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WO2024119413A9 - Procédé de préparation basé sur un procédé en une étape pour nanosphère d'acide nucléique double - Google Patents

Procédé de préparation basé sur un procédé en une étape pour nanosphère d'acide nucléique double Download PDF

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Publication number
WO2024119413A9
WO2024119413A9 PCT/CN2022/137336 CN2022137336W WO2024119413A9 WO 2024119413 A9 WO2024119413 A9 WO 2024119413A9 CN 2022137336 W CN2022137336 W CN 2022137336W WO 2024119413 A9 WO2024119413 A9 WO 2024119413A9
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nucleic acid
acid template
double
molecule
stranded
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WO2024119413A1 (fr
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杨晋
曹硕
吴慧
徐崇钧
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MGI Tech Co Ltd
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MGI Tech Co Ltd
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Priority to CN202280098401.8A priority Critical patent/CN119654421A/zh
Priority to PCT/CN2022/137336 priority patent/WO2024119413A1/fr
Publication of WO2024119413A1 publication Critical patent/WO2024119413A1/fr
Publication of WO2024119413A9 publication Critical patent/WO2024119413A9/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
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B50/00—Methods of creating libraries, e.g. combinatorial synthesis
    • C40B50/06—Biochemical methods, e.g. using enzymes or whole viable microorganisms
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B50/00—Methods of creating libraries, e.g. combinatorial synthesis
    • C40B50/14—Solid phase synthesis, i.e. wherein one or more library building blocks are bound to a solid support during library creation; Particular methods of cleavage from the solid support
    • C40B50/18—Solid phase synthesis, i.e. wherein one or more library building blocks are bound to a solid support during library creation; Particular methods of cleavage from the solid support using a particular method of attachment to the solid support

Definitions

  • the present invention relates to the field of gene sequencing. Specifically, the present invention relates to a one-step method for preparing double nucleic acid nanospheres.
  • the present invention aims to solve at least one of the technical problems existing in the prior art to at least a certain extent.
  • paired-end sequencing is a very important sequencing technology.
  • PE sequencing PE sequencing for short
  • the sequence information of the 5' and 3' ends of the DNA chain can be obtained under short-read sequencing. Because the sequence information of these two ends has the characteristics of pairing, it can be confirmed that they come from the same DNA chain, which is of great significance for genome assembly and variation detection (such as insertion/deletion).
  • paired-end sequencing more sequence information can be obtained in one sequencing, which helps to shorten the sequencing time and reduce the sequencing cost.
  • the mainstream double-end sequencing technologies include the following:
  • Illumina (as shown in Figure 1): After completing single-end sequencing, the single-end sequencing chain (denoted as Read1) is extended; then the sequencing chain is washed away, and the template chain is combined with another primer (denoted as P5') on the surface of the chip, and the complementary chain of the template chain is obtained by extending from the P5' primer. Then the template chain is washed away, and only the P5' extended chain is retained, which is used as the double-end template chain. After hybridization with the primers, double-end sequencing is performed (denoted as Read2). For Illumina's double-end sequencing technology, its main disadvantage is that the steps are relatively complicated, involving multiple steps such as hybridization, extension, denaturation, and removal of the template chain, which is time-consuming;
  • MGI multiple displacement amplification
  • the sequencing chain will "float" above the DNB, at which time the sequencing primer of Read2 can be hybridized to complete the double-end sequencing.
  • the steps are also more complicated, involving primer hybridization, extension, chain displacement, end blocking and other steps, which takes a long time; at the same time, the MDA reaction system is more sensitive to temperature, and certain requirements are put forward for the storage, transportation and use of reagents.
  • Double-end sequencing using DNA nanoballs as sequencing units generally includes the following steps: (1) Obtaining a sequencing library. (2) Preparing a DNA sense strand nanoball from the library. (3) Hybridizing specific primers to sequence the DNA sense strand. (4) Obtaining the DNA antisense strand through multiple strand displacement amplification reactions. (5) Hybridizing specific primers to sequence the DNA antisense strand.
  • the traditional double-end sequencing method requires obtaining the DNA antisense strand through multiple strand displacement amplification reactions before performing antisense strand sequencing. The steps are cumbersome and time-consuming.
  • the present invention provides a method for preparing DNA nanoballs, which contain both DNA sense chain nanoballs and DNA antisense chain nanoballs, and can directly sequence the sense chain or antisense chain without multiple strand displacement amplification reactions, thereby obtaining real sequence information of double-end sequencing.
  • the present invention proposes a double-ended cyclization primer.
  • the double-ended cyclization primer includes a first nucleic acid single-stranded molecule and a second nucleic acid single-stranded molecule, and the 5' end of the first nucleic acid single-stranded molecule is connected to the 5' end of the second nucleic acid single-stranded molecule by a connector.
  • the cyclization primer obtained after connecting the first nucleic acid single-stranded molecule and the second nucleic acid single-stranded molecule together by a connector can be simultaneously complementary to the end joint part sequence of the sense strand and the antisense strand of the double-stranded nucleic acid template molecule to perform a cyclization reaction.
  • nucleic acid sense strand nanospheres and nucleic acid antisense strand nanospheres can be obtained at the same time, and then the sense strand and the antisense strand can be sequenced, without the need to perform multiple strand displacement amplification reactions, and can save the steps of hybridization, depolymerization, and template removal, which shortens the time for subsequent sequencing.
  • the present invention proposes a method for preparing the double-terminal circularized primer described above.
  • the method is to perform a ligation reaction on a first nucleic acid single-stranded molecule and a second nucleic acid single-stranded molecule so as to obtain the double-terminal circularized primer; wherein the 5' end of the first nucleic acid single-stranded molecule is connected to a first modification group, the 5' end of the second nucleic acid single-stranded molecule is connected to a second modification group, and the first modification group and the second modification group are suitable for a ligation reaction; the 3' end of the first nucleic acid single-stranded molecule and the 3' end of the second nucleic acid single-stranded molecule are not suitable for a ligation reaction; the first nucleic acid single-stranded molecule and the second nucleic acid single-stranded molecule cannot be complementary to each other.
