot clearly detect the specific interval sequence of mitochondrial deletions. Long-fragment PCR can detect mitochondrial deletions, but it needs to be sequenced by second-generation sequencing or third-generation sequencing to clarify the specific interval sequence of mitochondrial deletions, and can also detect mitochondrial point mutations. Second-generation sequencing and third-generation sequencing, including sequencing of long-fragment PCR amplification products and targeted sequencing, can detect point mutations and fragment deletions in mitochondrial DNA, but due to the short sequencing read length of second-generation sequencing, there are limitations in detecting low-proportion fragment deletions. Although the third-generation sequencing has no limitations in detecting low-proportion fragment deletions, both long-fragment PCR amplification products and targeted sequencing have PCR amplification links, and the mutation ratios of point mutations and fragment deletions in the detection results are significantly different from the actual ratios. None of the above detection technologies can detect mitochondrial DNA depletion. Mitochondrial DNA depletion is generally detected by fluorescent quantitative PCR, using nuclear genes as a reference and detecting the mitochondrial DNA copy number.

[0005] Therefore, it is very meaningful to develop a simple and effective detection method to achieve simultaneous detection of mitochondrial DNA nucleotide mutations, fragment deletions, and copy numbers. Summary of the invention

[0006] The purpose of the present invention is to provide a primer set, a kit and a detection method for detecting human mitochondrial DNA mutation and copy number, which can detect different types of mutations such as human mitochondrial DNA point mutations and large fragment deletions, as well as mitochondrial DNA copy number associated with human mitochondrial DNA depletion.

[0007] In a first aspect of the present invention, a primer set for detecting mutations and copy number of human mitochondrial DNA is provided, comprising: primer Y1, primer Y2, primer Y3, primer Y4 and primer Y5;

[0008] The nucleotide sequence of the primer Y1 is the sequence of the primer P5 with the UMI sequence and primer P5 added to the 5' end of the primer P1;

[0009] The nucleotide sequence of the primer Y2 is a barcode sequence added to the 5' end of the primer P2 to distinguish different samples;

[0010] The nucleotide sequence of the primer Y3 is the sequence of the primer P5 with the UMI sequence and primer P5 added to the 5' end of the primer P3;

[0011] The nucleotide sequence of the primer Y4 is a barcode sequence added to the 5' end of the primer P4 to distinguish different samples;

[0012] The nucleotide sequence of the primer Y5 is a barcode sequence added to the 5' end of the primer P5 to distinguish different samples;

[0013] The primers P1 and P2 are a pair of specific primers for amplifying human mitochondrial DNA, which can amplify the 16569bp full-length sequence of human mitochondria; the primers P3 and P4 are a pair of specific primers for amplifying human TBP gene, which can amplify the 16638bp human TBP gene sequence; and the primer P5 is a universal primer.

[0014] Preferably, the nucleotide sequences of the primers P1 and P2 are shown in SEQ ID NOs: 1-2.

[0015] Preferably, the nucleotide sequences of the primers P3 and P4 are shown in SEQ ID NOs: 3-4.

[0016] Preferably, the nucleotide sequence of the primer P5 is as shown in SEQ ID NO:5.

[0017] Preferably, the nucleotide sequences of the primers Y1 and Y3 are shown in SEQ ID NOs: 6-7.

[0018] Preferably, the length of the UMI (unique molecular identifier) ​​sequence is 12 nt, and the length of the Barcode (DNA barcode) sequence is 16 nt.

[0019] In the present invention, there is no limitation on the Barcode sequence, and the Barcode sequences (including Barcode-F and Barcode-R) may be consistent or inconsistent; the Barcode sequence may be a Barcode designed by PacBio or a self-designed Barcode, and a double Barcode with a Barcode primer or a Barcode adapter may be used to achieve more Barcode combinations; a protective base may or may not be added to the 5' end of the Barcode sequence, and those skilled in the art may select as needed; preferably, a 3-5 nt protective base is added to the 5' end of the Barcode sequence, and the sequence of the protective base is GGTAG.

