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WO2023240564A1 - Réactif d'excision contenant un tampon glycine-naoh et son utilisation dans le séquençage d'acides nucléiques - Google Patents

Réactif d'excision contenant un tampon glycine-naoh et son utilisation dans le séquençage d'acides nucléiques Download PDF

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Publication number
WO2023240564A1
WO2023240564A1 PCT/CN2022/099260 CN2022099260W WO2023240564A1 WO 2023240564 A1 WO2023240564 A1 WO 2023240564A1 CN 2022099260 W CN2022099260 W CN 2022099260W WO 2023240564 A1 WO2023240564 A1 WO 2023240564A1
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Prior art keywords
sequencing
excision
reagent
excision reagent
blocking group
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Ceased
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PCT/CN2022/099260
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English (en)
Chinese (zh)
Inventor
贾曼
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MGI Tech Co Ltd
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MGI Tech Co Ltd
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Priority to CN202280096939.5A priority Critical patent/CN119343466A/zh
Priority to PCT/CN2022/099260 priority patent/WO2023240564A1/fr
Publication of WO2023240564A1 publication Critical patent/WO2023240564A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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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 invention belongs to the field of sequencing, and specifically relates to an excision reagent containing glycine-NaOH buffer, a kit containing the same and its application in polynucleotide sequencing.
  • First-generation sequencing is a sequencing technology based on the Sanger method.
  • second-generation sequencing is based on massively parallel sequencing technology, which can simultaneously complete the synthesis of complementary strands of the sequencing template and the acquisition of sequence data.
  • Third-generation sequencing is based on single-molecule sequencing and massively parallel sequencing (MPS) technology.
  • Next-generation sequencing is the current mainstream sequencing technology.
  • second-generation sequencing technology includes three consecutive steps: 1. Add dNTPs to the sequencing reaction system; 2. Detect the base type of dNTP added to the sequencing system; 3. Remove various enzymes and fluorescence in the reaction. Label or dNTP 3' end hindering group and other elution reactions. This series of consecutive steps achieves "sequencing while synthesising".
  • the current mainstream mcMPS (multi-copy base signal acquisition unit MPS) technology is based on the "discontinuous polymerization sequencing method" to realize the reading of nucleotide sequences.
  • MPS multi-copy base signal acquisition unit
  • the “base extension” of different nucleic acid molecules within the “unit” is not completely synchronized. There may be individual nucleic acid molecules that do not undergo the expected polymerization extension or that have more than one unexpected polymerization extension reaction.
  • MGISEQ-2000RS high-throughput rapid sequencing reagent set is fast and efficient, and can complete sequencing requirements in a short time. It is more suitable for samples with small data volume and urgent samples, especially for tumor detection.
  • MGISEQ-2000RS high-throughput sequencing kit uses combined probe anchored polymerization technology (cPAS) to polymerize DNA molecular anchors and fluorescent probes on DNA nanospheres (DNB), and uses a high-resolution imaging system to detect light The signal is collected, and the optical signal is digitally processed to obtain high-quality and high-accuracy sample sequence information.
  • cPAS combined probe anchored polymerization technology
  • DDB DNA nanospheres
  • the MGISEQ-2000RS high-throughput sequencing kit will also cause runon and lag phenomena during the sequencing process. Therefore, the rapid and effective removal of the 3' end blocking group is crucial to the speed and accuracy of sequencing. Importantly, the kit components need to be optimized to increase the speed and completeness of resection.
  • the present invention provides an excision reagent containing glycine-NaOH buffer and its application in nucleic acid sequencing.
  • an alkaline buffer solution is more conducive to the excision, that is, to speed up the excision and the completeness of the excision, thereby reducing the lead and lag phenomena in the sequencing process, especially hysteresis.
  • the Tris-HCl buffer system is used as the excision solution, which cannot meet the strong alkaline requirements, that is, the pH is greater than 9.0.
