WO2024138714A1 - Procédé de purification fine pour complexe d'acide nucléique, kit de purification fine et application - Google Patents

Procédé de purification fine pour complexe d'acide nucléique, kit de purification fine et application Download PDF

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Publication number
WO2024138714A1
WO2024138714A1 PCT/CN2022/144250 CN2022144250W WO2024138714A1 WO 2024138714 A1 WO2024138714 A1 WO 2024138714A1 CN 2022144250 W CN2022144250 W CN 2022144250W WO 2024138714 A1 WO2024138714 A1 WO 2024138714A1
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nucleic acid
modified
sequencing
protein
complex
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PCT/CN2022/144250
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English (en)
Chinese (zh)
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王欧
刘珍君
师虓
郭斐
孔超娣
孙昊
曾涛
董宇亮
章文蔚
徐讯
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深圳华大生命科学研究院
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Priority to PCT/CN2022/144250 priority Critical patent/WO2024138714A1/fr
Publication of WO2024138714A1 publication Critical patent/WO2024138714A1/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
    • C12Q1/6869—Methods for sequencing

Definitions

  • the present invention relates to nanopore sequencing technology, and in particular to a purification method, a purification kit and applications of a nucleic acid complex.
  • Nanopore sequencing is an emerging sequencing technology in recent years. It has the characteristics of long read length, high throughput, no need for additional modification, and easy operation. It has been widely used in basic theoretical research in life sciences and biomedical clinical practice.
  • Nanopore sequencing is a sequencing technology based on electrical signals.
  • a (protein or solid) nanopore inserted in a membrane separates two electrolyte chambers. When voltage is applied between the two electrolyte chambers, a stable perforation current is generated. Different molecules entering the nanopore will hinder the flow of ions, which is called the nanopore signal.
  • the nanopore signal When ssDNA passes through the nanopore, the magnitude of the current obstruction will vary due to the different bases.
  • the adapter complex can help the DNA sequence to be tested to be effectively captured by the nanopore and start sequencing after applying voltage.
  • the motor protein on the adapter complex can effectively reduce and control the perforation movement of nucleic acid molecules, improve the accuracy of sequencing, and maintain sequencing speed and sequencing uniformity.
  • the preparation of this complex requires the combination of motor protein and a polynucleotide, and its purity is crucial to the stability of nanopore sequencing.
  • Components that fail to adsorb to the magnetic beads can be washed with a cleaning reagent containing high salt and polyethylene glycol, and then the polyethylene glycol is removed and the salt concentration of the solution is reduced to obtain the linker complex bound to the surface of the magnetic beads, thereby achieving a purification effect.
  • the current method for preparing the adapter complex can purify some non-target components, such as motor proteins that are not bound to polynucleotides, excess cross-linking agents, and other reaction solvents when preparing the complex.
  • some non-target components such as motor proteins that are not bound to polynucleotides, excess cross-linking agents, and other reaction solvents when preparing the complex.
  • the polynucleotide adapter that is not bound to the protein will still be retained.
  • This adapter with only polynucleotides can also be connected to the library, but the library product cannot be sequenced normally. Therefore, in the actual sequencing process, this byproduct will have an adverse effect on nanopore sequencing and directly affect the sequencing effect.
  • the present invention aims to provide a purification method, a purification kit and an application of a nucleic acid complex, so as to further improve the purity of the nucleic acid complex.
  • a method for purifying a nucleic acid complex comprises a polynucleotide fragment and a protein bound to the polynucleotide fragment, or the nucleic acid complex is a polynucleotide fragment in which at least part of the nucleotides are modified and lose the ability to complement and pair with nucleic acids or nucleic acid analogs.
  • the purification method comprises the following steps: S1, solidifying a nucleic acid or nucleic acid analog complementary to the protein binding sequence on the polynucleotide fragment or the nucleotide before the modified nucleotide is modified, to obtain a solid phase capture element; S2, incubating the nucleic acid complex to be purified with the solid phase capture element, and the polynucleotide fragments that are not bound to the protein or are not successfully modified are captured and removed by the solid phase capture element through base complementarity, to obtain a purified nucleic acid complex.
  • the protein includes a motor protein.
  • the protein comprises a helicase.
  • the nucleic acid complex includes a sequencing adapter.
  • the nucleic acid complex includes a nanopore sequencing adapter.