[0020] Preferably, the nucleotide sequence of the primer Y4 is a barcode sequence having the same sequence as that of the primer Y2 added to the 5' end of the primer P4.

[0021] Preferably, the nucleotide sequence of the primer Y5 is a barcode sequence having a different sequence from that of the primer Y2 added to the 5' end of the primer P5.

[0022] In a specific embodiment, the Barcode sequence is selected from any one of the following groups:

[0023] (A) bc1193-F: 5'-ATACGCGCGCGCATGC-3' (SEQ ID NO: 8);

[0024] bc1193-R: 5'-GCATGCGCGCGCGTAT-3' (SEQ ID NO: 9);

[0025] (B) bc1194-F: 5'-CTCTGACTCGCGTCGA-3' (SEQ ID NO: 10);

[0026] bc1194-R: 5'-TCGACGCGAGTCAGAG-3' (SEQ ID NO: 11).

[0027] The second aspect of the present invention provides a kit for detecting mutations and copy numbers of human mitochondrial DNA, comprising: the above-mentioned primer set.

[0028] Preferably, it also includes: one or more of a DNA extraction system, a PCR reaction buffer, ddH2O, and a DNA polymerase.

[0029] The third aspect of the present invention provides a method for detecting human mitochondrial DNA mutation and copy number for non-diagnostic purposes, comprising the following steps:

[0030] S1. Extract DNA from the sample to be tested.

[0031] Specifically, the sample to be tested can be any one of peripheral blood, dried blood spots, muscle tissue, urine or oral swab samples.

[0032] S2. Use the above-mentioned primer set or the above-mentioned kit to add UMI to the DNA extracted in step S1.

[0033] Preferably, in step S2, the amplification reaction mixture used includes: 12.5 μl of 2×PCR Buffer for KOD FXNeo, 5 μl of 2mM dNTPs, 0.5 μl of KOD FX Neo (1U / μl), 2.5 μl of primer Y1 with a concentration of 10 μM, 2.5 μl of primer Y3 with a concentration of 10 μM, and 1 μl of ddH2O; 1 μl of DNA is added to 24 μl of the amplification reaction mixture to perform PCR amplification.

[0034] Preferably, in step S2, the PCR amplification procedure comprises the following steps:

[0035] In a PCR instrument, first denature at 98°C for 1 minute; then run the following cycles 6 times: anneal at 70°C for 5 seconds (1°C lower per cycle); extend at 72°C for 10 minutes; and store at 4°C.

[0036] The PCR amplification product was purified using 0.8× purification magnetic beads before the next step of reaction.

[0037] S3. Use the above primer set or the above kit to perform PCR amplification on the product to which the UMI is added in step S2 to generate a PCR product with a barcode sequence.

[0038] Preferably, in step S3, the amplification reaction mixture used includes: 25 μl of 2×PCR Buffer for KOD FXNeo, 10 μl of 2mM dNTPs, 1 μl of KOD FX Neo (1U / μl), 1 μl of primer Y2 with a concentration of 10 μM, 1 μl of primer Y4 with a concentration of 10 μM, and 2 μl of primer Y5 with a concentration of 10 μM; 10 μl of the purified product of step S2 is added to 40 μl of the amplification reaction mixture to perform PCR amplification.

[0039] Preferably, in step S3, the PCR amplification procedure comprises the following steps:

[0040] In a PCR instrument, first denature at 94°C for 2 minutes; then run the following cycle 30 times: denature at 98°C for 10 seconds, anneal and extend at 68°C for 9 minutes; finally extend at 68°C for 10 minutes and store at 4°C.

[0041] The PCR amplification product was purified using 0.8× purification magnetic beads before the next step of reaction.

[0042] S4. Construction of PacBio sequencing library.

[0043] Preferably, step S4 comprises the following steps: the PCR purified product obtained in step S3 is purified by using Prep kit 3.0 (PacBio, Cat#102-141-700) was used to construct the PacBio library. According to the concentration of the library, an appropriate volume of the library was reacted with the binding reagent (PacBio, Cat#101-820-200) and primers (PacBio, Cat#100-970-100) to prepare the final machine-ready library for PacBio library sequencing.

