WO2023077720A1 - Procédé de détection de l'activité d'une enzyme de métabolisation d'acide nucléique - Google Patents

Procédé de détection de l'activité d'une enzyme de métabolisation d'acide nucléique Download PDF

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Publication number
WO2023077720A1
WO2023077720A1 PCT/CN2022/086204 CN2022086204W WO2023077720A1 WO 2023077720 A1 WO2023077720 A1 WO 2023077720A1 CN 2022086204 W CN2022086204 W CN 2022086204W WO 2023077720 A1 WO2023077720 A1 WO 2023077720A1
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WIPO (PCT)
Prior art keywords
nucleic acid
metabolizing enzyme
acid metabolizing
activity
detection method
Prior art date
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Ceased
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PCT/CN2022/086204
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English (en)
Chinese (zh)
Inventor
周蓉
王攀
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Me Instruments Inc
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Me Instruments Inc
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Publication of WO2023077720A1 publication Critical patent/WO2023077720A1/fr
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    • 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
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]
    • 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
    • C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase
    • C12Q1/485—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase involving kinase

Definitions

  • the invention relates to the field of biotechnology, in particular to a method for detecting the activity of nucleic acid metabolizing enzymes.
  • Nucleic acid metabolism including DNA and RNA synthesis and degradation, is fundamental to all nucleic acid research and related life science fields of study.
  • Standard activity assays for nucleic acid metabolizing enzymes involved in DNA replication and repair are by assays that measure DNA or RNA product synthesis or degradation of radioactive or fluorescently labeled nucleic acid substrates.
  • the method of isotope labeling and the fluorescent dye method combined with DNA are widely used in DNA quantification, and then applied in the activity detection method of nucleic acid metabolizing enzymes to measure the synthesis activity or degradation activity of nucleic acid metabolizing enzymes.
  • polyacrylamide gel electrophoresis is widely used to analyze substrates, intermediates
  • PAGE polyacrylamide gel electrophoresis
  • further characterization and standard determination of nucleases are carried out.
  • the analysis method of polyacrylamide gel electrophoresis has the disadvantages of long experiment time, low efficiency and low throughput. Such shortcomings limit the scope of nuclease analysis.
  • the invention provides a method for detecting the activity of nucleic acid metabolizing enzymes.
  • the technical scheme adopted by the present invention to solve its technical problems is: a kind of nucleic acid metabolizing enzyme activity detection method, it is characterized in that: its steps are as follows:
  • steps S2 and S3 making a standard curve, and substituting the elongation efficiency calculated in S4 into the standard curve to obtain the activity of the nucleic acid metabolizing enzyme to be determined; wherein the PCR reaction conditions in steps S2 and S3 are that the temperature is 10°C-90°C, and the time is 10s- 60min.
  • the template is a single-stranded nucleic acid fragment with a fragment length of 50-150 bp.
  • the length of the primer is 15-25 bp.
  • the reaction conditions of the step S2 are a temperature of 45-65° C. and a time of 1 min.
  • the reaction conditions of the step S3 are a temperature of 72° C. and a time of 20 min.
  • the preparation method of the standard curve is: in step S2, the nucleic acid metabolizing enzyme to be determined is changed to a nucleic acid metabolizing enzyme of known activity that is diluted into different concentrations according to the activity gradient, and then sequentially undergoes steps S2, S3, and S4 The initial slope of each gradient unit activity was obtained, and a standard curve was made.
  • the nucleic acid metabolizing enzyme to be determined is diluted into different concentrations of nucleic acid metabolizing enzyme according to the concentration gradient, and then each concentration of nucleic acid metabolizing enzyme is successively passed through steps S2, S3, and S4 to obtain the activity of each concentration unit The initial slope and substituted into the standard curve yielded relative activity units.
  • the capillary electrophoresis analysis is calculated based on the type of fluorescent dye and the peak size of the separation spectrum.
  • the nucleic acid metabolizing enzymes include high-throughput nucleic acid metabolizing enzymes.