  • This invention applies the glycine-sodium hydroxide buffer system to the excision reagent for sequencing for the first time to speed up the excision efficiency and improve the sequencing quality.
  • the use of this kind of reagent in sequencing kits will improve the sequencing quality of the product and increase the product value and market share.
  • an excision reagent which includes a glycine-sodium hydroxide buffer.
  • the glycine-sodium hydroxide buffer further contains a 3' end blocking group cleaving agent.
  • the 3' end blocking group excluding agent is, for example, THPP, TCEP or DTT.
  • the excision reagent is located in hole No. 9 of the sequencing reagent tank.
  • the pH range of the glycine-sodium hydroxide buffer is greater than 9, specifically 9-12, more specifically 9.6-10.6, such as 9.6, 10 or 10.6.
  • the pH of the glycine-sodium hydroxide buffer is 10.6.
  • the concentration of the 3' end blocking group cleaving agent, such as THPP is 5-30mM, such as 5mM, 10mM, 20mM or 30mM.
  • the concentration of the 3'-end blocking group excluding agent is 20mM.
  • the second aspect of the present invention provides a sequencing kit, which includes the excision reagent according to the first aspect of the present invention.
  • the sequencing kit further includes one or more of DNA nanospheres, sequencing slides, MDA polymerase, dNTPs and scanning reagents.
  • the third aspect of the present invention provides a method for excluding the 3' end blocking group of a polynucleotide.
  • the method uses the excision reagent described in the first aspect of the present invention to excise the 3' end blocking group. Cut off the group, exposing the 3' terminal hydroxyl group.
  • the conditions for resection are as follows; the temperature is 30-60°C, and/or the time is 1-5 s.
  • the temperature is 30-40°C, 40-50°C or 50-60°C, such as 30°C, 40°C, 50°C or 60°C.
  • the temperature is 50°C and the time is 2 seconds.
  • the fourth solution of the present invention provides a sequencing method, which method includes the following steps:
  • a sequencing reagent such as a dNTP molecule and DNA polymerase
  • the method includes the following steps:
  • the cycle is performed 50-400 times, such as 50, 100, 150, 200 or 400 times.
  • the scanning reagent is located in hole No. 10 of the sequencing reagent tank.
  • the fifth aspect of the present invention provides an excision reagent as described in the first aspect of the present invention or a sequencing kit as described in the second aspect of the present invention for use in polynucleotide sequencing or preparation of polynucleosides.
  • Application of acid sequencing reagents are described in the first aspect of the present invention or a sequencing kit as described in the second aspect of the present invention for use in polynucleotide sequencing or preparation of polynucleosides.
  • the reagents and raw materials used in the present invention are all commercially available.
  • This invention applies the glycine-sodium hydroxide buffer system to the excision reagent for sequencing for the first time to speed up the excision efficiency and improve the sequencing quality.
  • the use of this kind of reagent in sequencing kits will improve the sequencing quality of the product and increase the value and market share of the product.
  • Figure 1 shows the effect of glycine-sodium hydroxide buffer system on Lag (lag) in sequencing.
  • Figure 2 shows the effect of the glycine-sodium hydroxide buffer system on Runon (lead) in sequencing.
  • Ammonium bicarbonate (analytical grade) N-ethylmaleimide, ultrapure water, Escherichia coli single-stranded circular DNA as template, standard library reagent V3.0, primer sequence: CAACTCCTTGGCTCACAGAACATGGCTACGATCCGACTT (SEQ ID NO: 1).
  • DNA polymerase and DNA nanospheres were both from MGI (MGISEQ-2000RS high-throughput sequencing kit PE150). The following experiments all use E. coli single-stranded circular DNA as a template, and use the MGISEQ-2000RS high-throughput sequencing kit to prepare DNA nanospheres and load them onto a sequencing slide for subsequent sequencing.
  • the nucleotide mixture with fluorescence modification and reversible blocking mentioned in this example includes: dATP-1, which refers to adenine nucleotide with both reversible blocking group modification and Cy5 fluorescence modification; dTTP- 1. It refers to thymine nucleotides with both reversible blocking group modification and ROX fluorescence modification; dGTP-1, which refers to guanine nucleotides with both reversible blocking group modification and Cy3 fluorescence modification; and dCTP-1, which refers to a cytosine nucleotide with both reversible blocking group modification and EF700 fluorescence modification.