[0044] S5. Perform PacBio library sequencing and perform bioinformatics analysis on the PacBio sequencing data to obtain human mitochondrial DNA mutation and copy number results.

[0045] Preferably, step S5 includes the following steps: the sequencing data obtained in step S4 is subjected to bioinformatics analysis of the mutation results such as point mutations and large fragment deletions of mitochondrial DNA, and the UMI types of mitochondrial DNA and the UMI types of TBP genes are obtained at the same time. DNA templates from different sources are distinguished according to different UMI types, and the correct DNA fragments are traced to the source, and more accurate mutation ratios of mitochondrial DNA point mutations, large fragment deletions and other mutation types are calculated, and false positive mutations caused by random errors in PCR amplification and sequencing processes can be distinguished. In addition, the number of mitochondrial DNA copies in a single cell in the sample is obtained according to the ratio between the TBP gene UMI type and the mitochondrial DNA UMI type.

[0046] Beneficial effects:

[0047] (1) The present invention uses a set of primers with specific molecular tags UMI added, based on long-fragment multiplex PCR amplification and PacBio third-generation high-throughput sequencing methods, to specifically, accurately and quickly detect mutation types such as mitochondrial DNA point mutations and large fragment deletions, and accurately detect the mitochondrial DNA copy number associated with human mitochondrial DNA depletion.

[0048] (2) The present invention can detect various mutation types of human mitochondrial DNA in one reaction system, including point mutations, large fragment deletions and other mutation types; by adding the human TBP gene as an internal reference gene, the mitochondrial copy number can be detected while detecting mitochondrial mutations, and the detection range is wide.

[0049] (3) The present invention combines the unique molecular tag UMI technology and PacBio's third-generation high-throughput sequencing technology. The UMI correction combined with the sequencing depth correction base accuracy is greater than 99.9%, which can more accurately interpret the gene mutations and mutation ratios within the detection range with high accuracy.

[0050] (4) The PacBio third-generation sequencing technology used in the present invention can provide 384 barcode sequences. More barcode sequences can be designed as needed, or double barcodes with barcode primers or barcode adapters can be used to achieve more barcode combinations, which can achieve high-throughput sample detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0052] Figure 1 This is a flow chart of the method for detecting human mitochondrial DNA mutation and copy number provided by the present invention.

[0053] Figure 2 This is a schematic diagram of the positions of primers P1 and P2 provided by the present invention and their amplification directions.

[0054] Figure 3 This is a schematic diagram of the positions of primers P3 and P4 provided by the present invention and their amplification directions.

[0055] Figure 4 This is the gel electrophoresis diagram of the amplification products of primers Y2 and Y5 provided by the present invention.

[0056] Figure 5 This is the gel electrophoresis diagram of the amplification products of primers Y4 and Y5 provided by the present invention.

[0057] Figure 6 The coverage of human mitochondrial DNA sequencing data provided by the present invention.

[0058] Figure 7 This is the coverage of the human TBP gene sequencing data provided by the present invention.

[0059] Figure 8 The present invention provides a comparison of the mitochondrial mutation ratio and mitochondrial copy number detected using the UMI technology and without the UMI technology. DETAILED DESCRIPTION

[0060] It should be noted that the following detailed descriptions are illustrative and are intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present application belongs.

[0061] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present application. As used herein, unless the context clearly indicates otherwise, the singular form also includes the plural form. In addition, it should be understood that when the terms "comprise" and / or "include" are used in this specification, it indicates the presence of features, steps, operations, devices, components and / or combinations thereof.

