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WO2023108865A1 - Paire d'amorces, kit et procédé de détection de la mutation du gène de la boucle mitochondriale - Google Patents

Paire d'amorces, kit et procédé de détection de la mutation du gène de la boucle mitochondriale Download PDF

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Publication number
WO2023108865A1
WO2023108865A1 PCT/CN2022/074506 CN2022074506W WO2023108865A1 WO 2023108865 A1 WO2023108865 A1 WO 2023108865A1 CN 2022074506 W CN2022074506 W CN 2022074506W WO 2023108865 A1 WO2023108865 A1 WO 2023108865A1
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Prior art keywords
primer pair
mitochondrial
detecting
volume
sequencing
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PCT/CN2022/074506
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English (en)
Chinese (zh)
Inventor
伍建
姬晓雯
武璇
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Mygenostics (chongqing) Gene Technology Co Ltd
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Mygenostics (chongqing) Gene Technology Co Ltd
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6869—Methods for sequencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material

Definitions

  • the invention relates to the field of molecular biology, in particular to a pair of primers, a kit and a detection method for detecting mutations of mitochondrial ring genes.
  • Mitochondria are important sites for energy metabolism, and mutations in the mitochondrial genome or genes encoding mitochondrial proteins on the nuclear genome may lead to disease.
  • Three typical phenotypes caused by mtDNA deletion are Kearns-Sayre syndrome (KSS), Pearson syndrome and progressive external ophthalmoplegia (PEO), and very rare Leigh syndrome.
  • KSS Kearns-Sayre syndrome
  • PEO progressive external ophthalmoplegia
  • the problem to be solved in the present invention is: how to provide a primer pair, a kit and a detection method for detecting the mutation of the mitochondrial ring gene, so as to solve the inconvenience and detection of the mutation of the mitochondrial ring gene at present. difficult question.
  • the present invention adopts the following technical solutions:
  • a pair of primers for detecting the mutation of the mitochondrial ring gene is the primer pair M4 or the primer pair M5;
  • the primer pair M4 is:
  • M4-F AAGTGAACTGTATCCGACATCTGGTTCC
  • M4-R TAGCTACCCCCAAGTGTTATGGGC
  • the primer pair M5 is:
  • M5-F ATAAAGCCTAAATAGCCCACACGTTCC
  • M5-R GACCCTGAAGTAGGAACCAGATGTCG.
  • the present invention also provides a kit for detecting the mutation of the mitochondrial loop gene, including the above-mentioned primer pair.
  • the kit includes: 2 parts by volume of PCR amplification solution, 4 parts by volume of 2.5M dNTP, 4 parts by volume of 5M betaine, 0.2 parts by volume of TaKaRa LA Taq HS, 7.8 parts by volume of nuclease-free Purified water and 1 part by volume of the primer pair described in claim 1.
  • the present invention also provides a non-disease diagnosis detection method for detecting mitochondrial loop gene mutation, comprising:
  • DNA is extracted, and the above-mentioned primer pair is used to amplify long fragments of mtDNA;
  • the product amplified in the step (1) is subjected to quality inspection, and a specificity is selected from the products amplified by the primer pair M4 and M5.
  • the step (2) is preferably carried out.
  • the quality inspection includes: using 0.8% agarose gel to perform quality inspection on the products amplified in the step (1), so that each sample has at least one comparison between the products amplified by the primer pair M4 and M5 Marker can observe a single specific band in the target area, if not, repeat the step (1).
  • the conditions for carrying out long-fragment amplification of mtDNA in the step (1) are:
  • performing quantification and quality control on the sequencing library in the step (2) includes: the concentration of the sequencing library is greater than 5 ng/ ⁇ L, the volume is greater than 10 ul, and the fragment length of the sequencing library is 200-500 bp.
  • the present invention also provides the application of the above-mentioned primer pair for detecting the mutation of the mitochondrial ring gene in the preparation of a reagent for detecting the mutation of the mitochondrial ring gene.
  • the present invention also provides the application of the above-mentioned kit for detecting mitochondrial ring gene mutations in the preparation of reagents for detecting mitochondrial ring gene mutations.
  • the beneficial effect of the present invention is that: the primer pair, kit and detection method for detecting the mutation of the mitochondrial ring gene provided by the present invention can quickly detect the mutation of the mitochondrial ring gene with a high degree of reliability.
  • Fig. 1 is an agarose gel diagram of mtDNA amplification of primer pair M4 and M5.
  • the design idea of the present invention first extract the genomic DNA (gDNA) in the whole blood sample, carry out specific mitochondrial genome (mtDNA) long fragment amplification on the extracted gDNA, then perform high-throughput sequencing on the mtDNA, and use biological information analysis software Analyze the sequencing data to obtain information about mtDNA variations (SV, Indel, SNPs).
  • gDNA genomic DNA
  • mtDNA mitochondrial genome
  • the blood sample was extracted using the Magnetic Bead Method Universal Genomic DNA Extraction Kit (DP705-02) produced by Tiangen Biochemical Technology (Beijing) Co., Ltd.
  • the following DNA extraction process is the standard process in the kit:
  • Reagent preparation Commercially available PCR kits were used for PCR.
  • the brand of the kit used in this example is TAKARA, and the name is TaKaRa LA Hot Start Version, the article number is RR042Q.
  • test samples need to be amplified by PCR with two pairs of primers M4 and M5 respectively, so two systems need to be prepared.
  • N number of samples to be tested + 1.
  • Amplification The amplification conditions are as follows:
  • each sample uses primers M4 and M5 for amplification products with at least one comparison marker that can observe a single marker in the target area Specific bands, if not the DNA was re-extracted and PCR amplified. From the amplification products of the samples using primers M4 and M5, a PCR amplification product with good specificity and high yield was selected for subsequent mitochondrial DNA purification. According to Figure 1, the amplification product of M5 was used for subsequent experiments.
  • the library preparation steps are as follows:
  • the library was prepared using a self-produced kit of our company, the name is Universal Library Preparation Kit (Illumina) (enzyme cutting method) V4.1, and the catalog number is MG0077/96.
  • nucleic acid fragmentation prepare the nucleic acid fragmentation reaction system according to the following table
  • Element volume 10 ⁇ Fragmentation Buffer V2 2.5 ⁇ l Fragmentation Mixed Enzyme V2 2.5 ⁇ l mt DNA amplification product (purified) 100-500ng Enzyme-free water Rehydrate to 25 ⁇ l
  • step temperature control time 1 4°C 1min 2 32°C 30min 3 65°C 30min 4 4°C HOLD
  • connection product The magnetic beads produced by the company are used for purification.
  • step 6.6 once, and thoroughly blot the residual liquid at the bottom of the tube;
  • Different Index biological labels
  • the biological labels used must match the precise blocking agent. If the UMI connector is used, the biological label needs to be used corresponding to the UMI connector, for example, Index I553 corresponds to A553.
  • the number of PCR cycles depends on the amount of DNA loaded and the quality of the DNA. It is recommended to use 6-9 cycles: 6 cycles are recommended for 500ng DNA loading; 9 cycles are recommended for 100ng DNA loading.
  • step 5.6 Repeat step 5.6 once, and thoroughly blot the residual liquid at the bottom of the tube;
  • the quantified products can be directly sequenced using a high-throughput sequencing platform.
  • High-throughput sequencing uses a sequencer and performs sequencing according to the standard operating procedures of the sequencer.
  • SNP, CNV, SV analysis and sample report comparison The analysis results of 8 samples and their comparison with the capture results are shown in the table below.
  • the detection results of 2 negative samples were still negative for SNPIndel and CNV, and the results were consistent; the detection results of 4 point mutation samples were consistent with the capture method; the results of the two algorithms for CNV analysis of 2 missing samples were inconsistent, and the CNRs were all detected deletions. It shows the deletion, but the specific location of the deletion is different; the SV analysis results of 2 missing samples show a high consistency with the capture results, and the reliability is high.

