WO2024119413A1 - 基于一步法的双核酸纳米球制备方法 - Google Patents

基于一步法的双核酸纳米球制备方法 Download PDF

Info

Publication number
WO2024119413A1
WO2024119413A1 PCT/CN2022/137336 CN2022137336W WO2024119413A1 WO 2024119413 A1 WO2024119413 A1 WO 2024119413A1 CN 2022137336 W CN2022137336 W CN 2022137336W WO 2024119413 A1 WO2024119413 A1 WO 2024119413A1
Authority
WO
WIPO (PCT)
Prior art keywords
nucleic acid
acid template
double
molecule
stranded
Prior art date
Application number
PCT/CN2022/137336
Other languages
English (en)
French (fr)
Inventor
杨晋
曹硕
吴慧
徐崇钧
Original Assignee
深圳华大智造科技股份有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 深圳华大智造科技股份有限公司 filed Critical 深圳华大智造科技股份有限公司
Priority to PCT/CN2022/137336 priority Critical patent/WO2024119413A1/zh
Publication of WO2024119413A1 publication Critical patent/WO2024119413A1/zh

Links

Images

Classifications

    • 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 5' end of the sense nucleic acid template strand and the 5' end of the antisense nucleic acid template strand have different joint 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 joint 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 3
  • 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 purification gel image of a dual splint oligo 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, nano-gold 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 generated by the first single-stranded nucleic acid molecule or the second single-stranded nucleic acid molecule itself, and to avoid the first single-stranded nucleic acid molecule and the second single-stranded nucleic acid 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 includes 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 dual 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 nanosphere 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

Landscapes

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

Abstract

提出了一种双端环化引物，包括第一核酸单链分子和第二核酸单链分子，第一核酸单链分子的5'端与第二核酸单链分子的5'端通过连接子相连。通过连接子将第一核酸单链分子和第二核酸单链分子连接在一起后所得到的环化引物可同时与双链核酸模板分子的正义链和反义链末端接头部分序列互补配对，进行环化反应，经滚环扩增后，可同时获得正义链核酸纳米球和反义链核酸纳米球，进而可对正义链和反义链进行测序，无需多重链置换扩增反应，能够省去杂交、解链、去掉模板等步骤，为后续的测序缩短了时间。

Description

基于一步法的双核酸纳米球制备方法 技术领域
本发明涉及基因测序领域。具体地，本发明涉及一种基于一步法的双核酸纳米球制备方法。
背景技术
随着测序技术的发展和社会认知度的提高，高通量基因测序已广泛应用于科学研究、精准医学、社会卫生和公共安全等各个领域。近年来，大规模平行测序(MPS)的成本不断下降，2022年上半年Ultima Genomics公司发布的UG100测序仪，号称可以实现SE300测序，每个WGS(全基因组测序，100Gb数据)的测序成本可降低到100美元；2022年9月30号，美国Illumina公司发布了最新款的NovaSeq X系列测序仪，可实现PE150测序，测序成本可达到200美元WGS。预期在未来5年内，主流MPS测序平台的测序成本将普遍进入100美元每个WGS的时代。随着测序成本降低到普通大众可以接受的水平，将显著促进高通量测序在传感染、肿瘤防治、新生儿疾病筛查等方面的应用。