[0062] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0063] Example 1

[0064] This example uses the UMI-PCR method to amplify human mitochondrial DNA sequences, including the following steps:

[0065] (1) Primer design

[0066] Primer3web version 4.1.0 software was used to design primer pairs P1 and P2 for amplifying human mitochondrial DNA sequences based on the full-length mitochondrial nucleotide sequence (e.g. Figure 2 ), amplified the full length of human mitochondrial DNA sequence 16569bp, specifically:

[0067] The nucleotide sequence of primer P1 is 5′-GATCACAGGTCTATCACCCTATTAAC-3′ (SEQ ID NO: 1);

[0068] The nucleotide sequence of primer P2 is 5′-CATCGTGATGTCTTATTTAAGGGGAA-3′ (SEQ ID NO: 2);

[0069] The nucleotide sequence of primer P5 is 5′-GGAAGGAGTTCCGAATAGTCAAGAC-3′ (SEQ ID NO: 5);

[0070] The nucleotide sequence of primer Y1 is 5′-GGAAGGAGTTCCGAATAGTCAAGACNN NNNNNNNNNNNNGATCACAGGTCTATCACCCTATTAAC-3′ (SEQ ID NO: 6);

[0071] There is no limitation on the Barcode sequence. When the samples to be tested are multiple different samples, each sample can use a different Barcode sequence. In this embodiment, there are two samples to be tested: sample 1 (peripheral blood) and sample 2 (dried blood spot). Sample 1 can use bc1193-F and bc1193-R of group (A), and sample 2 can use bc1194-F and bc1194-R of group (B);

[0072] When the barcode sequence is bc1193-F and bc1193-R of group (A):

[0073] The nucleotide sequence of primer Y2 is 5′-GGTAGGCATGCGCGCGCGTATCATCGTGATGTCTTATTTAAGGGGAA-3′ (SEQ ID NO: 12);

[0074] The nucleotide sequence of primer Y5 is 5′-GGTAGATACGCGCGCGCATGCGGAAGGAGTTCCGAATAGTCAAGAC-3′ (SEQ ID NO: 13);

[0075] When the barcode sequence is bc1194-F or bc1194-R from group (B):

[0076] The nucleotide sequence of primer Y2 is 5′-GGTAGTCGACGCGAGTCAGAGCATCGTGATGTCTTATTTAAGGGGAA-3′ (SEQ ID NO: 14);

[0077] The nucleotide sequence of primer Y5 is 5′-GGTAGCTCTGACTCGCGTCGAGGAAGGAGTTCCGAATAGTCAAGAC-3′ (SEQ ID NO: 15).

[0078] (2) DNA extraction

[0079] This example uses a blood genomic DNA extraction kit (EE121) produced by Beijing Quanshijin Biotechnology Co., Ltd. to extract genomic DNA from peripheral blood and dried blood spots:

[0080] Take 250 μl of EDTA anticoagulated whole blood and add it to a 2 ml EP centrifuge tube.

[0081] Add 500 μl BB3 and 20 μl Proteinase K, vortex to mix, and incubate at room temperature for 10 min.

[0082] After brief centrifugation, all the liquid was transferred to the adsorption column in the collection tube, centrifuged at 12,000 rpm for 1 min, and the filtrate was discarded.

[0083] The adsorption column was placed in a collection tube, 500 μl of CB3 was added, centrifuged at 12,000 rpm for 1 min, and the filtrate was discarded.

[0084] The adsorption column was placed in a collection tube, 500 μl of WB3 was added, and the mixture was centrifuged at 12,000 rpm for 1 min, and the filtrate was discarded.

[0085] The adsorption column was placed in a collection tube, 500 μl of WB3 was added, and the mixture was centrifuged at 12,000 rpm for 1 min, and the filtrate was discarded.

[0086] The adsorption column was placed in a collection tube and centrifuged at 12,000 rpm for 2 min.

[0087] Place the adsorption column in a clean 1.5 ml EP centrifuge tube and leave it open for 2 minutes.

[0088] Add 70 μl of EB (Elution Buffer) preheated at 70°C to the center of the adsorption column and let it stand at room temperature for 1 min.

[0089] Centrifuge at 12,000 rpm for 1 min to elute the DNA and discard the adsorption column.