  • the solid phase carrier in the solid phase capture element includes magnetic beads and/or polyethylene porous plates.
  • nucleic acid or a nucleic acid analog complementary to a protein binding sequence on a polynucleotide fragment or a nucleotide before modification of the modified nucleotide is immobilized by covalent binding and/or affinity binding.
  • nucleic acid or nucleic acid analog complementary to the protein binding sequence on the polynucleotide fragment or the nucleotide before the modified nucleotide is modified is immobilized on the solid phase carrier by binding the streptavidin magnetic beads to the biotin-modified nucleic acid or nucleic acid analog chain.
  • nucleic acid or nucleic acid analog that is complementary to the protein binding sequence on the polynucleotide fragment or the nucleotide before the modified nucleotide is modified includes a 3'-labeled modified Biotin base or a 5'-labeled modified Biotin base.
  • nucleic acid complex at least part of the nucleotides are modified with polyethylene glycol.
  • a nucleic acid complex purification kit which comprises a solid phase capture element and a buffer reagent, wherein the solid phase capture element comprises a nucleic acid or nucleic acid analog that is fixed by a solid phase carrier and contains a nucleotide complementary to a protein binding sequence on a polynucleotide fragment of the nucleic acid complex or a modified nucleotide before modification.
  • FIG1 shows a purification process of affinity magnetic beads + complementary nucleic acid chains according to one embodiment of the present application, wherein polyA is a biotin-modified nucleic acid chain with a complementary sequence, and AD3 is a free linker that needs to be removed;
  • the present invention utilizes base complementarity to immobilize a section of nucleic acid or nucleic acid analogs that are complementary to the motor protein binding sequence on the polynucleotide adapter.
  • base complementarity After incubation with the adapter complex, the polynucleotide that is not bound to the motor protein will be immobilized through base complementarity, while the polynucleotide bound to the protein cannot be immobilized because the complementary sequence is bound by the motor protein, thereby separating the free polynucleotide and the polynucleotide bound to the protein, thereby achieving the purpose of further purifying the complex.
  • nucleic acids or nucleic acid analogs complementary to the protein binding sequence on the polynucleotide fragment or the nucleotides before the modified nucleotides are modified are immobilized by covalent binding and/or affinity binding methods; for example, nucleic acids or nucleic acid analogs complementary to the protein binding sequence on the polynucleotide fragment or the nucleotides before the modified nucleotides are modified include 3'-labeled modified Biotin bases or 5'-labeled modified Biotin bases; the nucleic acid complex is at least partially modified with polyethylene glycol.
  • Buffer C 20 mM Tris-HCl pH 7.5, 80 mM NaCl
  • Amino acid sequence of helicase DDA (SEQ ID NO: 4):
  • Buffer F 20 mM HEPES (pH 7.2), 50 mM NaCl
  • Buffer G 50mM HEPES (Ph2.0), 1000mM KCl, 4mM ATP, 20mM MgCl2
  • TMAD oxidant N, N, N′, N′-tetramethylazodicarbonamide
  • EDTA EDTA
  • Buffer J 50 mM Tris (pH 7.5), 2.5 M NaCl, 20% PEG8000
  • Buffer K 50 mM Tris (pH 7.5), 20 mM NaCl
  • Magnetic beads (VAHTSTM DNA Clean Beads #N411-03) were used to purify and remove free proteins, crosslinkers, non-specific binding of non-target complexes, etc. in the reaction system.
  • the specific operation steps are as follows: first take the magnetic beads out of the refrigerator in advance, shake and mix, and then place at room temperature. First, wash the magnetic beads with magnetic bead washing buffer Buffer H, so that the buffer in the commercial magnetic beads is replaced with the appropriate pH high salt buffer used in our purification conditions.
  • magnetic bead equilibration buffer Buffer I to equilibrate the magnetic beads, and then mix the equilibrated magnetic beads with the above-constructed optimized helicase DDA and linker AD complex at twice the input amount, place them in a low-absorption centrifuge tube, and mix and combine on a rotating shaker at room temperature for 1 hour. Then place the centrifuge tube on the magnetic rack for 10 minutes, wait until the magnetic beads are completely adsorbed to the side of the magnetic rack, and the solution becomes completely clear, and then carefully remove the supernatant. Then use the magnetic bead equilibration buffer Buffer J to wash the mixed solution of magnetic beads and complexes.
  • Buffer L 5 mM Tris (pH 7.5), 0.5 mM EDTA, 1 M NaCl
  • Magnetic beads (Dynabeads_M280_Streptavidin) from the refrigerator in advance, shake and mix, place at room temperature, place in a low-absorption centrifuge tube and then place on the magnetic rack. After the magnetic beads are completely adsorbed to the side of the magnetic rack and the solution becomes completely clear, carefully remove the supernatant. Then wash the magnetic beads twice with an equal volume of magnetic bead washing buffer Buffer L, and then soak the balanced magnetic beads in 100ul Buffer L. According to the instructions of the commercial magnetic beads, the mass concentration of the magnetic beads is 10mg/ml.