发明内容
本发明旨在至少在一定程度上解决现有技术中存在的技术问题至少之一。
在WGS应用当中，双末端测序(Paired-end sequencing，简称为PE测序)是非常重要的测序技术手段，通过该技术，可以在短读长测序下，获得DNA链5’和3’末端的序列信息，因这两个末端的序列信息具有配对的特征，可以确认来自于同一条DNA链，对于基因组组装以及变异检测(如***/缺失)等方面具有重要意义。同时，通过双末端测序，也可以实现一次测序即获取更多序列信息，有助于缩短测序时间，降低测序成本。
目前市面上的MPS测序仪产品中，主流的双末端测序技术包括以下几种：
1、Illumina公司(如图1所示)：在完成单末端测序以后，对单末端测序链(记为Read1)进行延伸；随后洗去测序链，以模板链与芯片表面的另一种引物(记为P5’)结合，并从P5’引物开始延伸获得模板链的互补链，再通过洗去模板链，只保留P5’延伸链，以它作为双末端模板链，杂交引物后进行双末端测序(记为Read2)。对于Illumina的双末端测序技术而言，其主要缺点是步骤较为复杂，涉及到杂交、延伸、解链、去掉模板链等多个步骤， 耗时较长；
2、华大智造(如图2所示)：在DNB(DNA纳米球)上完成单末端测序后，与模板链上的接头杂交引物，通过MDA(多重置换扩增)反应，进行Read1的延伸，在一个DNB上，同时会有多个测序链向前延伸，在前一条测序链的3’末端遇到后一条测序链的5’端时，会将后一条测序链从5’端开始，从模板链上置换下来。因MDA反应在不同的测序链上前进速度接近，因此虽然测序链的5’端会被置换下来，但3’端依然与模板链互补。MDA反应结束后，测序链会“漂”在DNB的上方，此时可以杂交Read2的测序引物，从而完成双末端测序。对于华大智造的双末端测序技术而言，同样步骤较为复杂，涉及到引物杂交、延伸、链置换、末端封闭等步骤，耗时较长；同时MDA反应体系对温度较为敏感，对于试剂储存、运输和使用均提出了一定的要求。
当前主流技术都需要在Read1测序完成后，进行单独的双末端测序模板链合成的步骤。
以DNA纳米球为测序单元进行的双端测序一般包括以下几个步骤：(1)获得测序文库。(2)由文库制备DNA正义链纳米球。(3)杂交特异性引物进行DNA正义链测序。(4)通过多重链置换扩增反应获得DNA反义链。(5)杂交特异性引物进行DNA反义链测序。传统的双端测序方式需要通过多重链置换扩增反应获得DNA反义链后方能进行反义链测序，步骤繁琐耗时长。
本发明提供了一种DNA纳米球的制备方法，这种DNA纳米球同时包含有DNA正义链纳米球和DNA反义链纳米球，可直接进行正义链或反义链的测序而无需多重链置换扩增反应，获得双端测序的真实序列信息。
因此，在本发明的一个方面，本发明提出了一种双端环化引物。根据本发明的实施例，所述双端环化引物包括第一核酸单链分子和第二核酸单链分子，所述第一核酸单链分子的5’端与第二核酸单链分子的5’端通过连接子相连。通过连接子将第一核酸单链分子和第二核酸单链分子连接在一起后所得到的环化引物可同时与双链核酸模板分子的正义链和反义链末端接头部分序列互补配对，进行环化反应，经滚环扩增后，可同时获得核酸正义链纳米球和核酸反义链纳米球，进而可对正义链和反义链进行测序，无需再进行多重链置换扩增反应，能够省去杂交、解链、去掉模板等步骤，为后续的测序缩短了时间。
在本发明的另一个方面，本发明提出了一种制备前面所述的双端环化引物的方法。根据本发明的实施例，所述方法为将第一核酸单链分子和第二核酸单链分子进行连接反应，以便获得所述双端环化引物；其中，所述第一核酸单链分子的5’端连接有第一修饰基团，所述第二核酸单链分子的5’端连接有第二修饰基团，所述第一修饰基团与第二修饰基团适于发生连接反应；所述第一核酸单链分子的3’端与第二核酸单链分子的3’端不适于发生连 接反应；所述第一核酸单链分子和第二核酸单链分子之间不能互补配对。通过在第一核酸单链分子及第二核酸单链分子的5’端连接修饰基团，能够使两条核酸单链连接起来，形成双端环化引物，该引物可同时与双链核酸模板分子的正义链和反义链末端接头部分序列互补配对，进行环化反应，经滚环扩增后，可同时获得核酸正义链纳米球和核酸反义链纳米球，进而可对正义链和反义链进行测序，无需再进行多重链置换扩增反应，能够省去杂交、解链、去掉模板等步骤，为后续的测序缩短了时间。
在本发明的又一个方面，本发明提出了一种获得环化核酸模板的方法。根据本发明的实施例，所述方法包括：将双端环化引物与双链核酸模板分子进行环化反应，所述双链核酸模板分子包括正义核酸模板链和反义核酸模板链，所述双端环化引物的两个3’端分别与正义核酸模板链和反义核酸模板链的5’端和3’端的至少部分核酸序列互补配对，以便获得环化核酸模板，所述环化核酸模板具有正义核酸模板链环和反义核酸模板链环；其中，所述双端环化引物包括第一核酸单链分子和第二核酸单链分子，所述第一核酸单链分子的5’端与第二核酸单链分子的5’端通过连接子相连，如前面所限定的或根据前面所述的方法制备获得；所述双链核酸分子的两端连接有不同的接头。根据本发明实施例的环化核酸模板进行环化反应，经滚环扩增后，可同时获得核酸正义链纳米球和核酸反义链纳米球，进而可对正义链和反义链进行测序，无需再经过传统的多重链置换扩增反应，可以避免在多重链置换扩增反应过程中，因Read1测序累积损伤造成Read2模板链合成失败、效率低或者产生偏好性等问题。