  • the fluorescently modified nucleotide derivatives and fluorescently modified nucleotide derivative analogs are nucleotide derivatives modified with protecting groups at the 3' hydroxyl and carrying fluorescent labeling groups on the bases.
  • the beneficial effects of the present invention are: the present invention is quick to operate, the activity detection result is accurate, the sensitivity is high, the detection of high flux is realized, and the characterization precision accurate to a single nucleotide can be realized.
  • high flux capillary electrophoresis fluorescent marker Nucleic acid substrates, intermediates and products are separated by size and charge, and/or detected by laser excitation, detection sample injection, gel electrophoresis and data acquisition processes are all automated, allowing a single experiment to be performed within one hour 96 samples can be detected, which is applicable to the detection of all templates with or without fluorescent dyes, and also solves the problems of multiple operation steps, complex design of multiple fluorescent labels, environmental pollution caused by isotope labeling, and limitation of instrument resolution in the prior art. restrictions etc.
  • Fig. 1 is a schematic diagram of the principle of the present invention
  • Fig. 2 is the electrophoretic spectrum that the embodiment of the present invention 1 obtains
  • "extending" means linking the modified nucleotide to the 5' phosphate group of the fluorescently modified nucleotide derivative by forming a phosphodiester bond with the 5' phosphate group of the fluorescently modified nucleotide derivative.
  • the free 3' hydroxyl, the second nucleotide to which the modified nucleotide is attached usually occurs at the 3' end of the polynucleotide chain.
  • Extension may be at low temperature and/or over a wider temperature range, may be in a short time and/or over a wider time range, reaction with fluorescently modified nucleotide derivatives or analogs ability. In the present invention, “extension” may be the ability to respond when a lower concentration of a fluorescently modified nucleotide or analogue is used as a substrate.
  • fluorescence-modified nucleotide derivative derivatives and “fluorescence-modified nucleotide derivative analogs” refer to those that have been modified by the protecting group at the 3' sugar hydroxyl group and carry fluorescent labels on the bases. Nucleotide derivatives of the group. Such nucleotide derivatives or analogs can act as chain reaction terminators, preventing the chain reaction from continuing after the addition of dNTPs. These terms are used interchangeably.
  • the template is preferably a single-stranded nucleic acid fragment with a length of 99 bp, and the primer length is preferably 16-19 bp;
  • the capillary electrophoresis instrument used is any instrument that can perform accounting capillary electrophoresis analysis, such as QSEP, CE etc.;
  • the diluent used to dilute the synthesized primers and templates during the reaction can be sterile purified water or 1XTE (pH8.0); Invention principle as shown in Figure 1,
  • TGATCCCGCGACGACTTT (SEQ ID NO.3) TGATCCCGCGACGACTTTG (SEQ ID NO.4) TGATCCCGCGACGACTTTGAATT (SEQ ID NO.5)
  • the elongation efficiency per unit time is obtained, as shown in Figure 2, and then by changing the concentration gradient of the added nucleic acid metabolizing enzyme, the concentration of the nucleic acid metabolizing enzyme at different concentrations can be obtained.
  • the extension efficiency of the nucleic acid metabolism enzyme can be calculated by substituting it into the standard curve.

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

Abstract

La présente invention concerne un procédé de détection de l'activité d'une enzyme métabolisant les acides nucléiques, comprenant les étapes suivantes : S1) Synthèse artificielle d'un fragment d'acide nucléique simple brin et d'une amorce complémentaire de l'extrémité 3', et formation d'une matrice au moyen d'une réaction de recuit ; S2) réaction de la matrice avec une solution tampon de réaction, un dérivé de nucléotide modifié par fluorescence ou un analogue de dérivé de nucléotide modifié par fluorescence, et une enzyme métabolisant l'acide nucléique à détecter, et purification en vue d'obtenir un produit primaire purifié ; S3) réaction du produit purifié primaire avec une solution tampon de réaction Taq, du dNTP et de l'ADN polymérase Taq, et purification pour obtenir un produit purifié secondaire ; S4) analyse du produit purifié secondaire à l'aide d'un instrument d'électrophorèse capillaire ; et S5) génération d'une courbe standard, et substitution du résultat de S4) dans la courbe standard pour obtenir un résultat de détection.
PCT/CN2022/086204 2021-11-03 2022-04-12 Procédé de détection de l'activité d'une enzyme de métabolisation d'acide nucléique Ceased WO2023077720A1 (fr)