  • the two single-stranded nucleic acids can be connected to form a double-terminal circularized primer, which can simultaneously complementarily pair with the terminal linker sequence of the sense chain and the antisense chain of the double-stranded nucleic acid template molecule to carry out a circularization reaction.
  • nucleic acid sense chain nanospheres and nucleic acid antisense chain nanospheres can be obtained at the same time, and then the sense chain and the antisense chain can be sequenced without the need for multiple strand displacement amplification reactions, and the steps of hybridization, denaturation, and template removal can be omitted, thereby shortening the time for subsequent sequencing.
  • the present invention proposes a method for obtaining a circularized nucleic acid template.
  • the method comprises: performing a circularization reaction on a double-terminal circularization primer and a double-stranded nucleic acid template molecule, wherein the double-stranded nucleic acid template molecule comprises a sense nucleic acid template strand and an antisense nucleic acid template strand, and the two 3' ends of the double-terminal circularization primer are respectively complementary to at least part of the nucleic acid sequences of the 5' end and the 3' end of the sense nucleic acid template strand and the antisense nucleic acid template strand, so as to obtain a circularized nucleic acid template, wherein the circularized nucleic acid template has a sense nucleic acid template chain loop and an antisense nucleic acid template chain loop; wherein the double-terminal circularization primer comprises a first nucleic acid single-stranded molecule and a second nucle
  • nucleic acid sense chain nanoballs and nucleic acid antisense chain nanoballs can be simultaneously obtained after rolling circle amplification, and then the sense chain and the antisense chain can be sequenced without going through the traditional multiple strand displacement amplification reaction. This can avoid problems such as failure of Read2 template chain synthesis, low efficiency or preference caused by cumulative damage to Read1 sequencing during the multiple strand displacement amplification reaction.
  • the present invention provides a circularized nucleic acid molecule obtained by the method described above.
  • the circularized nucleic acid molecule obtained by this method can simultaneously obtain nucleic acid sense strand nanospheres and nucleic acid antisense strand nanospheres after rolling circle amplification, and then the sense strand and the antisense strand can be sequenced, without going through the traditional multiple strand displacement amplification reaction, and can avoid the problems of Read2 template strand synthesis failure, low efficiency or bias caused by Read1 sequencing cumulative damage during the multiple strand displacement amplification reaction.
  • the present invention proposes a cyclized nucleic acid molecule.
  • the cyclized nucleic acid molecule comprises: the double-ended cyclization primer as described above and a sense nucleic acid template strand and an antisense nucleic acid template strand, wherein the sense nucleic acid template strand and the antisense nucleic acid template strand are reversely complementary, and the two ends of the sense nucleic acid template strand and the antisense nucleic acid template strand are connected with a joint sequence, and the joint sequences of the 5' end of the sense nucleic acid template strand and the 5' end of the antisense nucleic acid template strand are different, and the joint sequences of the 3' end of the sense nucleic acid template strand and the 3' end of the antisense nucleic acid template strand are different; wherein the two 3' ends of the double-ended cyclization primer are complementary to at least part of the nucleic acid sequence of the 5
  • the circularized nucleic acid molecule can simultaneously obtain nucleic acid sense chain nanoballs and nucleic acid antisense chain nanoballs after rolling circle amplification, and then the sense chain and the antisense chain can be sequenced without going through the traditional multiple strand displacement amplification reaction. It can avoid the problems of failure of Read2 template chain synthesis, low efficiency or preference caused by cumulative damage to Read1 sequencing during the multiple strand displacement amplification reaction.
  • the present invention proposes a nucleic acid sequencing chip.
  • the nucleic acid sequencing chip includes: a plurality of nucleic acid sample binding sites and a plurality of nucleic acid molecule complexes fixed to the nucleic acid sample binding sites, wherein the nucleic acid molecule complexes include a sense nucleic acid template strand, an antisense nucleic acid template strand and a double-ended cyclization primer, the double-ended cyclization primer is connected to the sense nucleic acid template strand, and the double-ended cyclization primer is connected to the antisense nucleic acid template strand.
  • the nucleic acid sequencing chip can sequence the sense strand and the antisense strand in any order, and can obtain nucleic acid double-end sequence information directly through double-end sequencing without undergoing traditional multiple strand displacement amplification reactions, which can greatly simplify the sequencing process, reduce sequencing time and sequencing costs.
  • the present invention proposes a method for preparing a dual nucleic acid nanosphere.
  • the method comprises: subjecting the aforementioned cyclized nucleic acid molecule to a rolling circle amplification process to obtain a dual nucleic acid nanosphere.
  • the dual nucleic acid nanosphere obtained by this method has both a nucleic acid sense strand nanosphere and a nucleic acid antisense strand nanosphere, and can obtain nucleic acid double-end sequence information directly through double-end sequencing without undergoing a traditional multiple strand displacement amplification reaction, which can greatly simplify the sequencing process, reduce sequencing time and sequencing costs.
  • the present invention provides a dual nucleic acid nanoball.
  • the dual nucleic acid nanoball is prepared by the method described above.
  • the dual nucleic acid nanoball contains both a nucleic acid sense strand nanoball and a nucleic acid antisense strand nanoball, and can directly sequence the sense strand or the antisense strand without multiple strand displacement amplification reactions, thereby obtaining true sequence information for double-end sequencing.
  • the present invention proposes a nucleic acid library.
  • the nucleic acid library includes the dual nucleic acid nanoballs described above.
  • the library has a large capacity and a high success rate of pairing with the nucleic acid to be tested.
  • the sense strand and the antisense strand can be sequenced at the same time, and the sequencing order can be adjusted as needed and sequenced separately.
  • a high-throughput sequencing library can be constructed simply and efficiently, and a sequencing plate can be efficiently used, thereby improving the success rate of library construction and reducing the loss of samples and reagents.
  • the present invention provides a gene sequencing method.
  • the aforementioned nucleic acid library is subjected to sequencing.
  • the nucleic acid library is used for sequencing, and the sense strand and the antisense strand can be sequenced simultaneously, or the template strand can be sequenced step by step, so that the sequencing information can be more complete, the target fragment can be captured efficiently, and accurate sequencing results can be obtained.