在本发明的又一个方面，本发明提出了一种环化核酸分子，是通过前面所述的方法获得的。通过此方法获得的环化核酸分子，经滚环扩增后，可同时获得核酸正义链纳米球和核酸反义链纳米球，进而可对正义链和反义链进行测序，能够不经过传统的多重链置换扩增反应，可以避免在多重链置换扩增反应过程中，因Read1测序累积损伤造成Read2模板链合成失败、效率低或者产生偏好性等问题。
在本发明的又一个方面，本发明提出了一种环化核酸分子。根据本发明的实施例，所述环化核酸分子包括：前面所述的双端环化引物以及正义核酸模板链和反义核酸模板链，所述正义核酸模板链和所述反义核酸模板链反向互补，所述正义核酸模板链和所述反义核酸模板链的两端连接有接头序列，所述正义核酸模板链的5’端和所述反义核酸模板链的5’端的接头序列不同，所述正义核酸模板链的3’端和所述反义核酸模板链的3’端的接头序列不同；其中，所述双端环化引物的两个3’端分别与正义核酸模板链或反义核酸模板链的5’端和3’端的至少部分核酸序列互补配对。所述环化核酸分子，经滚环扩增后，可同时获得核酸正义链纳米球和核酸反义链纳米球，进而可对正义链和反义链进行测序，能够不经过 传统的多重链置换扩增反应，可以避免在多重链置换扩增反应过程中，因Read1测序累积损伤造成Read2模板链合成失败、效率低或者产生偏好性等问题。
在本发明的又一个方面，本发明提出了一种核酸测序芯片。根据本发明的实施例，所述核酸测序芯片包括：多个核酸样本结合位点和多个固定于所述核酸样本结合位点的核酸分子复合物，其中，所述核酸分子复合物包含正义核酸模板链、反义核酸模板链和双端环化引物，所述双端环化引物与所述正义核酸模板链相连，所述双端环化引物与所述反义核酸模板链相连。所述核酸测序芯片可以任何顺序对正义链和反义链进行测序，可以不经过传统的多重链置换扩增反应，直接通过双端测序获得核酸双端序列信息，可以极大简化测序流程、降低测序时间和测序成本。
在本发明的又一个方面，本发明提出了一种制备双核酸纳米球的方法，根据本发明的实施例，所述方法包括：将前面所述的环化核酸分子进行滚环扩增处理，以便获得双核酸纳米球。通过此方法获得的双核酸纳米球同时具有核酸正义链纳米球和核酸反义链纳米球，可以不经过传统的多重链置换扩增反应，直接通过双端测序获得核酸双端序列信息，可以极大简化测序流程、降低测序时间和测序成本。
在本发明的又一个方面，本发明提出了一种双核酸纳米球。根据本发明的实施例，所述双核酸纳米球由前面所述的方法制备得到。所述双核酸纳米球同时包含有核酸正义链纳米球和核酸反义链纳米球，可直接进行正义链或反义链的测序而无需多重链置换扩增反应，获得双端测序的真实序列信息。
在本发明的又一个方面，本发明提出了一种核酸文库。根据本发明的实施例，所述核酸文库包括前面所述的双核酸纳米球。所述文库容量大，与待测核酸配对的成功率高。使用本发明构建的核酸文库，进而可同时对正义链和反义链进行测序，也可以根据需要调整测序顺序，分别测序。能够简单、高效地构建高通量测序文库，高效的利用测序板，提高了文库构建的成功率，降低了样品和试剂的损耗。
在本发明的又一个方面，本发明提出了一种基因测序方法。根据本发明的实施例，对前面所述的核酸文库进行测序处理。采用所述核酸文库进行测序，可同时对正义链和反义链进行测序，也可以分步对模板链进行测序，能够使测序的信息较完整，高效地捕获目的片段，获得精确的测序结果，对于片段较短的核酸模板链而言，在双末端测序的尾端产生序列重叠，可以通过互相校正，降低因测序累积导致的错误率。
本发明的附加方面和优点将在下面的描述中部分给出，部分将从下面的描述中变得明显，或通过本发明的实践了解到。
附图说明
本发明的上述和/或附加的方面和优点从结合下面附图对实施例的描述中将变得明显和容易理解，其中：
图1是根据本发明实施例的Illumina双末端测序技术示意图；
图2是根据本发明实施例的华大智造双末端测序技术示意图；
图3是根据本发明实施例的用于制备DNA纳米球的双链DNA分子，在以下实施例中称为特殊DNA文库(Dual splint dsDNA文库)的构建过程图；
图4是根据本发明实施例的双端环化引物(Dual splint oligo)PAGE纯化照胶图；
图5是根据本发明实施例的DNA纳米球的制备及加载原理图。
具体实施方式
在本发明的一个方面，本发明提出了一种双端环化引物。根据本发明的实施例，所述双端环化引物包括第一核酸单链分子和第二核酸单链分子，所述第一核酸单链分子的5’端与第二核酸单链分子的5’端通过连接子相连。通过连接子将第一核酸单链分子和第二核酸单链分子连接在一起后所得到的环化引物可同时与双链核酸模板分子的正义链和反义链末端接头部分序列互补配对，进行环化反应，经滚环扩增后，可获得核酸正义链纳米球和核酸反义链纳米球，进而可同时对正义链和反义链进行测序，无需多重链置换扩增反应，能够省去杂交、解链、去掉模板等步骤，为后续的测序缩短了时间。
根据本发明的实施例，所述第一核酸单链分子和第二核酸单链分子分别独立的选自DNA或RNA分子。
根据本发明的实施例，所述的双端环化引物的连接子包括选自-S-S-、-NH-、-C-S-C-的至少之一。在这些键能的作用下，使得两条核酸单链分子能够连接在一起。
需要说明的是，本申请所述“连接子”为“第一修饰基团”和“第二修饰基团”进行加成反应而产生的结构。其中，所述“第一修饰基团”和“第二修饰基团”是指位于第一核酸单链分子和第二核酸单链分子的末端可以进行加成反应的分子，以此实现第一核酸单链分子和第二核酸单链分子之间的连接。因此并不局限于“-S-S-、-NH-、-C-S-C-”这三种连接子。
