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WO2023138206A1 - Amplification du signal moléculaire de l'adn et procédé de séquençage de l'acide nucléique fondés sur un support en phase solide - Google Patents

Amplification du signal moléculaire de l'adn et procédé de séquençage de l'acide nucléique fondés sur un support en phase solide Download PDF

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WO2023138206A1
WO2023138206A1 PCT/CN2022/133646 CN2022133646W WO2023138206A1 WO 2023138206 A1 WO2023138206 A1 WO 2023138206A1 CN 2022133646 W CN2022133646 W CN 2022133646W WO 2023138206 A1 WO2023138206 A1 WO 2023138206A1
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dna
phase carrier
solid
amplification
solid phase
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Chinese (zh)
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周魏
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Shenzhen Archean Tech Co Ltd
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Shenzhen Archean Tech Co Ltd
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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
    • 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 application belongs to the technical field of genetic engineering, and relates to a method for DNA molecular signal amplification and nucleic acid sequencing based on a solid-phase carrier.
  • High-throughput sequencing technology also known as “next-generation” sequencing technology (“Next-generation” sequencing technology)
  • Next-generation sequencing technology is marked by the ability to sequence hundreds of thousands to several million DNA molecules in parallel at a time and generally having a short read length.
  • the steps of high-throughput sequencing include sample preparation, library construction, sequencing reaction, and data analysis.
  • the library template can be amplified thousands of times by PCR, so as to reach the amount of the library on the machine, so as to complete the subsequent determination of the genomic DNA sequence; in the sequencing stage, the signal of a single DNA molecule is difficult to detect, and it is necessary to amplify the single DNA molecule to form a cluster (i.e., cluster) or ball (DNB) to obtain a signal of sufficient strength before sequencing can be detected.
  • cluster i.e., cluster
  • DDB ball
  • the way to amplify DNA molecular signals in the market during the sequencing stage is to form a cluster of single DNA molecules through amplification, that is, cluster.
  • the accumulation of errors in the process of bridge PCR amplification forming clusters will reduce the fidelity of DNA sequence replication, affecting the accuracy and sensitivity of low-depth sequencing variation and low-frequency mutation detection. Due to the PCR amplification of library construction and sequencing, it will bring a higher duplicate rate, resulting in a waste of DNA data, thereby increasing the cost of sequencing.
  • duplicates cannot be removed, which will affect the accuracy of transcriptome expression, especially the accuracy of small and medium expression transcripts.
  • CN113774120A discloses a DNB paired-end sequencing method. The process is to first amplify in a PCR machine, quantify the DNA nanospheres, and finally load the DNA nanospheres into the chip and fix the DNB in the chip in a certain way.
  • the present application provides a method for DNA molecular signal amplification and nucleic acid sequencing based on a solid-phase carrier, which can efficiently and cost-effectively amplify DNA molecular signals during the sequencing process while improving sequencing accuracy.
  • the present application provides a method for amplifying DNA molecular signals based on a solid-phase carrier, the method comprising:
  • the carboxylated DNA primers are immobilized on the surface aminated solid phase carrier, and the circular DNA library and amplification reagents are added to perform rolling circle amplification;
  • the DNA primers are combined with the circular DNA library through base complementation.
  • DNA primers containing carboxyl groups are immobilized on the solid-phase carrier containing amino groups on the surface through the condensation reaction of carboxyl groups and amino groups, and a solid-phase carrier for efficiently loading DNA primers can be obtained. Then, the circularized library and DNA primers are combined and fixed on the solid-phase carrier through base complementarity.
  • the field of high-throughput sequencing is of great significance.
  • the material of the solid phase carrier includes any one of glass, silica gel, ceramics, plastic or metal.
  • the carboxylated DNA primer comprises a 5' end carboxylated DNA primer.
  • said amplification reagents include DNA polymerase, dNTPs and buffer.
  • the DNA polymerase comprises phi29 DNA polymerase.
  • phi29 DNA polymerase has DNA strand displacement activity and continuous DNA synthesis ability, can synthesize DNA with a length of more than 70kb, and can perform isothermal DNA amplification in vitro that does not depend on thermal cycles.
  • the condensing agent used in the condensation reaction includes 2-succinimidyl-1,1,3,3-tetramethyluronium tetrafluoroborate and diisopropylethylamine.
  • the molar concentration ratio of 2-succinimidyl-1,1,3,3-tetramethyluronium tetrafluoroborate (TSTU) and diisopropylethylamine (DIEA) in the condensing agent is 1:(1-4), including but not limited to 1:1.2, 1:1.5, 1:1.8, 1:2, 1:2.5, 1:2.8, 1:3, 1:3.5 or 1:3.8, preferably 1:2 .
  • 2-succinimidyl-1,1,3,3-tetramethyluronium tetrafluoroborate is used to promote the condensation reaction between the carboxyl group in the DNA primer and the amino group on the solid-phase carrier, which significantly increases the loading rate of the DNA primer on the solid-phase carrier.
  • Diisopropylethylamine can react with the by-product generated by the reaction between the solid-phase carrier and TSTU loaded with the primer, thereby consuming the by-product and promoting the reaction to proceed in the positive direction, further improving the DNA primer loading rate of the solid-phase carrier.
  • the molar concentration ratio of imido-1,1,3,3-tetramethyluronium tetrafluoroborate and diisopropylethylamine is 1:2, which can further increase the DNA primer loading rate of the solid phase carrier.
  • a blocking step is also included after the rolling circle amplification.
  • the blocking includes adding a blocking reagent to a solid phase carrier for blocking.