Applications Claiming Priority (2)

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CN202111296052.4A CN114250281B (zh) 2021-11-03 2021-11-03 一种核酸代谢酶活性检测方法
CN202111296052.4 2021-11-03

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CN (1) CN114250281B (fr)
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Cited By (1)

* Cited by examiner, † Cited by third party
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CN117778523A (zh) * 2024-02-26 2024-03-29 苏州近岸蛋白质科技股份有限公司 一种Poly(A)聚合酶活性测定方法

Families Citing this family (1)

* Cited by examiner, † Cited by third party
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CN114250281B (zh) * 2021-11-03 2024-12-17 深圳铭毅智造科技有限公司 一种核酸代谢酶活性检测方法

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CA2233701A1 (fr) * 1996-08-12 1998-02-19 Clas Kallander Methode d'analyse
US20060057596A1 (en) * 2004-09-16 2006-03-16 Keener William K Activity-based assay for ricin-like toxins
WO2008024052A1 (fr) * 2006-08-24 2008-02-28 Rönnerbol International Ab Procédé et kit permettant la détermination d'une activité enzymatique impliquée dans la production métabolique d'un désoxynucléoside triphosphate et leur utilisation
US20120070838A1 (en) * 2010-08-20 2012-03-22 Life Technologies Corporation Polymerase Assay with a FRET Substrate
WO2015058104A1 (fr) * 2013-10-18 2015-04-23 The University Of Utah Research Foundation Procédés pour déterminer une activité de polymérase
CN114250281A (zh) * 2021-11-03 2022-03-29 深圳铭毅智造科技有限公司 一种核酸代谢酶活性检测方法

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CA2867916C (fr) * 2012-03-22 2020-05-26 Lgc Genomics Limited Systeme de detection de reaction en chaine de la polymerase utilisant des oligonucleotides comprenant un groupe phosphorothioate
CN104293917B (zh) * 2013-06-17 2016-03-02 北京大学 用于具有3’-5’外切活性的核酸酶检测的硫代探针
CN107541508A (zh) * 2016-06-24 2018-01-05 广州康昕瑞基因健康科技有限公司 模板‑引物核酸分子、聚合酶活性测定方法及试剂盒
CN106987643A (zh) * 2017-05-05 2017-07-28 广州和实生物技术有限公司 一种 Taq DNA 聚合酶活性检测方法
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CA2233701A1 (fr) * 1996-08-12 1998-02-19 Clas Kallander Methode d'analyse
US20060057596A1 (en) * 2004-09-16 2006-03-16 Keener William K Activity-based assay for ricin-like toxins
WO2008024052A1 (fr) * 2006-08-24 2008-02-28 Rönnerbol International Ab Procédé et kit permettant la détermination d'une activité enzymatique impliquée dans la production métabolique d'un désoxynucléoside triphosphate et leur utilisation
US20120070838A1 (en) * 2010-08-20 2012-03-22 Life Technologies Corporation Polymerase Assay with a FRET Substrate
WO2015058104A1 (fr) * 2013-10-18 2015-04-23 The University Of Utah Research Foundation Procédés pour déterminer une activité de polymérase
CN114250281A (zh) * 2021-11-03 2022-03-29 深圳铭毅智造科技有限公司 一种核酸代谢酶活性检测方法

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CN117778523A (zh) * 2024-02-26 2024-03-29 苏州近岸蛋白质科技股份有限公司 一种Poly(A)聚合酶活性测定方法
CN117778523B (zh) * 2024-02-26 2024-05-28 苏州近岸蛋白质科技股份有限公司 一种Poly(A)聚合酶活性测定方法

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