根据本发明的实施例，所述连接子通过支持物连接基团与固体支持物相连，所述支持物连接基团包括选自亲和素或链霉亲和素-生物素基团、氨基-NHS酯或醛基或羟甲基膦或羧基基团、羟基-异氰酸盐基团、丙烯酰胺-硅烷基基团、叠氮-炔烃基团和巯基-马莱酰亚胺 或卤代乙酰基或硫代磺酸盐基团的至少之一。因此，能够同时将大量所述双端环化引物固定于固体支持物上，为之后与大量双链核酸模板分子同时进行环化反应，获得大量环化核酸模板奠定基础。
根据本发明的实施例，所述固体支持物包括选自：磁珠、凝胶珠、玻璃珠、玻片、纳米金颗粒、多聚物体和芯片的至少之一。
需要说明的是，在所述第一核酸单链分子与第一修饰基团(或所述第二核酸单链分子与第二修饰基团)之间引入不影响第一修饰基团与第二修饰基团连接的第三修饰基团，可以实现双端环化引物与固体支持物表面第四修饰基团的连接，所述固体支持物可以选自磁珠、凝胶珠、玻璃珠、多聚物体、玻片、芯片、纳米金颗粒等，所述双端环化引物与固相载体间的连接反应基团可以是亲和素/链霉亲和素-生物素、氨基-NHS酯/醛基/羟甲基膦/羧基、羟基-异氰酸盐、丙烯酰胺-硅烷基，也可以是叠氮-炔烃、巯基-马莱酰亚胺/卤代乙酰基/硫代磺酸盐等。但需要注意的是，第一修饰基团与第二修饰基团之间的连接方式与第三修饰基团与第四修饰基团之间的连接方式不同，主要是为了避免第一核酸单链分子或第二核酸单链分子自身产生的连接，以及避免第一核酸单链分子与第二核酸单链分子发生多个位点的连接而失去与固相载体的连接能力。
需要说明的是，本申请所述第三修饰基团和第四修饰基团分别选自亲和素或链霉亲和素和生物素基团、氨基和NHS酯或醛基或羟甲基膦或羧基基团、羟基和异氰酸酯基团、丙烯酰胺和硅烷基基团、叠氮和炔烃基团、巯基和马莱酰亚胺或卤代乙酰基或硫代磺酸盐基团。
在本发明的另一个方面，本发明提出了一种制备前面所述的双端环化引物的方法。根据本发明的实施例，将第一核酸单链分子和第二核酸单链分子进行连接反应，以便获得所述双端环化引物；其中，所述第一核酸单链分子的5’端连接有第一修饰基团，所述第二核酸单链分子的5’端连接有第二修饰基团，所述第一修饰基团与第二修饰基团适于发生连接反应；所述第一核酸单链分子的3’端与第二核酸单链分子的3’端不适于发生连接反应；所述第一核酸单链分子和第二核酸单链分子之间不能互补配对。通过在第一核酸单链分子及第二核酸单链分子的5’端连接修饰基团，能够使两条核酸单链连接起来，形成双端环化引物，该引物可同时与双链核酸模板分子的正义链和反义链末端接头部分序列互补配对，进行环化反应，经滚环扩增后，可同时获得核酸正义链纳米球和核酸反义链纳米球，进而可对正义链和反义链进行测序，无需多重链置换扩增反应，能够省去杂交、解链、去掉模板等步骤，为后续的测序缩短了时间。
需要说明的是，本申请中所述的“连接反应”为能使所述第一核酸单链分子和第二核酸 单链分子连接在一起的反应，即只要可以实现两条核酸单链分子5’端的连接就可以，所述连接反应可以为偶联反应、亲和反应或其他化学反应。
根据本发明的实施例，所述制备前面所述的双端环化引物的方法还可以进一步包括如下附加技术特征至少之一：
根据本发明的实施例，所述第一修饰基团与第二修饰基团分别独立的选自化学分子或生物大分子；其中，所述化学小分子之间适于通过共价键结合，所述生物大分子之间适于进行连接反应。
根据本发明的实施例，所述第一修饰基团与第二修饰基团分别独立的选自二苄基环辛炔(DBCO)、叠氮化物(Azide)、马莱酰亚胺(Maleimide)和巯基(-SH)的至少之一。根据本发明实施例的上述修饰基团之间可在一定的条件下发生连接反应，使得第一核酸单链分子和第二核酸单链分子的5’端连接在一起。
需要说明的是，本申请所述叠氮化物(Azido)与二苯并环辛炔(DBCO)的加成反应属于应变促进叠氮-炔烃的环化加成反应(strain-promoted azide-alkyne cycloaddition,SPAAC)，利用环化加成反应成为环辛炔(例如OCT、BCN、DBCO、DIBO和DIFO)释放的焓形成稳定的***，反应高效且无需催化物。除此之外，本申请也可使用其他通过化学共价键结合两个或多个分子或生物大分子的生物偶联体系，如含氨基的分子与NHS酯化合物形成胺键进行偶联、含巯基的分子与马莱酰亚胺化合物形成硫醚键进行偶联等。
根据本发明的实施例，所述第一修饰基团与第二修饰基团分别为二苄基环辛炔(DBCO)和叠氮化物(Azide)，所述连接反应是在温度为37℃、溶剂为PBS、pH为7.4的条件下进行48小时。其中，二苄基环辛炔(DBCO)和叠氮化物(Azide)的无铜点击化学反应是一种环张力促进的叠氮-炔基环加成反应，不需要使用有毒的铜离子作为催化剂，反应高效且快速。
根据本发明的实施例，所述溶剂进一步包括DMSO。其中，DMSO有助于修饰基团之间的连接反应。
根据本发明的实施例，所述DMSO在连接反应体系中的体积分数为10％。加入此体积的DMSO能够使修饰基团之间的连接更加充分。
根据本发明的实施例，所述方法进一步包括所述双端环化引物通过第三修饰基团和第四修饰基团与固体支持物相连，所述第三修饰基团与第一修饰基团和第二修饰基团相连，所述第四修饰基团与所述固体支持物相连。因此，能够同时将大量所述双端环化引物固定于固体支持物上，为之后与大量双链核酸模板分子同时进行环化反应，获得大量环化核酸模板奠定基础。
根据本发明的实施例，所述第三修饰基团和第四修饰基团之间适于形成如下支持物连接基团的至少之一：亲和素或链霉亲和素-生物素基团、氨基-NHS酯或醛基或羟甲基膦或羧基基团、羟基-异氰酸盐基团、丙烯酰胺-硅烷基基团、叠氮-炔烃基团和巯基-马莱酰亚胺或卤代乙酰基或硫代磺酸盐基团。当所述第三修饰基团和第四修饰基团之间形成所述的支持物连接基团时，能够使双端环化引物更好的固定于固体支持物上。
根据本发明的实施例，所述固体支持物包括选自磁珠、凝胶珠、玻璃珠、玻片、纳米金颗粒、多聚物体或和芯片的至少之一。