  • the blocking reagent comprises bovine serum albumin reagent.
  • the solid-phase carrier is blocked after the amplification reaction is completed, which is beneficial for the amplified DNA and phi29 enzyme to be more firmly immobilized on the carrier.
  • the DNA molecular signal amplification method based on a solid phase carrier comprises the following steps:
  • Amination treatment is carried out to the solid phase carrier
  • the reagent for amination treatment includes 3-aminopropyltriethoxysilane.
  • the present application provides a method for nucleic acid sequencing based on a solid-phase carrier, and the method for nucleic acid sequencing based on a solid-phase carrier includes:
  • the nucleic acid sequencing includes mixing the amplification product with sequencing primers to perform a sequencing reaction.
  • the solid-phase carrier-based DNA molecular signal amplification method described in the first aspect is used to amplify the DNA to be tested, so that the DNA is amplified into a linear helical structure. All amplified templates are the original insert fragments, and the errors generated by PCR will not accumulate. At the same time, the reaction is not strictly dependent on the PCR instrument.
  • the DNA molecular signal is automatically amplified in the chip, which can reduce the burden on operators and realize automatic preparation.
  • the DNA primers are immobilized on the solid-phase carrier, the DNA primers are combined with the circular DNA library, and the DNA is amplified into a linear helical structure by rolling circle amplification (Rolling circle replication, RCA), so that all the amplified templates are the original insert fragments, avoiding the accumulation of errors in the process of amplifying into clusters due to bridge PCR, and also reducing the high duplicate ratio caused by bridge PCR, thereby reducing the waste of DNA data and saving sequencing costs.
  • Rolling circle replication, RCA rolling circle replication
  • the preparation method for DNA molecular signal amplification in a solid-phase carrier chip does not strictly rely on PCR equipment for reaction and DNA nanosphere quality control operations, and can automatically load DNA molecules into the chip, and DNA molecular signals are automatically amplified in the chip, which can reduce the burden on operators and realize automatic preparation.
  • Figure 1 is a diagram of the structure of hydroxyl groups on the glass surface.
  • FIG. 2 is a schematic diagram of the surface of 3-aminopropyltriethoxysilane (APTES) modified silica, a represents physical adsorption, b represents condensation, and c represents the main structure after heating and curing.
  • APTES 3-aminopropyltriethoxysilane
  • Fig. 3 is a schematic diagram of the condensation reaction between the amino group on the surface of the solid phase carrier and the primer with carboxyl group.
  • Fig. 4 is a schematic diagram of DNA amplification and immobilization of a sample to be sequenced in a solid phase carrier.
  • Fig. 5 is a picture of DNA amplification products observed under a fluorescence microscope.
  • a glass substrate is used as a solid phase carrier and amination treatment is performed on it.
  • Amino groups were connected to the glass substrate by soaking method.
  • the hydroxyl groups on the surface of glass were mainly isolated hydroxyl groups, dihydroxyl groups and hydrogen-bonded hydroxyl groups ( Figure 1).
  • 3-Aminopropyltriethoxysilane (APTES) was used to absorb and react on the surface of silica under anhydrous conditions, and heat and solidify at 55°C.
  • the 5' end carboxylated DNA primer was immobilized on the glass solid phase carrier prepared in Example 1.
  • the glass solid-phase carrier loaded with DNA primers prepared in Example 2 was used to amplify DNA molecular signals.
  • TE buffer as the solvent for the circular DNA library, take 120 fmol of the circular DNA library and dissolve it in the TE buffer, so that the final volume is 80 ⁇ L, and inject it into the glass solid-phase carrier loaded with DNA primers prepared in Example 2.
  • the primers in the library and the carrier form a double strand through the principle of base complementarity.
  • After standing at room temperature for 30 minutes, add the amplification reaction reagents for rolling circle amplification (the schematic diagram of the amplification is shown in Figure 4).
  • the rolling circle amplification reaction system is shown in Table 1.
  • the rolling circle amplification reaction conditions are: 30 °C, 45min, after the end of the amplification, use 1% BSA reagent to block.
  • Example 3 the amplification product of Example 3 was sequenced and analyzed.
  • the rolling circle amplification method based on the solid-phase carrier of the present application is performed on a solid carrier, and sequencing primers are added after amplification, and the genes located in the genome can be sequenced by sequencing while synthesizing SBS, that is, single-end sequencing (SE testing) or double-end sequencing (PE testing).
  • SE testing single-end sequencing
  • PE testing double-end sequencing
  • this application immobilizes DNA primers on a solid-phase carrier, combines the DNA primers with a circular DNA library, and uses rolling circle amplification to amplify the DNA into a linear helical structure, so that all amplified templates are the original insert fragments, avoiding the accumulation of errors in the process of amplifying bridge PCR into a cluster, improving the accuracy of sequencing, and reducing the high duplicate ratio caused by bridge PCR, thereby reducing the waste of DNA data and saving sequencing costs.
  • Molecules are automatically loaded into the chip, and DNA molecular signals are automatically amplified in the chip, which can reduce the burden on operators and realize automatic preparation.
  • the present application illustrates the detailed method of the present application through the above-mentioned examples, but the present application is not limited to the above-mentioned detailed method, that is, it does not mean that the application must rely on the above-mentioned detailed method to be implemented.
  • Those skilled in the art should understand that any improvement to the present application, the equivalent replacement of each raw material of the product of the present application, the addition of auxiliary components, the selection of specific methods, etc., all fall within the scope of protection and disclosure of the present application.