  • the solution was placed on a magnetic rack, and after the magnetic beads were completely adsorbed to the side of the magnetic rack and the solution became completely clear, the supernatant was carefully removed, and the supernatant was collected and 2 ⁇ L of the purified product was quantified using the Qubit ssDNA detection kit (Thermofisher, Q10212), which was recorded as value b. Then the magnetic beads combined with the modified bases were washed twice with Buffer L, and after each thorough mixing, the magnetic beads were completely adsorbed to the side of the magnetic rack, and the solution became completely clear, and the supernatant was carefully removed.
  • the Qubit ssDNA detection kit Thermofisher, Q10212
  • the supernatant was collected for the last time, and 2 ⁇ L of the purified product was quantified using the Qubit ssDNA detection kit (Thermofisher, Q10212), which was recorded as value c. Then, an appropriate amount of the complex obtained in Example 3 was added to the magnetic beads combined with the modified bases, mixed and incubated at room temperature for 1 hour, and then placed on the side of the magnetic stand. After the solution became completely clear, the supernatant was aspirated, and 2 ⁇ L of the purified product was quantified using the Qubit dsDNA HS kit (Thermofisher, Q32854).
  • (1-b/a)*400pmol is the binding capacity of 1mg magnetic beads and biotin modified bases in this method.
  • the value is 302.4pmol, which is greater than the reported capacity of the commercial magnetic beads for biotin markers (200pmol/mg), and the value c is very low and not within the detection range. Therefore, it can be explained that the magnetic beads have fully combined with the biotin modified bases and have successfully removed the remaining free bases in the solution.
  • the comparison of the gel images of the crude and pure products of the complex shows that after purification, the free linkers in the complex solution are significantly reduced.
  • Magnetic beads (Dynabeads_M280_Streptavidin) from the refrigerator in advance, shake and mix, place at room temperature, place in a low-absorption centrifuge tube and then place on the magnetic rack. After the magnetic beads are completely adsorbed to the side of the magnetic rack and the solution becomes completely clear, carefully remove the supernatant. Then wash the magnetic beads twice with an equal volume of magnetic bead washing buffer Buffer L, and then soak the balanced magnetic beads in 100ul Buffer L. According to the instructions of the commercial magnetic beads, the mass concentration of the magnetic beads is 10mg/ml.
  • the solution was placed on a magnetic rack, and after the magnetic beads were completely adsorbed to the side of the magnetic rack and the solution became completely clear, the supernatant was carefully removed, and the supernatant was collected and 2 ⁇ L of the purified product was quantified using the Qubit ssDNA detection kit (Thermofisher, Q10212), which was recorded as value b. Then the magnetic beads combined with the modified bases were washed twice with Buffer L, and after each thorough mixing, the magnetic beads were completely adsorbed to the side of the magnetic rack, and the solution became completely clear, and the supernatant was carefully removed.
  • the Qubit ssDNA detection kit Thermofisher, Q10212
  • the supernatant was collected for the last time, and 2 ⁇ L of the purified product was quantified using the Qubit ssDNA detection kit (Thermofisher, Q10212), which was recorded as value c. Then, an appropriate amount of the complex obtained in Example 3 was added to the magnetic beads combined with the modified bases, mixed and incubated at room temperature for 1 hour, and then placed on the side of the magnetic stand. After the solution became completely clear, the supernatant was aspirated, and 2 ⁇ L of the purified product was quantified using the Qubit dsDNA HS kit (Thermofisher, Q32854).
  • (1-b/a)*400pmol is the binding capacity of 1mg magnetic beads and biotin-modified bases in this method.
  • the value is 278.8pmol, which is greater than the reported capacity of the commercial magnetic beads for biotin markers (200pmol/mg), and the value c is very low and not within the detection range. Therefore, it can be explained that the magnetic beads have fully combined with the biotin-modified bases and have successfully removed the remaining free bases in the solution.
  • the comparison of the gel images of the crude and pure products of the complex shows that after purification, the free linkers in the complex solution are significantly reduced.
  • genomic DNA extracted from Escherichia coli was used to prepare a library for sequencing.
  • Pipette 240 ⁇ L of magnetic beads and add them to the reacted sample. Mix by flicking the tube wall with your hand, or gently pipette at least 6 times with a widened pipette tip until completely mixed. The last time should ensure that all the liquid and magnetic beads in the pipette tip are injected into the tube. Incubate at room temperature for 5 minutes on a rotary mixer. Centrifuge the DNA LoBind Microcentrifuge Tube (Eppendorf, 0030108051) for a short time and place it on a magnetic rack. Let it stand for 2-5 minutes until the liquid is clear. Use a pipette to carefully aspirate the supernatant and discard it.