  • dATP-1 which refers to adenine nucleotide with both reversible blocking group modification and Cy5 fluorescence modification
  • dTTP- 1 It refers to thymine nucleotides with both reversible blocking group modification and ROX fluorescence modification
  • dGTP-1 which refers to
  • Control group 1 Use the MGISEQ-2000RS high-throughput sequencing kit and perform SE50 sequencing on the MGISEQ-2000RS sequencing platform according to the experimental process.
  • the nucleosides with fluorescence modification and reversible blocking are sequentially polymerized on the MGISEQ-2000RS sequencing platform.
  • Acid mixture then use an elution reagent to elute the free nucleotides, collect signals under a photographing solution, use an excision reagent to remove the protecting group, and use an elution reagent for washing. Then the Lag and Runon decreases of each cycle and the sequencing error rate curve of each cycle are calculated to evaluate the sequencing quality.
  • Experimental Group 1 Use the MGISEQ-2000RS high-throughput sequencing kit, then remove the reagent in the No. 9 hole in the kit and replace it with the buffer solution containing the excision reagent of the present invention, and sequentially polymerize the fluorescence modification and The nucleotide mixture is reversibly blocked, and then the free nucleotides are eluted using elution reagents, so that polymerization and leveling can be performed while the signal is being collected. At this time, the polymerized nucleotide mixture is reversibly blocked.
  • the modified nucleotides were subjected to SE50 sequencing on the MGISEQ-2000RS sequencing platform according to the same experimental process as the control group, and then the Lag and Runon decreases in each cycle and the sequencing error rate curve in each cycle were calculated to evaluate the sequencing quality.
  • glycine and sodium hydroxide Accurately weigh a certain amount of glycine and sodium hydroxide, and configure them into glycine-sodium hydroxide buffer solutions with different pH values according to Table 1, X mL 0.2mol/L glycine + Y mL 0.2mol/L sodium hydroxide, and dilute with water to 200 mL, then weigh a certain amount of THPP (tris(3-hydroxypropyl)phosphine) and dissolve it in the above buffer solution, and prepare a 20 mM THPP solution for later use.
  • THPP tris(3-hydroxypropyl)phosphine
  • Step 1 Load the DNA nanospheres onto the prepared sequencing slide
  • Step 2 Pump the prepared dNTP molecule mixture into the sequencing slide, and use MDA polymerase to add the dNTP molecules to the parent strand of DNA;
  • Step 3 Determine the type of base by taking photos with scanning reagent
  • Step 4 Use the excision reagent to expose the 3’ hydroxyl group, the temperature is 50°C, and the excision time is 2 seconds;
  • DNA nanospheres, MDA polymerase, dNTPs and scanning reagents can be derived from the MGISEQ-2000RS high-throughput sequencing kit or other similar kits. Wherein, the scanning reagent is located in hole No. 10 of the sequencing reagent tank.
  • the alkaline solution of glycine-sodium hydroxide is beneficial to improving sequencing Q30 and reducing Lag and Runon.
  • the glycine-sodium hydroxide buffer system can also be used in combination with TCEP and DTT excision reagents to achieve good sequencing results.
  • DNA sequencing is a very complex process, and various reagents in each link include many ingredients.
  • the inventor also tried to use a boric acid-sodium borate buffer system, but compared with the glycine-sodium hydroxide buffer system at the same pH value Shows poor sequencing quality. This may be caused by the boric acid-sodium borate buffer not being suitable for the entire reagent environment. There may be the following reasons: 1. Poor biocompatibility, destroying DNA nanospheres; 2. The pH value is thermally unstable; 3. Mixed with the excision reagent. Chemical reactions occur with other ingredients; 4. Poor buffering capacity, etc.; but the specific reasons are unknown.
  • the inventor also tried a carbonic acid-sodium carbonate (pH 10.6) buffer system, but it was only effective in the strategy of using the azide series as the exposed hydroxyl group at the 3' end, and was ineffective in other cleavable blocking groups.
  • the carbonate-sodium carbonate system buffer solution has problems such as instability and easy decomposition when heated, and is not suitable for long-term storage as a commercial product. This also shows that when selecting the buffer composition for sequencing, it is not only the buffer efficiency that affects the sequencing effect, but also other comprehensive factors such as its composition and thermal stability.