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  • Organic Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
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Abstract

Amplification du signal moléculaire de l'ADN et procédé de séquençage d'acide nucléique fondé sur un support en phase solide, le procédé comprenant les étapes suivantes : immobilisation d'une amorce d'ADN carboxylée sur un support en phase solide à surface aminée au moyen d'une réaction de condensation des groupes carboxyle et amino; et ajout d'une banque d'ADN annulaire et d'un réactif d'amplification au support en phase solide pour effectuer une amplification en cercle roulant, l'amorce d'ADN étant liée à la banque d'ADN annulaire d'une manière complémentaire sur le plan des bases. Une amorce d'ADN est immobilisée sur un support en phase solide, et l'ADN est amplifié en une structure d'hélice linéaire au moyen d'une amplification en cercle roulant, afin d'éviter l'accumulation d'erreurs causée par l'amplification PCR en pont abouttisant à la génération de grappes, d'améliorer la précision du séquençage, de réduire le taux de duplication relativement élevé causé par la PCR en pont, et de réduire le coût du séquençage. En outre, grâce à l'amplification isotherme de l'ADN, les exigences en matière d'équipement sont faibles, la charge de travail incombant aux opérateurs peut être allégée et la préparation est automatisée.
PCT/CN2022/133646 2022-01-21 2022-11-23 Amplification du signal moléculaire de l'adn et procédé de séquençage de l'acide nucléique fondés sur un support en phase solide Ceased WO2023138206A1 (fr)

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CN202210084194.2A CN114350773B (zh) 2022-01-21 2022-01-21 一种基于固相载体的dna分子信号扩增及核酸测序的方法
CN202210084194.2 2022-01-21

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CN114350773B (zh) * 2022-01-21 2025-02-07 深圳太古语科技有限公司 一种基于固相载体的dna分子信号扩增及核酸测序的方法

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CN1995369A (zh) * 2006-12-14 2007-07-11 东南大学 一种固相制备核酸分子克隆的方法
US20090117621A1 (en) * 2005-07-20 2009-05-07 Jonathan Mark Boutell Methods of nucleic acid amplification and sequencing
CN102604934A (zh) * 2012-03-31 2012-07-25 盛司潼 一种基于固相载体进行扩增及进行核酸测序的方法
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CN114350773A (zh) * 2022-01-21 2022-04-15 深圳太古语科技有限公司 一种基于固相载体的dna分子信号扩增及核酸测序的方法

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JPH07203998A (ja) * 1994-01-26 1995-08-08 Hamamatsu Photonics Kk 核酸塩基配列決定方法
CN102766688B (zh) * 2012-04-17 2014-04-02 盛司潼 一种检测基因序列的方法
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Publication number Priority date Publication date Assignee Title
US20090117621A1 (en) * 2005-07-20 2009-05-07 Jonathan Mark Boutell Methods of nucleic acid amplification and sequencing
CN1995369A (zh) * 2006-12-14 2007-07-11 东南大学 一种固相制备核酸分子克隆的方法
CN102604934A (zh) * 2012-03-31 2012-07-25 盛司潼 一种基于固相载体进行扩增及进行核酸测序的方法
CN104357549A (zh) * 2014-09-25 2015-02-18 徐州医学院 一种基于dna芯片的数字化定量检测核酸的方法
CN104830985A (zh) * 2015-05-06 2015-08-12 东南大学 基于固相滚环扩增和颗粒团聚的多重核酸可视化检测方法及试剂盒
CN114350773A (zh) * 2022-01-21 2022-04-15 深圳太古语科技有限公司 一种基于固相载体的dna分子信号扩增及核酸测序的方法

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