  • sequence overlap is generated at the tail end of double-end sequencing, and the error rate caused by sequencing accumulation can be reduced by mutual correction.
  • FIG1 is a schematic diagram of Illumina double-end sequencing technology according to an embodiment of the present invention.
  • FIG2 is a schematic diagram of MGI's double-end sequencing technology according to an embodiment of the present invention.
  • FIG3 is a diagram of the construction process of a double-stranded DNA molecule for preparing a DNA nanoball according to an embodiment of the present invention, which is referred to as a special DNA library (Dual splint dsDNA library) in the following embodiments;
  • Figure 4 is a PAGE gel image of dual splint oligo purification according to an embodiment of the present invention.
  • FIG. 5 is a schematic diagram of the preparation and loading principle of DNA nanospheres according to an embodiment of the present invention.
  • the present invention proposes a double-ended circularization primer.
  • the double-ended circularization primer includes a first nucleic acid single-stranded molecule and a second nucleic acid single-stranded molecule, and the 5' end of the first nucleic acid single-stranded molecule is connected to the 5' end of the second nucleic acid single-stranded molecule by a connector.
  • the circularization primer obtained after connecting the first nucleic acid single-stranded molecule and the second nucleic acid single-stranded molecule together by a connector can be simultaneously complementary to the end joint part sequence of the sense strand and the antisense strand of the double-stranded nucleic acid template molecule to perform a circularization reaction.
  • nucleic acid sense strand nanospheres and nucleic acid antisense strand nanospheres can be obtained, and then the sense strand and the antisense strand can be sequenced at the same time, without the need for multiple strand displacement amplification reactions, and can save the steps of hybridization, depolymerization, and template removal, thereby shortening the time for subsequent sequencing.
  • the first single-stranded nucleic acid molecule and the second single-stranded nucleic acid molecule are independently selected from DNA or RNA molecules.
  • the linker of the double-ended circularized primer includes at least one selected from -S-S-, -NH-, and -C-S-C-. Under the action of these bond energies, two single-stranded nucleic acid molecules can be connected together.
  • the "connector” described in the present application is a structure produced by the addition reaction of the "first modification group” and the “second modification group”.
  • the "first modification group” and the “second modification group” refer to molecules located at the ends of the first nucleic acid single-stranded molecule and the second nucleic acid single-stranded molecule that can undergo addition reaction, thereby realizing the connection between the first nucleic acid single-stranded molecule and the second nucleic acid single-stranded molecule. Therefore, it is not limited to the three types of connectors "-S-S-, -NH-, -C-S-C-".
  • the linker is connected to the solid support via a support linking group
  • the support linking group includes at least one selected from avidin or streptavidin-biotin group, amino-NHS ester or aldehyde group or hydroxymethylphosphine or carboxyl group, hydroxyl-isocyanate group, acrylamide-silyl group, azide-alkyne group and sulfhydryl-maleimide or haloacetyl or thiosulfonate group.
  • a large number of the double-ended cyclized primers can be fixed on the solid support at the same time, laying the foundation for the subsequent cyclization reaction with a large number of double-stranded nucleic acid template molecules to obtain a large number of cyclized nucleic acid templates.
  • the solid support comprises at least one selected from the group consisting of: magnetic beads, gel beads, glass beads, glass slides, nanogold particles, polymer objects and chips.
  • the connection between the double-ended circularized primer and the fourth modification group on the surface of the solid support can be achieved.
  • the solid support can be selected from magnetic beads, gel beads, glass beads, polymers, glass slides, chips, nanogold particles, etc.
  • connection reaction group between the double-ended circularized primer and the solid phase carrier can be avidin/streptavidin-biotin, amino-NHS ester/aldehyde group/hydroxymethylphosphine/carboxyl group, hydroxyl-isocyanate, acrylamide-silane group, or azide-alkyne, thiol-maleimide/haloacetyl group/thiosulfonate, etc.
  • connection mode between the first modification group and the second modification group is different from the connection mode between the third modification group and the fourth modification group, mainly to avoid the connection between the first nucleic acid single-stranded molecule or the second nucleic acid single-stranded molecule itself, and to avoid the first nucleic acid single-stranded molecule and the second nucleic acid single-stranded molecule from connecting at multiple sites and losing the ability to connect to the solid phase carrier.
  • the third modification group and the fourth modification group described in the present application are respectively selected from avidin or streptavidin and biotin groups, amino and NHS ester or aldehyde or hydroxymethylphosphine or carboxyl groups, hydroxyl and isocyanate groups, acrylamide and silane groups, azide and alkyne groups, thiol and maleimide or haloacetyl or thiosulfonate groups.
  • the present invention proposes a method for preparing the double-terminal circularized primer described above.
  • a first nucleic acid single-stranded molecule and a second nucleic acid single-stranded molecule are subjected to a ligation reaction to obtain the double-terminal circularized primer; wherein the 5' end of the first nucleic acid single-stranded molecule is connected to a first modification group, the 5' end of the second nucleic acid single-stranded molecule is connected to a second modification group, and the first modification group and the second modification group are suitable for a ligation reaction; the 3' end of the first nucleic acid single-stranded molecule and the 3' end of the second nucleic acid single-stranded molecule are not suitable for a ligation reaction; the first nucleic acid single-stranded molecule and the second nucleic acid single-stranded molecule cannot be complementary to each other.
  • the two single-stranded nucleic acids can be connected to form a double-terminal circularized primer, which can simultaneously complementarily pair with the terminal linker sequences of the sense strand and the antisense strand of the double-stranded nucleic acid template molecule to carry out a circularization reaction.
  • nucleic acid sense strand nanospheres and nucleic acid antisense strand nanospheres can be obtained at the same time, and then the sense strand and the antisense strand can be sequenced without the need for multiple strand displacement amplification reactions, and the steps of hybridization, denaturation, and template removal can be omitted, thereby shortening the time for subsequent sequencing.
  • connection reaction is a reaction that can connect the first single-stranded nucleic acid molecule and the second single-stranded nucleic acid molecule together, that is, as long as the 5' ends of the two single-stranded nucleic acid molecules can be connected, the connection reaction can be a coupling reaction, affinity reaction or other chemical reaction.