在本发明的又一个方面，本发明提出了一种获得环化核酸模板的方法。根据本发明的实施例，所述方法包括：将双端环化引物与双链核酸模板分子进行环化反应，所述双链核酸模板分子包括正义核酸模板链和反义核酸模板链，所述双端环化引物的两个3’端分别与正义核酸模板链和反义核酸模板链的5’端和3’端的至少部分核酸序列互补配对，以便获得环化核酸模板，所述环化核酸模板具有正义核酸模板链环和反义核酸模板链环；其中，所述双端环化引物包括第一核酸单链分子和第二核酸单链分子，所述第一核酸单链分子的5’端与第二核酸单链分子的5’端通过连接子相连；所述双端环化引物如前面所限定或根据前面所述的方法制备获得；所述双链核酸模板分子的两端连接有不同的接头。根据本发明实施例的环化核酸模板进行环化反应，经滚环扩增后，可同时获得核酸正义链纳米球和核酸反义链纳米球，进而可对正义链和反义链进行测序，无需经过传统的多重链置换扩增反应，可以避免在多重链置换扩增反应过程中，因Read1测序累积损伤造成Read2模板链合成失败、效率低或者产生偏好性等问题。
根据本发明的实施例，所述获得环化核酸模板的方法还可以进一步包括如下附加技术特征至少之一：
根据本发明的实施例，所述双端环化引物包括第一游离3’端和第二游离3’端，所述第一游离3’端与正义核酸链5’端和3’端的接头的至少部分互补配对，所述第二游离3’端与反义核酸链5’端和3’端的接头的至少部分互补配对；或所述第二游离3’端与正义核酸链5’端和3’端的接头的至少部分互补配对，所述第一游离3’端与反义核酸链5’端和3’端的接头的至少部分互补配对。因此，正义核酸链与反义核酸链能够通过与双端环化引物互补配对，从而正义核酸链与反义核酸链各自成环。
根据本发明的实施例，所述双链核酸模板分子的两端连接有接头，所述正义核酸模板链的5’端与所述反义核酸模板链的5’端所连接接头的序列不同。因此，能够使所述正义核酸模板链的5’端与所述反义核酸模板链的5’端分别与双端环化引物的两个游离3’端结合，连接成环。
根据本发明的实施例，所述双链核酸模板分子的5’端具有游离磷酸基团，所述双链核酸模板分子的3’端具有游离羟基基团。
需要说明的是，所述“游离磷酸基团”或“游离羟基基团”中的游离是指所述磷酸基团或羟基基团连接在所述双链核酸模板分子的5’端或3’端末端，而不再与其他核苷酸分子或基团相连。
根据本发明的实施例，进一步包括：对所述环化核酸模板进行滚环扩增反应，获得同时具有多个拷贝的正义链和反义链的核酸分子。
根据本发明的实施例，进行所述滚环扩增反应之前，将所述环化核酸模板加载在测序芯片上；将加载在所述测序芯片上的环化核酸模板进行所述滚环扩增反应，获得同时具有多个拷贝的正义链和反义链的核酸分子。所述核酸分子可对正义链和反义链进行测序，无需经过传统的多重链置换扩增反应，可以避免在多重链置换扩增反应过程中，因Read1测序累积损伤造成Read2模板链合成失败、效率低或者产生偏好性等问题。
根据本发明的实施例，所述双链核酸模板分子是通过如下方式制备获得：将核酸双链模板进行3’末端加A处理，所述核酸双链模板有且仅有一个5’末端具有游离磷酸基团，其余末端具有游离羟基基团；将3’末端加A处理产物与第一接头进行第一连接处理；将第一连接处理产物进行第一5’末端磷酸化处理；将第一5’末端磷酸化处理产物与第二接头进行第二连接处理；以及将第二连接处理产物进行第二5’末端磷酸化处理，以便获得所述双链核酸模板分子。根据本发明实施例的方法可以在双链核酸模板分子的两端分别加上不同的接头，且所获得的双链核酸模板分子解链后所获得的两条单链可以特异性的进行杂交。
需要说明的是，在所述“第二连接处理”中，需要补全在第一连接处理过程中所造成的缺口。在第一连接处理时，仅使一个(因为仅有一个)5’-P与相邻的3’-OH连接形成磷酸二酯键，同端另一边的5'-OH与3'-OH无法连接而导致缺口出现。在第二连接处理时，在5’末端磷酸化处理后，缺口处的5’-OH转变为5’-P，并在之后的连接酶作用下与3’-OH进行连接形成另一个磷酸二酯键，此谓补全。
根据本发明的实施例，所述3’末端加A处理是在Taq聚合酶存在、温度为72℃的条件下进行20min。
根据本发明的实施例，所述3’末端加A处理是在Taq聚合酶存在的条件下进行PCR反应。
根据本发明的实施例，所述PCR反应的扩增轮数不多于8。通过控制PCR的扩增轮数能够减少突变产生。
根据本发明的实施例，所述第一连接处理和第二连接处理是在T4核酸连接酶的作用 下进行的。采用T4核酸连接酶能够高效的催化平端双链核酸模板分子的5’-P末端和3’-OH末端之间以磷酸二酯键结合，进而将接头与所述核酸双链模板的5’端相连。
根据本发明的实施例，所述第一5’末端磷酸化处理和第二5’末端磷酸化处理是在多聚磷酸激酶的作用下进行的。采用多聚磷酸酶能够在第一和第二5’的末端引入磷酸基团。
根据本发明的实施例，3’末端加A处理之后，第一连接处理之前进一步包括将3’末端加A处理产物进行第一纯化处理。
根据本发明的实施例，第一连接处理之后，第一5’末端磷酸化处理之前进一步包括第一连接处理产物进行第二纯化处理。
根据本发明的实施例，第一5’末端磷酸化处理之后，第二连接处理之前进一步包括第一5’末端磷酸化处理产物进行第三纯化处理。
根据本发明的实施例，所述第一纯化处理、第二纯化处理以及第三纯化处理分别独立地选自磁珠纯化。通过采用此纯化方式，能够减少纯化时间，使纯化之后的第一纯化处理产物、第二纯化处理产物以及第三纯化处理产物效率高，不会造成核酸机械损伤，而且重复性极好。
根据本发明的实施例，在所述第一纯化处理、第二纯化处理以及第三纯化处理中，如在200μl PCR管中反应为例，待纯化核酸的投入量不低于1500ng，洗脱液体积不高于50μl。进而可有效提高核酸的浓度。根据本发明的实施例，所述第一接头和第二接头预先进行预退火处理。进而所述第一接头和第二接头以双链的形式与所述核酸双链模板的两端相连。