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Abstract

L'invention concerne un réactif d'excision contenant un tampon glycine-NaOH et son utilisation dans le séquençage d'acide nucléiques. La présente invention concerne également un kit de séquençage contenant le réactif d'excision, un procédé d'excision d'un groupe de blocage à l'extrémité 3' d'un polynucléotide, et un procédé de séquençage. Lors du séquençage du polynucléotide selon le procédé, l'efficacité de l'excision du groupe de blocage à l'extrémité 3' peut être améliorée, le Q30 de séquençage est effectivement augmenté, le Lag et le Runon sont réduits, et la qualité du séquençage est améliorée.
PCT/CN2022/099260 2022-06-16 2022-06-16 Réactif d'excision contenant un tampon glycine-naoh et son utilisation dans le séquençage d'acides nucléiques Ceased WO2023240564A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
CN202280096939.5A CN119343466A (zh) 2022-06-16 2022-06-16 含甘氨酸-NaOH缓冲液的切除试剂及其在核酸测序中的应用
PCT/CN2022/099260 WO2023240564A1 (fr) 2022-06-16 2022-06-16 Réactif d'excision contenant un tampon glycine-naoh et son utilisation dans le séquençage d'acides nucléiques

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PCT/CN2022/099260 WO2023240564A1 (fr) 2022-06-16 2022-06-16 Réactif d'excision contenant un tampon glycine-naoh et son utilisation dans le séquençage d'acides nucléiques

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1991017264A1 (fr) * 1990-05-02 1991-11-14 Imperial Chemical Industries Plc Analyse de sequences specifiques d'acide nucleique
CN107002067A (zh) * 2014-09-11 2017-08-01 宝生物工程株式会社 利用热稳定性错配内切核酸酶的方法
US20170268048A1 (en) * 2014-08-20 2017-09-21 Huawei Yang Test kit and method for testing target nucleic acid in sample
WO2019080725A1 (fr) * 2017-10-25 2019-05-02 深圳华大生命科学研究院 Méthode de séquençage d'acide nucléique et kit de séquençage d'acide nucléique
CN114317787A (zh) * 2021-12-29 2022-04-12 成都大学 吸附磁珠及其试剂盒和用途以及检测金黄色葡萄球菌的方法

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1991017264A1 (fr) * 1990-05-02 1991-11-14 Imperial Chemical Industries Plc Analyse de sequences specifiques d'acide nucleique
US20170268048A1 (en) * 2014-08-20 2017-09-21 Huawei Yang Test kit and method for testing target nucleic acid in sample
CN107002067A (zh) * 2014-09-11 2017-08-01 宝生物工程株式会社 利用热稳定性错配内切核酸酶的方法
WO2019080725A1 (fr) * 2017-10-25 2019-05-02 深圳华大生命科学研究院 Méthode de séquençage d'acide nucléique et kit de séquençage d'acide nucléique
CN114317787A (zh) * 2021-12-29 2022-04-12 成都大学 吸附磁珠及其试剂盒和用途以及检测金黄色葡萄球菌的方法

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
GATES FREDERICK T, LINN STUART: "Endonuclease V of Escherichia coli", JBC - THE JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 252, no. 5, 10 March 1977 (1977-03-10), pages 1647 - 1653, XP093117709 *

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