  • the method for preparing the double-ended circularized primer described above may further include at least one of the following additional technical features:
  • the first modification group and the second modification group are independently selected from chemical molecules or biological macromolecules; wherein the chemical small molecules are suitable for being combined by covalent bonds, and the biological macromolecules are suitable for being connected by reaction.
  • the first modifying group and the second modifying group are independently selected from at least one of dibenzylcyclooctyne (DBCO), azide (Azide), maleimide (Maleimide) and sulfhydryl (-SH).
  • DBCO dibenzylcyclooctyne
  • Azide azide
  • Maleimide maleimide
  • -SH sulfhydryl
  • the above-mentioned modifying groups can undergo a connection reaction under certain conditions, so that the 5' ends of the first nucleic acid single-stranded molecule and the second nucleic acid single-stranded molecule are connected together.
  • azide azide
  • DBCO dibenzocyclooctyne
  • SPAAC strain-promoted azide-alkyne cycloaddition
  • the present application can also use other bio-coupling systems that combine two or more molecules or biomacromolecules through chemical covalent bonds, such as coupling of amino-containing molecules with NHS ester compounds to form amine bonds, coupling of sulfhydryl-containing molecules with maleimide compounds to form thioether bonds, etc.
  • the first modification group and the second modification group are dibenzylcyclooctyne (DBCO) and azide (Azide), respectively, and the connection reaction is carried out at a temperature of 37°C, a solvent of PBS, and a pH of 7.4 for 48 hours.
  • the copper-free click chemistry reaction of dibenzylcyclooctyne (DBCO) and azide (Azide) is a ring strain-promoted azide-alkynyl cycloaddition reaction, which does not require the use of toxic copper ions as a catalyst, and the reaction is efficient and fast.
  • the solvent further comprises DMSO, wherein DMSO facilitates the connection reaction between the modification groups.
  • the volume fraction of DMSO in the connection reaction system is 10%. Adding this volume of DMSO can make the connection between the modification groups more complete.
  • the method further comprises that the double-ended circularization primer is connected to a solid support via a third modification group and a fourth modification group, wherein the third modification group is connected to the first modification group and the second modification group, and the fourth modification group is connected to the solid support. Therefore, a large number of the double-ended circularization primers can be fixed on the solid support at the same time, laying the foundation for the subsequent simultaneous circularization reaction with a large number of double-stranded nucleic acid template molecules to obtain a large number of circularized nucleic acid templates.
  • the third modification group and the fourth modification group are suitable for forming at least one of the following support linking groups: avidin or streptavidin-biotin group, amino-NHS ester or aldehyde group or hydroxymethylphosphine or carboxyl group, hydroxyl-isocyanate group, acrylamide-silyl group, azide-alkyne group and thiol-maleimide or haloacetyl or thiosulfonate group.
  • the support linking group is formed between the third modification group and the fourth modification group, the double-ended cyclized primer can be better fixed on the solid support.
  • the solid support comprises at least one selected from magnetic beads, gel beads, glass beads, glass slides, nanogold particles, polymer objects or chips.
  • the present invention proposes a method for obtaining a circularized nucleic acid template.
  • the method comprises: performing a circularization reaction on a double-terminal circularization primer and a double-stranded nucleic acid template molecule, wherein the double-stranded nucleic acid template molecule comprises a sense nucleic acid template strand and an antisense nucleic acid template strand, and the two 3' ends of the double-terminal circularization primer are respectively complementary to at least part of the nucleic acid sequences of the 5' end and the 3' end of the sense nucleic acid template strand and the antisense nucleic acid template strand, so as to obtain a circularized nucleic acid template, wherein the circularized nucleic acid template has a sense nucleic acid template chain loop and an antisense nucleic acid template chain loop; wherein the double-terminal circularization primer comprises a first nucleic acid single-stranded molecule and a second nucle
  • nucleic acid sense chain nanoballs and nucleic acid antisense chain nanoballs can be simultaneously obtained after rolling circle amplification, and then the sense chain and the antisense chain can be sequenced without going through a traditional multiple strand displacement amplification reaction. This can avoid problems such as failure of Read2 template chain synthesis, low efficiency, or preference caused by cumulative damage to Read1 sequencing during the multiple strand displacement amplification reaction.
  • the method for obtaining a circularized nucleic acid template may further include at least one of the following additional technical features:
  • the double-ended circularization primer includes a first free 3' end and a second free 3' end, wherein the first free 3' end is at least partially complementary to the joint of the 5' end and 3' end of the sense nucleic acid chain, and the second free 3' end is at least partially complementary to the joint of the 5' end and 3' end of the antisense nucleic acid chain; or the second free 3' end is at least partially complementary to the joint of the 5' end and 3' end of the sense nucleic acid chain, and the first free 3' end is at least partially complementary to the joint of the 5' end and 3' end of the antisense nucleic acid chain. Therefore, the sense nucleic acid chain and the antisense nucleic acid chain can be complementary to the double-ended circularization primer, so that the sense nucleic acid chain and the antisense nucleic acid chain are each ringed.
  • the two ends of the double-stranded nucleic acid template molecule are connected with a linker, and the sequences of the linkers connected to the 5' end of the sense nucleic acid template strand and the 5' end of the antisense nucleic acid template strand are different. Therefore, the 5' end of the sense nucleic acid template strand and the 5' end of the antisense nucleic acid template strand can be respectively combined with the two free 3' ends of the double-ended circularization primer to form a ring.
  • the 5' end of the double-stranded nucleic acid template molecule has a free phosphate group, and the 3' end of the double-stranded nucleic acid template molecule has a free hydroxyl group.
  • free in the “free phosphate group” or “free hydroxyl group” means that the phosphate group or hydroxyl group is connected to the 5’ end or 3’ end of the double-stranded nucleic acid template molecule, and is no longer connected to other nucleotide molecules or groups.
  • the method further comprises: performing a rolling circle amplification reaction on the circularized nucleic acid template to obtain a nucleic acid molecule having multiple copies of a sense strand and an antisense strand.