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  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Physics & Mathematics (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Biophysics (AREA)
  • Analytical Chemistry (AREA)
  • Biomedical Technology (AREA)
  • Immunology (AREA)
  • Plant Pathology (AREA)
  • Pathology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

La présente invention concerne une paire d'amorces, un kit et un procédé de détection de la mutation du gène de la boucle mitochondriale. La paire d'amorces est une paire d'amorces M4 ou une paire d'amorces M5, la paire d'amorces M4 étant : M4-F : AAGTGAACTGTATCCGACATCTGGTTCC, et M4-R: TAGCTACCCCCAAGTGTTATGGGC ; et la paire d'amorces M5 est : M5-F : ATAAAGCCTAAATAGCCCACACGTTCC, et M5-R : GACCCTGAAGTAGGAACCAGATGTCG. Une banque de séquençage est préparée en extrayant de l'ADN, en effectuant une amplification à long fragment sur l'ADNmt à l'aide de la paire d'amorces, et en effectuant une fragmentation, une réparation terminale, une liaison, une purification et un enrichissement par PCR sur le produit amplifié ; la quantification et le contrôle qualité sont effectués sur la banque de séquençage ; la banque de séquençage quantifiée est soumise à un séquençage haut débit à l'aide d'un séquenceur de gènes pour obtenir des données de séquençage ; et les données de séquençage sont analysées à l'aide d'un logiciel d'analyse de bioinformation pour obtenir des informations relatives à la mutation de l'ADNmt.
PCT/CN2022/074506 2021-12-14 2022-01-28 Paire d'amorces, kit et procédé de détection de la mutation du gène de la boucle mitochondriale Ceased WO2023108865A1 (fr)

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CN202111529140.4A CN114350778A (zh) 2021-12-14 2021-12-14 用于检测线粒体环基因突变的引物对、试剂盒及检测方法
CN202111529140.4 2021-12-14

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Cited By (1)

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CN118792393A (zh) * 2024-06-18 2024-10-18 北京百安济民医学科技有限公司 一种用于人线粒体dna突变检测和拷贝数检测的引物组、试剂盒及检测方法

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CN106701903A (zh) * 2015-11-17 2017-05-24 安诺优达基因科技(北京)有限公司 一种用于检测线粒体异质性的试剂盒及检测方法

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CN111621552A (zh) * 2019-06-13 2020-09-04 中国科学院广州生物医药与健康研究院 检测mtDNA突变的方法和系统
WO2021055431A1 (fr) * 2019-09-16 2021-03-25 Cornell University Séquençage d'adn mitochondrial humain par amplification ciblée de sondes multiplex (mtdna-stamp)
CN111172157A (zh) * 2020-01-22 2020-05-19 福州福瑞医学检验实验室有限公司 人类单细胞线粒体高通量测序文库的构建方法以及用于文库构建的试剂盒

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN118792393A (zh) * 2024-06-18 2024-10-18 北京百安济民医学科技有限公司 一种用于人线粒体dna突变检测和拷贝数检测的引物组、试剂盒及检测方法

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