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Abstract

L'invention concerne un procédé de purification fine pour un complexe d'acide nucléique, un kit de purification fine et une application. Le complexe d'acide nucléique comprend un fragment polynucléotidique et une protéine se liant au fragment polynucléotidique, ou le complexe d'acide nucléique est un fragment polynucléotidique dont au moins une partie du nucléotide est modifiée pour perdre la capacité d'appariement complémentaire d'un acide nucléique ou d'un analogue d'acide nucléique. Le procédé de purification comprend les étapes suivantes : S1, l'immobilisation d'un acide nucléique ou d'un analogue d'acide nucléique comprenant un nucléotide complémentaire d'une séquence de liaison de protéine ou d'un nucléotide modifié avant la modification sur un fragment de polynucléotide, pour obtenir un élément de capture de phase solide ; S2, l'incubation d'un complexe d'acide nucléique à purifier et de l'élément de capture de phase solide, et la capture et l'élimination, par l'élément de capture de phase solide et au moyen d'une complémentation de bases, d'un fragment de polynucléotide qui ne se lie pas à la protéine ou n'est pas modifié avec succès, pour obtenir un complexe d'acide nucléique purifié. Un complexe lieur purifié au moyen de la complémentation de séquence est plus pur qu'un complexe ne subissant pas le procédé de purification, et une banque construite en utilisant le complexe comme matière première présente un meilleur effet de séquençage.
PCT/CN2022/144250 2022-12-30 2022-12-30 Procédé de purification fine pour complexe d'acide nucléique, kit de purification fine et application WO2024138714A1 (fr)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20180340157A1 (en) * 2015-11-25 2018-11-29 Genia Technologies, Inc. Purification of polymerase complexes
CN110088299A (zh) * 2016-09-29 2019-08-02 牛津纳米孔技术公司 通过纳米孔指导的核酸检测方法
CN113462764A (zh) * 2021-09-01 2021-10-01 北京齐碳科技有限公司 用于表征双链靶多核苷酸的类发夹衔接子、构建体和方法
US20210381041A1 (en) * 2018-05-28 2021-12-09 Roche Sequencing Solutions, Inc. Enzymatic Enrichment of DNA-Pore-Polymerase Complexes
CN113862264A (zh) * 2021-12-03 2021-12-31 北京齐碳科技有限公司 用于靶多核苷酸测序的衔接子、构建体、方法和应用

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20180340157A1 (en) * 2015-11-25 2018-11-29 Genia Technologies, Inc. Purification of polymerase complexes
CN110088299A (zh) * 2016-09-29 2019-08-02 牛津纳米孔技术公司 通过纳米孔指导的核酸检测方法
US20210381041A1 (en) * 2018-05-28 2021-12-09 Roche Sequencing Solutions, Inc. Enzymatic Enrichment of DNA-Pore-Polymerase Complexes
CN113462764A (zh) * 2021-09-01 2021-10-01 北京齐碳科技有限公司 用于表征双链靶多核苷酸的类发夹衔接子、构建体和方法
CN113862264A (zh) * 2021-12-03 2021-12-31 北京齐碳科技有限公司 用于靶多核苷酸测序的衔接子、构建体、方法和应用

Non-Patent Citations (1)

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Title
WANG YUNHAO; ZHAO YUE; BOLLAS AUDREY; WANG YURU; AU KIN FAI: "Nanopore sequencing technology, bioinformatics and applications", NATURE BIOTECHNOLOGY, NATURE PUBLISHING GROUP US, NEW YORK, vol. 39, no. 11, 1 November 2021 (2021-11-01), New York, pages 1348 - 1365, XP037616214, ISSN: 1087-0156, DOI: 10.1038/s41587-021-01108-x *

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