根据本发明的实施例，所述双端环化引物预先经过预纯化处理。
根据本发明的实施例，所述预纯化处理是通过尿素聚丙烯酰胺凝胶电泳或高效液相色谱进行的。
根据本发明的实施例，退火处理之后进一步包括将退火处理产物进行第三连接处理，进而所述正义核酸模板链和反义核酸模板链各自连接成环，以便获得所述环化核酸模板分子。
需要说明的是，所述“第三连接处理”是指所述正义核酸模板链的5’端与3’端相连与所述反义核酸模板链的5’端与3’端相连。
根据本发明的实施例，所述第三连接处理是在T4核酸连接酶的作用下进行的。采用T4核酸连接酶能够高效的催化平端核酸的5’-P末端和3’-OH末端之间以磷酸二酯键结合，进而将正义核酸模板链的5’端与3’端相连，反义核酸模板链的5’端与3’端相连。
根据本发明的实施例，所述双链核酸模板分子是通过如下方式制备获得：将核酸双链模板进行末端修复和加A处理，处理产物与两种接头进行连接处理，以便获得所述双链核 酸模板分子。根据本发明实施例的方法可以在双链核酸模板分子的两端分别加上不同的接头，且所获得的双链核酸模板分子解链后所获得的两条单链可以特异性的进行杂交。
根据本发明的实施例，所述接头为部分互补的双链接头，两种接头的非连接端核酸序列互不相同。
根据本发明的实施例，所述双端环化引物是固定在固体支持物、高分子多聚物或芯片上的，使双端环化引物与双链核酸模板分子接触，在连接酶存在的情况下进行杂交连接环化反应。因此，能够使大量的双链核酸模板分子与所述双端环化引物结合，使所形成的大量的环化核酸模板同时固定于固体支持物、高分子多聚物或芯片上，为后续同时测序获得大量核酸双端序列信息奠定基础。
在本发明的又一个方面，本发明提出了一种环化核酸分子，是通过前面所述的方法获得的。通过此方法获得的环化核酸分子，经滚环扩增后，可获得核酸正义链纳米球和核酸反义链纳米球，进而可同时对正义链和反义链进行测序，能够不经过传统的多重链置换扩增反应，可以避免在多重链置换扩增反应过程中，因Read1测序累积损伤造成Read2模板链合成失败、效率低或者产生偏好性等。
在本发明的又一个方面，本发明提出了一种环化核酸分子。根据本发明的实施例，所述环化核酸分子包括：包括：前面所述的双端环化引物以及正义核酸模板链和反义核酸模板链，所述正义核酸模板链和所述反义核酸模板链反向互补，所述正义核酸模板链和所述反义核酸模板链的两端连接有接头序列，所述正义核酸模板链的5’端和所述反义核酸模板链的5’端的接头序列不同，所述正义核酸模板链的3’端和所述反义核酸模板链的3’端的接头序列不同；其中，所述双端环化引物的两个3’端分别与正义核酸模板链或反义核酸模板链的5’端和3’端的至少部分核酸序列互补配对。所述环化核酸分子经滚环扩增后，可同时获得核酸正义链纳米球和核酸反义链纳米球，进而可对正义链和反义链进行测序，能够不经过传统的多重链置换扩增反应，可以避免在多重链置换扩增反应过程中，因Read1测序累积损伤造成Read2模板链合成失败、效率低或者产生偏好性等问题。
根据本发明的实施例，所述双端环化引物以及正义核酸模板链和反义核酸模板链是固定在固体支持物、多聚物或游离在溶液中。
根据本发明的实施例，所述固体支持物包括选自磁珠、凝胶珠、玻璃珠、玻片、纳米金颗粒和芯片的至少之一。
在本发明的又一个方面，本发明提出了一种核酸测序芯片。根据本发明的实施例，所述核酸测序芯片包括：多个核酸样本结合位点和多个固定于所述核酸样本结合位点的核酸分子复合物，其中，所述核酸分子复合物包含正义核酸模板链、反义核酸模板链和双端环 化引物，所述双端环化引物与所述正义核酸模板链相连，所述双端环化引物与所述反义核酸模板链相连。所述核酸测序芯片可同时对正义链和反义链进行测序，可以不经过传统的多重链置换扩增反应，直接通过双端测序获得核酸双端序列信息，可以极大简化测序流程、降低测序时间和测序成本。
根据本发明的实施例，所述正义核酸模板链和所述反义核酸模板链为单链环状结构，所述双端环化引物的两个3’端分别与正义核酸模板链或反义核酸模板链的5’端和3’端的至少部分核酸序列互补配对。
根据本发明的实施例，所述核酸分子复合物包括多个正义核酸链拷贝和多个反义核酸链拷贝。进而可直接进行正义链和反义链的测序而无需多重链置换扩增反应，获得双端测序的真实序列信息。
根据本发明的实施例，所述多个正义核酸链拷贝和多个反义核酸链拷贝是通过将所述正义核酸模板链、反义核酸模板链进行滚环扩增处理后获得的。
需要说明的是，“核酸分子复合物”可以包括正义核酸模板链和反义核酸模板链，也可以包括正义核酸模板链和反义核酸模板链经滚环扩增后的多个正义核酸链拷贝和多个反义核酸链拷贝。其中当“核酸分子复合物”包括经正义核酸模板链和反义核酸模板链经滚环扩增后形成的多个正义核酸链拷贝和多个反义核酸链拷贝时，此时的“核酸分子复合物”与“双核酸纳米球”具有相同的结构；本申请所述的“多个正义核酸链拷贝”与“核酸正义链纳米球”、“多个反义核酸链拷贝”与“核酸反义链纳米球”具有相同的结构。
根据本发明的实施例，所述正义核酸模板链和所述反义核酸模板链的两端连接有接头序列，所述正义核酸模板链的5’端和所述反义核酸模板链的5’端的接头序列不同，所述正义核酸模板链的3’端和所述反义核酸模板链的3’端的接头序列不同。因此，能够使所述正义核酸模板链的5’端与所述反义核酸模板链的5’端分别与双端环化引物的两个游离3’端结合，连接成环。
根据本发明的实施例，所述正义核酸模板链和所述反义核酸模板链的一端或两端进一步包括标签序列，或者样本标签序列和分子标签序列等可以区分样本来源的标签信息和统计核酸分子数量的分子标签信息等。