[0090] Use nanodrop to measure DNA concentration and conduct subsequent experiments. DNA can be temporarily stored at 2-8℃ or stored at -18℃ for a long time.

[0091] (3) Add unique molecular label UMI

[0092] KOD FX Neo DNA Polymerase (Code No. KFX-101) from TOYOBO CO., LTD. was used to add a unique molecular tag UMI to the sample DNA using the above primer set.

[0093] The PCR amplification reaction mixture was prepared according to the amount of DNA in the test sample. The amounts of reagents in the amplification reaction mixture were shown in Table 1.

[0094] Table 1

[0095] Reagents Volume / μL 2×KODFXneoBuffer 12.5×n dNTPMix(2mMeach) 5×n Primer Y1 2.5×n KODFXneo (1.0U / μl) 0.5×n <![CDATA[ddH2O]]> 3.5×n

[0096] Where n = number of samples tested + 1.

[0097] Add 1 μl DNA (50 ng) to 24 μl PCR amplification reaction mixture of each sample to be tested.

[0098] Carry out PCR amplification program in a PCR instrument. The amplification program is as follows:

[0099] In a PCR instrument, first denature at 98°C for 1 minute; then run the following cycles 6 times: anneal at 70°C for 5 seconds (1°C lower per cycle); extend at 72°C for 10 minutes; and store at 4°C.

[0100] The product was purified using 0.8× purification magnetic beads.

[0101] (4) PCR amplification

[0102] The UMI product in the previous step is PCR amplified using the above primer set to generate a series of PCR products with Barcode sequences.

[0103] PCR amplification used KOD FX Neo DNA Polymerase (Cat. No. KFX-101) from TOYOBO CO., LTD.

[0104] The PCR amplification reaction mixture was prepared according to the amount of DNA in the test sample. The amounts of reagents in the amplification reaction mixture were shown in Table 2.

[0105] Table 2

[0106] Reagents Volume / μL 2×KODFXneoBuffer 25×n dNTPMix(2mMeach) 10×n Primer Y5 1×n Primer Y2 1×n KODFXneo (1.0U / μl) 1×n <![CDATA[ddH2O]]> 2×n

[0107] Where n = number of samples tested + 1.

[0108] Add 10 μl of the UMI product from the previous step to 40 μl of the PCR amplification reaction mixture of each sample to be tested.

[0109] Carry out PCR amplification program in a PCR instrument. The amplification program is as follows:

[0110] In a PCR instrument, first denature at 98°C for 1 minute; then run 30 cycles as follows: denature at 98°C for 15 seconds, anneal and extend at 68°C for 10 minutes; finally extend at 68°C for 10 minutes; and store at 4°C.

[0111] After amplification, 2ul of each sample was taken and tested on a 1% DNA gel. The results were as follows: Figure 4 As shown in Figure 2, mitochondrial DNA can be effectively amplified using different samples as templates.

[0112] The product was purified using 0.8× purification magnetic beads.

[0113] Example 2

[0114] This example uses the UMI-PCR method to amplify the human TBP gene DNA sequence, including the following steps:

[0115] (1) Primer design

[0116] Primer3web version 4.1.0 software was used to design primer pairs P3 and P4 (such as Figure 3 ). Since PCR amplification has a strong amplification advantage for short fragments, in order to avoid the problem of high amplification efficiency of short fragments in the PCR amplification process, the primers for amplifying the human TBP gene are designed to amplify a fragment of the size equivalent to the size of the amplified mitochondrial DNA sequence, specifically:

[0117] The nucleotide sequence of primer P3 is 5′-TAAATCCTGCCGTCATTATCTCTTG-3′ (SEQ ID NO: 3);

[0118] The nucleotide sequence of primer P4 is 5′-CGGCAATGATAACAACTGAATCTTC-3′ (SEQ ID NO: 4);

[0119] The nucleotide sequence of primer P5 is 5′-GGAAGGAGTTCCGAATAGTCAAGAC-3′ (SEQ ID NO: 5);