  • the circularized nucleic acid template is loaded on a sequencing chip; the circularized nucleic acid template loaded on the sequencing chip is subjected to the rolling circle amplification reaction to obtain a nucleic acid molecule having multiple copies of a sense strand and an antisense strand at the same time.
  • the nucleic acid molecule can sequence the sense strand and the antisense strand without undergoing a traditional multiple strand displacement amplification reaction, and can avoid problems such as failure of synthesis of the Read2 template strand, low efficiency, or generation of bias due to accumulated damage to the Read1 sequencing during the multiple strand displacement amplification reaction.
  • the double-stranded nucleic acid template molecule is prepared by the following method: the nucleic acid double-stranded template is subjected to 3' end A treatment, the nucleic acid double-stranded template has and only has one 5' end with a free phosphate group, and the other ends have free hydroxyl groups; the 3' end A treatment product is subjected to a first connection treatment with a first connector; the first connection treatment product is subjected to a first 5' end phosphorylation treatment; the first 5' end phosphorylation treatment product is subjected to a second connection treatment with a second connector; and the second connection treatment product is subjected to a second 5' end phosphorylation treatment, so as to obtain the double-stranded nucleic acid template molecule.
  • different connectors can be added to both ends of the double-stranded nucleic acid template molecule, and the two single strands obtained after the double-stranded nucleic acid template molecule is melted can be specifically hybridized.
  • the gap caused by the first connection process needs to be completed.
  • the first connection process only one (because there is only one) 5'-P is connected to the adjacent 3'-OH to form a phosphodiester bond, and the 5'-OH on the other side of the same end cannot be connected to the 3'-OH, resulting in a gap.
  • the second connection process after the 5' end is phosphorylated, the 5'-OH at the gap is converted to 5'-P, and then connected to the 3'-OH under the action of the ligase to form another phosphodiester bond, which is called completion.
  • the 3' end A addition treatment is performed in the presence of Taq polymerase at a temperature of 72°C for 20 minutes.
  • the 3' end A addition treatment is performed by performing a PCR reaction in the presence of Taq polymerase.
  • the number of amplification rounds of the PCR reaction is no more than 8.
  • the generation of mutations can be reduced.
  • the first connection process and the second connection process are performed under the action of T4 nucleic acid ligase.
  • T4 nucleic acid ligase can efficiently catalyze the phosphodiester bond between the 5'-P end and the 3'-OH end of the blunt-ended double-stranded nucleic acid template molecule, thereby connecting the adapter to the 5' end of the double-stranded nucleic acid template.
  • the first 5' end phosphorylation treatment and the second 5' end phosphorylation treatment are carried out under the action of polyphosphate kinase.
  • Polyphosphatase can be used to introduce phosphate groups at the first and second 5' ends.
  • the first connection treatment further includes subjecting the 3' end treated with A to a first purification treatment.
  • the first ligation treatment product is further subjected to a second purification treatment.
  • the first 5' end phosphorylation treatment product is further subjected to a third purification treatment.
  • the first purification treatment, the second purification treatment and the third purification treatment are independently selected from magnetic bead purification.
  • the purification time can be reduced, and the first purification treatment product, the second purification treatment product and the third purification treatment product after purification are highly efficient, will not cause mechanical damage to the nucleic acid, and have excellent repeatability.
  • the input amount of the nucleic acid to be purified is not less than 1500ng, and the volume of the elution solution is not more than 50 ⁇ l.
  • the concentration of the nucleic acid can be effectively increased.
  • the first connector and the second connector are pre-annealed in advance. Then, the first connector and the second connector are connected to the two ends of the nucleic acid double-stranded template in the form of double strands.
  • the double-terminal circularization primer is pre-purified.
  • the pre-purification treatment is performed by urea polyacrylamide gel electrophoresis or high performance liquid chromatography.
  • the annealing treatment further includes subjecting the annealing product to a third connection treatment, whereby the sense nucleic acid template strand and the antisense nucleic acid template strand are each connected into a ring, so as to obtain the circularized nucleic acid template molecule.
  • connection process refers to connecting the 5' end and 3' end of the sense nucleic acid template chain to the 5' end and 3' end of the antisense nucleic acid template chain.
  • the third connection process is performed under the action of T4 nucleic acid ligase.
  • T4 nucleic acid ligase can efficiently catalyze the phosphodiester bond between the 5'-P end and the 3'-OH end of the blunt-ended nucleic acid, thereby connecting the 5' end of the sense nucleic acid template strand to the 3' end, and connecting the 5' end of the antisense nucleic acid template strand to the 3' end.
  • the double-stranded nucleic acid template molecule is prepared by the following method: the double-stranded nucleic acid template is subjected to end repair and A addition treatment, and the treatment product is connected with two adapters to obtain the double-stranded nucleic acid template molecule.
  • different adapters can be added to both ends of the double-stranded nucleic acid template molecule, and the two single strands obtained after the double-stranded nucleic acid template molecule is melted can be specifically hybridized.
  • the linker is a partially complementary double-stranded linker, and the nucleic acid sequences of the non-linking ends of the two linkers are different from each other.
  • the double-ended circularization primer is fixed on a solid support, a high molecular weight polymer or a chip, so that the double-ended circularization primer is in contact with a double-stranded nucleic acid template molecule, and a hybridization ligation circularization reaction is performed in the presence of a ligase. Therefore, a large number of double-stranded nucleic acid template molecules can be combined with the double-ended circularization primer, so that a large number of circularized nucleic acid templates formed are simultaneously fixed on a solid support, a high molecular weight polymer or a chip, laying a foundation for subsequent simultaneous sequencing to obtain a large amount of nucleic acid double-end sequence information.
  • the present invention provides a circularized nucleic acid molecule obtained by the method described above.
  • the circularized nucleic acid molecule obtained by this method can obtain nucleic acid sense strand nanospheres and nucleic acid antisense strand nanospheres after rolling circle amplification, and then the sense strand and the antisense strand can be sequenced at the same time, without going through the traditional multiple strand displacement amplification reaction, and can avoid the failure of Read2 template strand synthesis, low efficiency or bias caused by Read1 sequencing cumulative damage during the multiple strand displacement amplification reaction.