根据本发明的实施例，当所述生物样本为RNA时，需要进行逆转录反应加接头或扩增建库步骤。
根据本发明的实施例，所述正义核酸模板链和所述反义核酸模板链的两端的接头序列是通过PCR扩增反应引入的。具体的，利用带有特异性序列和通用序列的引物对或引物组针对核酸样本的目标区域进行扩增反应，获得两端带有接头序列的双链核酸片段。
在本发明的又一个方面，本发明提出了一种制备双核酸纳米球的方法，根据本发明的实施例，所述方法包括：将前面所述的环化核酸分子进行滚环扩增处理，以便获得双核酸纳米球。通过此方法获得的双核酸纳米球同时具有核酸正义链纳米球和核酸反义链纳米球，可以不经过传统的多重链置换扩增反应，直接通过双端测序获得核酸双端序列信息，可以极大简化测序流程、降低测序时间和测序成本。
根据本发明的实施例，所述滚环扩增处理是在DNB酶I MIX和DNB酶II的作用下、温度为30℃的条件下进行40min。
需要说明的是，本申请所述的“DNB酶I MIX”为Make DNB enzyme MIX Ⅰ及Make DNB enzyme Ⅱ，两者均来自于华大智造的产品，是MGISEQ-2000RS高通量测序试剂套装(FCL PE100)中的组分。
在本发明的又一个方面，本发明提出了一种双核酸纳米球。根据本发明的实施例，所述双核酸纳米球由前面所述的方法制备得到。所述双核酸纳米球同时包含有核酸正义链纳米球和核酸反义链纳米球，可直接进行正义链或反义链的测序而无需多重链置换扩增反应，获得双端测序的真实序列信息。
根据本发明的实施例，所述双核酸纳米球包含核酸正义链纳米球和核酸反义链纳米球。
在本发明的又一个方面，本发明提出了一种核酸文库。根据本发明的实施例，所述核酸文库包括前面所述的双核酸纳米球。所述文库容量大，与待测核酸配对的成功率高。使用本发明构建的核酸文库，进而可同时对正义链和反义链进行测序，能够简单、高效地构建高通量测序文库，高效的利用测序板，提高了文库构建的成功率，降低了样品和试剂的损耗。
在本发明的又一个方面，本发明提出了一种基因测序方法。根据本发明的实施例，对前面所述的核酸文库进行测序处理。采用所述核酸文库进行测序，可同时对正义链和反义链进行测序，能够使测序的信息较完整，高效地捕获目的片段，获得精确的测序结果，对于片段较短的核酸模板链而言，在双末端测序的尾端产生序列重叠，可以通过互相校正，降低因测序累积导致的错误率。
根据本发明的实施例，所述测序是在MGI-SEQ2000平台上进行的。
根据本发明的实施例，本发明的技术优势具有以下几个方面：
(1)本发明提供了一种制备双链核酸模板分子的方法，这种方法可以在双链核酸模板分子的两端分别加上不同的各类接头，且文库解链后的两条单链可以特异性的进行杂交。
(2)本发明提供了一种由双链核酸模板分子和双端环化引物组合，进行滚环扩增，并获得双核酸纳米球，进行单末端或者双末端测序的方法。
(3)本发明提供了一种制备双端环化引物的方法，可以通过具有相互反应能力的生物化学分子将不同引物的5’端或3’端进行任意连接，获得正向或反向的新引物。
(4)本发明制备的双链核酸模板分子的正义核酸模板链和反义核酸模板链可以分别以末端连接的方式封闭成环，获得环状文库，因此本方法可以用于环状文库的制备。
(5)本发明提供的双链核酸模板分子制备方法可以对任意的接头进行连接，使双链两端获得不同类型的接头以满足测序需要。
(6)本发明在基于一步法制备双核酸纳米球的过程中，也可在环化后直接进行消化，获得正义链和反义链环状文库，其后再使用可同时与正义链及反义链环状文库互补配对的引物进行抓取，随后进行滚环扩增反应。
(7)本发明中连接引物的方法可以应用于任意多种引物，实现多条多种不同引物的连接。
下面详细描述本发明的实施例。下面描述的实施例是示例性的，仅用于解释本发明，而不能理解为对本发明的限制。实施例中未注明具体技术或条件的，按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。
为了使本发明的目的、技术方案及优点更加清楚明白，以下结合实施例，对本发明进行进一步详细说明。应当理解，此处所描述的具体实施例仅用以解释本发明，并不用于限定本发明。对于本领域的技术人员来说，本发明可以有各种更改和变化，DNA纳米球制备中所涉及的各流程、各种反应试剂和反应条件可以根据需要进行调整和改变。因此对于本领域技术人员来说，在不脱离本发明的构思和原则之内，还可做出若干简单替换，这些均应包含在本发明的保护范围之内。
实施例中构建DNA纳米球使用的各引物均由生工生物工程有限股份公司合成，各引物序列结构如表1所示：
表1
Figure PCTCN2022137336-appb-000001
Figure PCTCN2022137336-appb-000002
实施例中使用的Ad Lig buffer(连接反应缓冲液)、Ad ligase(T4 DNA连接酶)及Ligation Enhancer(连接增强液)均来自华大智造MGIEasy酶切PCR-Free DNA文库制备试剂盒，磁珠纯化试剂来自于华大智造MGIEasy DNA纯化磁珠试剂盒，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)、rTaq DNA Polymerase(Cat#1000004443)均采购自华大智造。
使用MGISEQ-2000测序仪进行测序时，测序仪及手持loader等设备均由武汉华大智造生产并提供，使用到的测序载片、测序引物、测序试剂、Make DNB enzyme MIX Ⅰ及Make DNB enzyme Ⅱ、stop run buffer均来自MGISEQ-2000RS高通量测序试剂套装(FCL PE100)(Cat#1000012554)。V3 PCR产物试剂由武汉华大智造生产并提供。