[0120] The nucleotide sequence of primer Y3 is 5′-GGAAGGAGTTCCGAATAGTCAAGACNN NNNNNNNNNNTAAATCCTGCCGTCATTATCTCTTG-3′ (SEQ ID NO: 7);

[0121] There is no limitation on the Barcode sequence. When the samples to be tested are multiple different samples, each sample can use a different Barcode sequence. In this embodiment, there are two samples to be tested: sample 1 (peripheral blood) and sample 2 (dried blood spot). Sample 1 can use bc1193-F and bc1193-R of group (A), and sample 2 can use bc1194-F and bc1194-R of group (B);

[0122] When the barcode sequence is bc1193-F and bc1193-R of group (A):

[0123] The nucleotide sequence of primer Y4 is 5′-GGTAGGCATGCGCGCGCGTATCGGCAATGATAACAACTGAATCTTC-3′ (SEQ ID NO: 16);

[0124] The nucleotide sequence of primer Y5 is 5′-GGTAGATACGCGCGCGCATGCGGAAGGAGTTCCGAATAGTCAAGAC-3′ (SEQ ID NO: 13);

[0125] When the barcode sequence is bc1194-F or bc1194-R from group (B):

[0126] The nucleotide sequence of primer Y4 is 5′-GGTAGTCGACGCGAGTCAGAGCGGCAATGATAACAACTGAATCTTC-3′ (SEQ ID NO: 17);

[0127] The nucleotide sequence of primer Y5 is 5′-GGTAGCTCTGACTCGCGTCGAGGAAGGAGTTCCGAATAGTCAAGAC-3′ (SEQ ID NO: 15).

[0128] (2) DNA extraction

[0129] The method is consistent with that of Example 1.

[0130] (3) Add unique molecular label UMI

[0131] KOD FX Neo DNA Polymerase (Code No. KFX-101) from TOYOBO CO., LTD. was used to add a unique molecular tag UMI to the sample DNA using the above primer set.

[0132] The PCR amplification reaction mixture was prepared according to the amount of DNA in the test sample. The amounts of reagents in the amplification reaction mixture were shown in Table 3.

[0133] Table 3

[0134] Reagents Volume / μL 2×KODFXneoBuffer 12.5×n dNTPMix(2mMeach) 5×n Primer Y3 2.5×n KODFXneo (1.0U / μl) 0.5×n <![CDATA[ddH2O]]> 3.5×n

[0135] Where n = number of samples tested + 1.

[0136] Add 1 μl DNA (50 ng) to 24 μl PCR amplification reaction mixture of each sample to be tested.

[0137] Carry out PCR amplification program in a PCR instrument. The amplification program is as follows:

[0138] In a PCR instrument, first denature at 98°C for 1 minute; then run the following cycles 6 times: anneal at 70°C for 5 seconds (1°C lower per cycle); extend at 72°C for 10 minutes; and store at 4°C.

[0139] The product was purified using 0.8× purification magnetic beads.

[0140] (4) PCR amplification

[0141] The UMI product in the previous step is PCR amplified using the above primer set to generate a series of PCR products with Barcode sequences.

[0142] PCR amplification used KOD FX Neo DNA Polymerase (Cat. No. KFX-101) from TOYOBO CO., LTD.

[0143] The PCR amplification reaction mixture was prepared according to the amount of DNA in the test sample. The amounts of reagents in the amplification reaction mixture were shown in Table 4.

[0144] Table 4

[0145] Reagents Volume / μL 2×KODFXneoBuffer 25×n dNTPMix(2mMeach) 10×n Primer Y5 1×n Primer Y4 1×n KODFXneo (1.0U / μl) 1×n <![CDATA[ddH2O]]> 2×n

[0146] Where n = number of samples tested + 1.

[0147] Add 10 μl of the UMI product from the previous step to 40 μl of the PCR amplification reaction mixture of each sample to be tested.