  • the present invention proposes a cyclized nucleic acid molecule.
  • the cyclized nucleic acid molecule includes: including: the double-ended cyclization primer as described above and a sense nucleic acid template strand and an antisense nucleic acid template strand, the sense nucleic acid template strand and the antisense nucleic acid template strand are reversely complementary, the two ends of the sense nucleic acid template strand and the antisense nucleic acid template strand are connected with a linker sequence, the 5' end of the sense nucleic acid template strand and the 5' end of the antisense nucleic acid template strand have different linker sequences, and the 3' end of the sense nucleic acid template strand and the 3' end of the antisense nucleic acid template strand have different linker sequences; wherein the two 3' ends of the double-ended cyclization primer are complementary to at least part of the nucleic acid sequence at the 5' end and
  • nucleic acid sense chain nanospheres and nucleic acid antisense chain nanospheres can be obtained at the same time, and then the sense chain and the antisense chain can be sequenced without going through the traditional multiple strand displacement amplification reaction. This can avoid problems such as failure of Read2 template chain synthesis, low efficiency or preference caused by cumulative damage to Read1 sequencing during the multiple strand displacement amplification reaction.
  • the double-ended circularized primer and the sense nucleic acid template strand and the antisense nucleic acid template strand are fixed on a solid support, a polymer or are free in a solution.
  • the solid support includes at least one selected from magnetic beads, gel beads, glass beads, glass slides, nanogold particles and chips.
  • the present invention proposes a nucleic acid sequencing chip.
  • the nucleic acid sequencing chip includes: a plurality of nucleic acid sample binding sites and a plurality of nucleic acid molecule complexes fixed to the nucleic acid sample binding sites, wherein the nucleic acid molecule complexes include a sense nucleic acid template strand, an antisense nucleic acid template strand and a double-ended circularization primer, the double-ended circularization primer is connected to the sense nucleic acid template strand, and the double-ended circularization primer is connected to the antisense nucleic acid template strand.
  • the nucleic acid sequencing chip can sequence the sense strand and the antisense strand at the same time, and can obtain nucleic acid double-end sequence information directly through double-end sequencing without the traditional multiple strand displacement amplification reaction, which can greatly simplify the sequencing process, reduce sequencing time and sequencing costs.
  • the sense nucleic acid template chain and the antisense nucleic acid template chain are single-stranded circular structures, and the two 3' ends of the double-ended circularization primer are complementary to at least part of the nucleic acid sequence at the 5' end and 3' end of the sense nucleic acid template chain or the antisense nucleic acid template chain, respectively.
  • the nucleic acid molecule complex includes multiple copies of sense nucleic acid strands and multiple copies of antisense nucleic acid strands, and thus the sense strands and antisense strands can be sequenced directly without multiple strand displacement amplification reactions, thereby obtaining true sequence information of double-end sequencing.
  • the multiple sense nucleic acid chain copies and the multiple antisense nucleic acid chain copies are obtained by subjecting the sense nucleic acid template chain and the antisense nucleic acid template chain to rolling circle amplification.
  • nucleic acid molecule complex may include a sense nucleic acid template strand and an antisense nucleic acid template strand, and may also include multiple sense nucleic acid strand copies and multiple antisense nucleic acid strand copies after rolling circle amplification of the sense nucleic acid template strand and the antisense nucleic acid template strand.
  • the "nucleic acid molecule complex” includes multiple sense nucleic acid strand copies and multiple antisense nucleic acid strand copies formed after rolling circle amplification of the sense nucleic acid template strand and the antisense nucleic acid template strand
  • the "nucleic acid molecule complex” at this time has the same structure as the “double nucleic acid nanoball”; the “multiple sense nucleic acid strand copies” and the “nucleic acid sense strand nanoball” and the “multiple antisense nucleic acid strand copies” described in this application have the same structure as the "nucleic acid antisense strand nanoball".
  • the two ends of the sense nucleic acid template strand and the antisense nucleic acid template strand are connected with a linker sequence
  • the 5' end of the sense nucleic acid template strand and the 5' end of the antisense nucleic acid template strand have different linker sequences
  • the 3' end of the sense nucleic acid template strand and the 3' end of the antisense nucleic acid template strand have different linker sequences. Therefore, the 5' end of the sense nucleic acid template strand and the 5' end of the antisense nucleic acid template strand can be respectively combined with the two free 3' ends of the double-ended circularization primer to form a ring.
  • one or both ends of the sense nucleic acid template chain and the antisense nucleic acid template chain further include a label sequence, or a sample label sequence and a molecular label sequence, etc., which can distinguish label information of the sample source and molecular label information for counting the number of nucleic acid molecules.
  • the biological sample is RNA
  • a reverse transcription reaction, linker addition or amplification library construction step is required.
  • the linker sequences at both ends of the sense nucleic acid template strand and the antisense nucleic acid template strand are introduced by PCR amplification reaction.
  • a primer pair or primer set with a specific sequence and a universal sequence is used to perform an amplification reaction on the target region of the nucleic acid sample to obtain a double-stranded nucleic acid fragment with linker sequences at both ends.
  • the present invention proposes a method for preparing a dual nucleic acid nanosphere.
  • the method comprises: subjecting the aforementioned cyclized nucleic acid molecule to a rolling circle amplification process to obtain a dual nucleic acid nanosphere.
  • the dual nucleic acid nanosphere obtained by this method has both a nucleic acid sense strand nanosphere and a nucleic acid antisense strand nanosphere, and can obtain nucleic acid double-end sequence information directly through double-end sequencing without undergoing a traditional multiple strand displacement amplification reaction, which can greatly simplify the sequencing process, reduce sequencing time and sequencing costs.
  • the rolling circle amplification process is carried out for 40 minutes under the action of DNB enzyme I MIX and DNB enzyme II at a temperature of 30°C.
  • NDB enzyme I MIX refers to Make DNB enzyme MIX I and Make DNB enzyme II, both of which are products from MGI and are components of the MGISEQ-2000RS high-throughput sequencing reagent set (FCL PE100).