[0148] Carry out PCR amplification program in a PCR instrument. The amplification program is as follows:

[0149] In a PCR instrument, first denature at 98°C for 1 minute; then run 30 cycles as follows: denature at 98°C for 15 seconds, anneal and extend at 68°C for 10 minutes; finally extend at 72°C for 10 minutes; and store at 4°C.

[0150] After amplification, 2ul of each sample was taken and tested on a 1% DNA gel. The results were as follows: Figure 5 As shown in Figure 2, TBP gene can be effectively amplified using different samples as templates.

[0151] The product was purified using 0.8× purification magnetic beads.

[0152] Example 3

[0153] This embodiment uses the UMI-PCR method to amplify human mitochondrial DNA sequence and TBP gene DNA sequence, including the following steps:

[0154] (1) Primer design

[0155] The method is consistent with that of Example 1 and Example 2.

[0156] (2) DNA extraction

[0157] The method is consistent with that of Example 1.

[0158] (3) Add unique molecular label UMI

[0159] KOD FX Neo DNA Polymerase (Cat. No. KFX-101) from TOYOBO CO., LTD. was used to add a unique molecular tag UMI to the sample DNA using the above primer set.

[0160] The PCR amplification reaction mixture was prepared according to the amount of DNA in the test sample. The amounts of reagents in the amplification reaction mixture were shown in Table 5.

[0161] Table 5

[0162] Reagents Volume / μL 2×KODFXneoBuffer 12.5×n dNTPMix(2mMeach) 5×n Primer Y1 2.5×n Primer Y3 2.5×n KODFXneo (1.0U / μl) 0.5×n <![CDATA[ddH2O]]> 1×n

[0163] Where n = number of samples tested + 1.

[0164] Add 1 μl DNA (50 ng) to 24 μl PCR amplification reaction mixture of each sample to be tested.

[0165] Carry out PCR amplification program in a PCR instrument. The amplification program is as follows:

[0166] In a PCR instrument, first denature at 98°C for 1 minute; then run the following cycles 6 times: anneal at 70°C for 5 seconds (1°C lower per cycle); extend at 72°C for 10 minutes; and store at 4°C.

[0167] The product was purified using 0.8× purification magnetic beads.

[0168] (4) PCR amplification

[0169] The UMI product in the previous step is PCR amplified using the above primer set to generate a series of PCR products with Barcode sequences.

[0170] PCR amplification used KOD FX Neo DNA Polymerase (Cat. No. KFX-101) from TOYOBO CO., LTD.

[0171] The PCR amplification reaction mixture was prepared according to the amount of DNA in the test sample. The amounts of reagents in the amplification reaction mixture were shown in Table 6.

[0172] Table 6

[0173] Reagents Volume / μL 2×KODFXneoBuffer 25×n dNTPMix(2mMeach) 10×n Primer Y5 1×n Primer Y2 1×n Primer Y4 1×n KODFXneo (1.0U / μl) 1×n <![CDATA[ddH2O]]> 1×n

[0174] Where n = number of samples tested + 1.

[0175] Add 10 μl of the UMI product from the previous step to 40 μl of the PCR amplification reaction mixture of each sample to be tested.

[0176] Carry out PCR amplification program in a PCR instrument. The amplification program is as follows:

[0177] In a PCR instrument, first denature at 98°C for 1 minute; then run 30 cycles as follows: denature at 98°C for 15 seconds, anneal and extend at 68°C for 10 minutes; finally extend at 72°C for 10 minutes; and store at 4°C.

[0178] After amplification, 2ul of each sample was taken and tested on a 1% DNA gel. Using different samples as templates, the TBP gene can be effectively amplified.

[0179] The product was purified using 0.8× purification magnetic beads.

[0180] Example 4

[0181] like Figure 1 As shown, this embodiment uses the method of the present invention to construct a PacBio sequencing library, including the following steps:

[0182] (1) Primer design

[0183] The method is consistent with Example 3.

[0184] (2) DNA extraction

[0185] The method is consistent with Example 3.

[0186] (3) Add unique molecular label UMI

[0187] The method is consistent with Example 3.