  • the present invention provides a dual nucleic acid nanoball.
  • the dual nucleic acid nanoball is prepared by the method described above.
  • the dual nucleic acid nanoball contains both a nucleic acid sense strand nanoball and a nucleic acid antisense strand nanoball, and can directly sequence the sense strand or the antisense strand without multiple strand displacement amplification reactions, thereby obtaining true sequence information for double-end sequencing.
  • the double nucleic acid nanoball comprises a nucleic acid sense chain nanoball and a nucleic acid antisense chain nanoball.
  • the present invention provides a nucleic acid library.
  • the nucleic acid library includes the dual nucleic acid nanoballs described above.
  • the library has a large capacity and a high success rate of pairing with the nucleic acid to be tested.
  • the nucleic acid library constructed using the present invention can then be sequenced for both the sense strand and the antisense strand at the same time, and a high-throughput sequencing library can be constructed simply and efficiently, and a sequencing plate can be efficiently used, thereby increasing the success rate of library construction and reducing the loss of samples and reagents.
  • the present invention provides a gene sequencing method.
  • the aforementioned nucleic acid library is subjected to sequencing.
  • the nucleic acid library is used for sequencing, and the sense strand and the antisense strand can be sequenced at the same time, so that the sequencing information can be more complete, the target fragment can be captured efficiently, and an accurate sequencing result can be obtained.
  • sequence overlap is generated at the tail end of double-end sequencing, and the error rate caused by sequencing accumulation can be reduced by mutual correction.
  • the sequencing is performed on the MGI-SEQ2000 platform.
  • the technical advantages of the present invention include the following aspects:
  • the present invention provides a method for preparing a double-stranded nucleic acid template molecule. This method can add different types of linkers to both ends of the double-stranded nucleic acid template molecule, and the two single strands after the library is unzipped can be specifically hybridized.
  • the present invention provides a method for performing rolling circle amplification by combining a double-stranded nucleic acid template molecule and a double-ended circularization primer, and obtaining double nucleic acid nanoballs for single-end or double-end sequencing.
  • the present invention provides a method for preparing a double-terminal circularized primer, which can arbitrarily connect the 5' end or 3' end of different primers through biochemical molecules with mutual reaction ability to obtain new forward or reverse primers.
  • the sense nucleic acid template strand and the antisense nucleic acid template strand of the double-stranded nucleic acid template molecule prepared by the present invention can be closed into a ring by end-connection to obtain a circular library. Therefore, this method can be used for the preparation of a circular library.
  • the method for preparing a double-stranded nucleic acid template molecule provided by the present invention can connect any linkers, so that different types of linkers are obtained at both ends of the double strand to meet sequencing requirements.
  • digestion can also be performed directly after cyclization to obtain the sense chain and antisense chain circular libraries, and then primers that can complementally pair with the sense chain and antisense chain circular libraries are used to capture them, followed by a rolling circle amplification reaction.
  • the method of connecting primers in the present invention can be applied to any variety of primers to achieve the connection of multiple different primers.
  • the primers used in the construction of DNA nanospheres in the examples were synthesized by Sangon Biotechnology Co., Ltd.
  • the sequence structures of the primers are shown in Table 1:
  • Ad Lig buffer (ligation reaction buffer), Ad ligase (T4 DNA ligase) and Ligation Enhancer (ligation enhancer) used in the examples are all from MGI MGIEasy enzyme cutting PCR-Free DNA library preparation kit
  • the magnetic bead purification reagent is from MGI MGIEasy DNA purification magnetic bead kit
  • T4 PNK (Cat#1000007884), T4 DNA Ligase (Cat#1000007877), 10*ligation buffer (Cat#1000007874), 10*phi29 buffer (Cat#1000004657), rTaq PCR ReadyMix (2x) (Cat#1000004656), and rTaq DNA Polymerase (Cat#1000004443) are all purchased from MGI.
  • the sequencer and handheld loader are all produced and provided by Wuhan MGI, and the sequencing slides, sequencing primers, sequencing reagents, Make DNB enzyme MIX I and Make DNB enzyme II, and stop run buffer are all from the MGISEQ-2000RS High-throughput Sequencing Reagent Set (FCL PE100) (Cat#1000012554).
  • FCL PE100 High-throughput Sequencing Reagent Set
  • V3 PCR product reagents are produced and provided by Wuhan MGI.
  • V3 PCR product (MGI, V3 PCR product reagent E. coli) as template, prepare the reaction system according to the conditions in Table 2, and use Taq enzyme to perform terminal A addition reaction by PCR.
  • the product was purified using 1 volume of magnetic beads (MGIEasy DNA Purification Magnetic Beads Kit) and named dA-tailed dsDNA.
  • the DNA concentration was determined on a Qubit 4 Fluorometer (ThermoFisher, Cat#Q33230) using the Qubit 1X dsDNA HS Assay Kit (ThermoFisher Cat#Q33230).
  • the reaction system was prepared according to the conditions in Table 4. The reaction conditions were: 95°C for 3 min, then the temperature was slowly lowered by 1°C every 2 min to 25°C, and then stored at 4°C.
  • the Y-shaped cyclized adapter 1 was obtained and named splint adapter 1.
  • the reaction system was prepared according to the conditions in Table 5. The reaction conditions were: 95°C for 3 min, then the temperature was slowly lowered to 25°C by decreasing 1°C every 2 min, and stored at 4°C.
  • the Y-shaped cyclized adapter 2 was obtained and named splint adapter 2.
  • dA-tailed dsDNA 52 ⁇ L splint adapter 1 (40 ⁇ M) 3 ⁇ L Ad Lig buffer 18 ⁇ L Ad ligase (T4 DNA ligase) 5 ⁇ L Ligation Enhancer 2 ⁇ L
  • the reaction conditions were 25°C for 10 min, followed by overnight storage at 4°C. The next day, the mixture was taken out at 4°C and placed at room temperature for 30 min to equilibrate to room temperature.
  • the product was purified using 0.8 times the volume of magnetic beads (MGIEasy DNA Purification Magnetic Beads Kit) and the purified product was named SA1-dA-tailed dsDNA.
  • the reaction conditions were 37°C for 1h.
  • the product was purified using 1 volume of magnetic beads (MGIEasy DNA Purification Magnetic Beads Kit) to obtain a purified product named 5P-SA1-dA-tailed dsDNA.
  • the DNA concentration was determined on a Qubit 4 Fluorometer (ThermoFisher, Cat#Q33230) using the Qubit 1X dsDNA HS Assay Kit (ThermoFisher, Cat#Q33230).
  • the reaction conditions were 25°C for 10 min, followed by overnight storage at 4°C. The next day, the mixture was taken out at 4°C and placed at room temperature for 30 min to equilibrate to room temperature.
  • the product was purified using 1 volume of magnetic beads (MGIEasy DNA Purification Magnetic Beads Kit) and the purified product was named SA2+SA1 dsDNA.
  • the reaction conditions were 37°C for 1h.
  • the product was purified using 1.5 times the volume of magnetic beads (MGIEasy DNA Purification Magnetic Beads Kit), and the purified product was named Dual splint dsDNA library.
  • the DNA concentration was measured on Qubit 4 Fluorometer (ThermoFisher, Cat#Q33230) using Qubit 1X dsDNA HS Detection Kit (ThermoFisher, Cat#Q33230), and the concentration was 6.36ng/ ⁇ L.
  • Two single-stranded DNA primers with 5'-ends modified with coupling chemical molecules are used, here a pair of primers modified with 5' azide (Azide) and 5' benzylcyclooctyne (DBCO) are used to prepare a reaction system according to the conditions in Table 10 for coupling.
  • Azide 5' azide
  • DBCO 5' benzylcyclooctyne
  • reaction conditions were 37°C water bath for 48 h.
  • Dual splint dsDNA library (6.36 ng/ ⁇ L) 9.5 ⁇ L
  • Dual splint oligo(0.28 ⁇ M) 0.6 ⁇ L
  • 10*phi29 buffer (annealing buffer) 2 ⁇ L ddH2O 7.9 ⁇ L
  • Double-end annealing product 20 ⁇ L T4 DNA ligase 1.6 ⁇ L 10*ligation buffer 2.4 ⁇ L
  • reaction conditions were 30°C for 30 min to obtain a double-end annealing cyclization product.
  • Double-end annealing cyclization product 24 ⁇ L Make DNB enzyme I MIX 20 ⁇ L Make DNB enzyme II 2 ⁇ L
  • the reaction conditions were 30°C for 40min, after which 10 ⁇ L stop run buffer was added to terminate the reaction.
  • Rolling circle amplification obtained DNA nanospheres with both positive and antisense strands.
  • the concentration of DNA nanospheres was determined on a Qubit 4 Fluorometer (ThermoFisher, Cat#Q33230) using the Qubit ssDNA detection kit (ThermoFisher, Cat#Q10212) and the concentration was 19.3ng/ ⁇ L.
  • the prepared DNA nanoballs were loaded onto the sequencing slide by the handheld loader, and the slide was placed in the sequencing chamber at room temperature for 30 minutes, and the MGISEQ-2000 standard PE100 sequencing kit was placed in it.
  • the PE100 sequencing script with the MDA process removed was used for on-machine testing. After obtaining the sequence information, it was compared with the Escherichia coli reference genome. The results are shown in Table 15. Without performing multiple strand displacement amplification reactions, the double-end PE information of the sequencing library was successfully obtained. The Q30 of the sequencing data after filtering reached 87.56%, and the alignment rate with the reference species genome sequence reached 86.73%, and the real double-end sequence information could be obtained.
  • Sequencing indicators DNA double-stranded nanoball sequencing results
  • Reference species Escherichia coli Sequencing cycle number 212 Filtered read length (M) 12.47 The read length that can be mapped to the reference genome (M) 10.81 Q30 after filtration (%) 87.56 One-strand sequencing lead ratio (%) 0.08 The proportion of double-strand sequencing ahead of time (%) 0.09 One-strand sequencing lag ratio (%) 0.13 Second-strand sequencing lag ratio (%) 0.12 Number of sequences with paired-end information 62229 The number of sequences that can be aligned to the reference genome 54651 Comparison rate (%) 86.73

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Abstract

La présente invention concerne une amorce de cyclisation à double extrémité qui comprend une première molécule simple brin d'acide nucléique et une deuxième molécule simple brin d'acide nucléique. L'extrémité 5' de la première molécule simple brin d'acide nucléique est reliée à l'extrémité 5' de la deuxième molécule simple brin d'acide nucléique au moyen d'un adaptateur. L'amorce de cyclisation obtenue en connectant la première molécule d'acide nucléique simple brin à la deuxième molécule d'acide nucléique simple brin au moyen de l'adaptateur peut être appariée de manière complémentaire avec les séquences des parties adaptatrices d'extrémité de queue des brins sens et antisens d'une molécule matrice d'acide nucléique double brin en même temps ; une réaction de cyclisation est effectuée ; après l'amplification en cercle roulant, une nanosphère d'acide nucléique de brin sens et une nanosphère d'acide nucléique de brin antisens peuvent être obtenues en même temps, puis les brins sens et antisens peuvent être séquencés ; des réactions d'amplification par déplacement de brins multiples ne sont pas nécessaires, de sorte que les étapes d'hybridation, de déroulement et d'élimination de la matrice et autres peuvent être supprimées, ce qui réduit le temps nécessaire au séquençage ultérieur.
PCT/CN2022/137336 2022-12-07 2022-12-07 Procédé de préparation basé sur un procédé en une étape pour nanosphère d'acide nucléique double Ceased WO2024119413A1 (fr)

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EP3225721B1 (fr) * 2014-11-26 2019-07-24 MGI Tech Co., Ltd. Procédé et réactif pour la construction d'une banque d'acide nucléique simple brin circulaire à double séquence de liaison
EP3783109B1 (fr) * 2015-03-31 2024-05-29 Illumina Cambridge Limited Surface de concatemerisation de matrices
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EP4208470A4 (fr) * 2020-10-22 2024-10-30 Singular Genomics Systems, Inc. Circularisation et amplification d'acide nucléique sur une surface
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