[0188] (4) PCR amplification

[0189] The method is consistent with Example 3.

[0190] (5) Construction of PacBio sequencing library

[0191] Pacific Biosciences PacBio library construction was performed using the PacBio prep kit 3.0 kit (PacBio, Cat#102-141-700).

[0192] Qubit 3.0 was used to quantify the sequencing library.

[0193] (6) PacBio sequencing and analysis

[0194] According to the concentration of the sequencing library in the previous step, an appropriate volume of the library was reacted with the binding reagent (PacBio, Cat#101-820-200) and primers (PacBio, Cat#100-970-100) to prepare the final machine-ready library for PacBio library sequencing. Bioinformatics analysis of PacBio sequencing data was performed to analyze the coverage of the mitochondrial DNA library (e.g. Figure 6 ) and TBP gene sequencing data coverage (such as Figure 7 ), mitochondrial point mutations, large fragment deletions and other mutation results, and the UMI types of mitochondrial DNA and the UMI types of TBP genes were obtained at the same time. Different DNA templates from different sources were distinguished according to different UMI types, and the correct DNA fragments were traced to calculate the accurate mutation ratios of mitochondrial DNA point mutations, large fragment deletions and other mutation types. In addition, according to the ratio between the TBP gene UMI type and the mitochondrial DNA UMI type, the mitochondrial DNA copy number in the sample (such as Figure 8 ).

[0195] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A primer set for detecting mutations and copy number of human mitochondrial DNA, characterized in that: include: Primer Y1, primer Y2, primer Y3, primer Y4 and primer Y5; The nucleotide sequence of the primer Y1 is the sequence of the primer P5 with the UMI sequence and primer P5 added to the 5' end of the primer P1; The nucleotide sequence of the primer Y2 is a barcode sequence added to the 5' end of the primer P2; The nucleotide sequence of the primer Y3 is the sequence of the primer P5 with the UMI sequence and primer P5 added to the 5' end of the primer P3; The nucleotide sequence of the primer Y4 is a Barcode sequence added to the 5' end of the primer P4; The nucleotide sequence of the primer Y5 is a barcode sequence added to the 5' end of the primer P5; The primers P1 and P2 are a pair of specific primers for amplifying human mitochondrial DNA, the primers P3 and P4 are a pair of specific primers for amplifying human TBP gene, and the primer P5 is a universal primer; The nucleotide sequence of the primer P1 is shown as SEQ ID NO: 1, and the nucleotide sequence of the primer P2 is shown as SEQ ID NO: 2; The nucleotide sequence of the primer P3 is shown in SEQ ID NO: 3, and the nucleotide sequence of the primer P4 is shown in SEQ ID NO: 4; The nucleotide sequence of the primer P5 is shown in SEQ ID NO: 5; The nucleotide sequence of the primer Y1 is shown as SEQ ID NO: 6, and the nucleotide sequence of the primer Y3 is shown as SEQ ID NO:

7.

2. The primer set according to claim 1, characterized in that A protective base is added to the 5' end of the Barcode sequence.

3. A kit for detecting mutations and copy number of human mitochondrial DNA, characterized in that: include: The primer set according to any one of claims 1 to 2.

4. The kit according to claim 3, characterized in that Also includes: One or more of DNA extraction system, PCR reaction buffer, ddH2O, and DNA polymerase.

5. A method for detecting human mitochondrial DNA mutation and copy number for non-diagnostic purposes, characterized in that: The steps include: S1, extracting DNA from the sample to be tested; S2. Add UMI to the DNA extracted in step S1 using the primer set described in any one of claims 1 to 2 or the kit described in any one of claims 3 to 4; S3. Perform PCR amplification on the product to which the UMI is added in step S2 using the primer set described in any one of claims 1 to 2 or the kit described in any one of claims 3 to 4 to generate a PCR product with a barcode sequence; S4, construction of PacBio sequencing library; S5. Perform PacBio library sequencing and perform bioinformatics analysis on the PacBio sequencing data to obtain human mitochondrial DNA mutation and copy number results.

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