实施例1
制备特殊双链DNA文库(Dual splint dsDNA文库)/双链核酸模板分子，如图3所示：
(1)以V3 PCR产物(华大智造，V3 PCR产物试剂E.coli)为模板，以表2为条件配制反应体系，使用Taq酶以PCR的方式进行末端加A反应。
表2
rTaq PCR ReadyMix(2x) 25μL
V3 PCR产物E.coli 1μL
引物PCR-F(20μM) 3μL
引物PCR-R(20μM) 3μL
rTaq DNA Polymerase 2μL
ddH 2O 16μL
在PCR仪上，以表3为条件进行扩增，获得3’末端加A产物。
表3
Figure PCTCN2022137336-appb-000003
使用1倍体积的磁珠(MGIEasy DNA纯化磁珠试剂盒)对产物进行纯化，将纯化所得产物命名为dA-tailed dsDNA。并用Qubit 1X dsDNA HS检测试剂盒(ThermoFisher  Cat#Q33230)在Qubit 4 Fluorometer(ThermoFisher，Cat#Q33230)上测定DNA浓度。
(2)以表4为条件配制反应体系，反应条件为：95℃3min，其后每2min降低1℃缓慢降低温度至25℃，置于4℃保存。获得Y型环化接头1，命名为splint adapter 1。
表4
Figure PCTCN2022137336-appb-000004
以表5为条件配制反应体系，反应条件为：95℃3min，其后每2min降低1℃缓慢降低温度至25℃，置于4℃保存。获得Y型环化接头2，命名为splint adapter 2。
表5
Figure PCTCN2022137336-appb-000005
(3)以表6为条件配制反应体系，进行dA-tailed dsDNA与splint adapter 1的连接。因dA-tailed dsDNA仅有一个5’末端为磷酸基，故此splint adapter 1只能与双链一端进行连接，双链另一端无法连接splint adapter 1。
表6
dA-tailed dsDNA 52μL
splint adapter 1(40μM) 3μL
Ad Lig buffer 18μL
Ad ligase(T4 DNA连接酶) 5μL
Ligation Enhancer 2μL
反应条件为25℃10min，其后于4℃放置过夜。第二天于4℃取出后室温放置30min平衡到室温，使用0.8倍体积的磁珠(MGIEasy DNA纯化磁珠试剂盒)对产物进行纯化，获得纯化产物命名为SA1-dA-tailed dsDNA。
(4)以表7为条件配制反应体系，进行SA1-dA-tailed dsDNA 5’羟基末端的磷酸化。
表7
SA1-dA-tailed dsDNA 50μL
T4 PNK(多聚磷酸激酶) 3μL
10*ligation buffer 6μL
ddH 2O 1μL
反应条件为37℃1h。反应结束后使用1倍体积的磁珠(MGIEasy DNA纯化磁珠试剂 盒)对产物进行纯化，获得纯化产物命名为5P-SA1-dA-tailed dsDNA。用Qubit 1X dsDNA HS检测试剂盒(ThermoFisher，Cat#Q33230)在Qubit 4 Fluorometer(ThermoFisher，Cat#Q33230)上测定DNA浓度。
(5)以表8为条件配制反应体系，将5P-SA1-dA-tailed dsDNA与splint adapter 2连接，并且补全连接splint adapter 1时未连接的缺口。
表8
5P-SA1-dA-tailed dsDNA 52μL
splint adapter 2(40μM) 3μL
Ad Lig buffer 18μL
Ad ligase(T4 DNA连接酶) 5μL
Ligation Enhancer 2μL
反应条件为25℃10min，其后于4℃放置过夜。第二天于4℃取出后室温放置30min平衡到室温，使用1倍体积的磁珠(MGIEasy DNA纯化磁珠试剂盒)对产物进行纯化，获得纯化产物命名为SA2+SA1 dsDNA。
(6)以表9为条件配制反应体系，使用多聚磷酸激酶对SA2+SA1 dsDNA进行5’羟基的磷酸化。
表9
SA1-dA-tailed dsDNA 50μL
T4PNK(多聚磷酸激酶) 3μL
10*ligation buffer 6μL
ddH 2O 1μL
反应条件为37℃1h。反应结束后使用1.5倍体积的磁珠(MGIEasy DNA纯化磁珠试剂盒)对产物进行纯化，获得纯化产物命名为Dual splint dsDNA文库。用Qubit 1X dsDNA HS检测试剂盒(ThermoFisher，Cat#Q33230)在Qubit 4 Fluorometer(ThermoFisher，Cat#Q33230)上测定DNA浓度，浓度为6.36ng/μL。
实施例2
制备双端环化引物(Dual splint oligo)：
(1)使用两种5’端分别修饰有可偶联化学分子的DNA单链引物，此处使用5’叠氮化物(Azide)修饰和5’苄基环辛炔(DBCO)修饰的一对引物以表10为条件配制反应体系，进行偶联。
表10
Azide splint oligo(5 OD) 干粉
DBCO splint oligo(5 OD) 干粉
DMSO(二甲基亚砜) 20μL
1*PBS buffer pH 7.4(磷酸盐缓冲溶液) 溶解干粉并定容至200μL
反应条件为37℃水浴48h。
(2)将所获得的偶联产物进行尿素聚丙烯酰胺凝胶电泳(PAEG)纯化，去除未偶联的Azide splint oligo和DBCO splint oligo：
1)制备尿素12％变性胶：将制胶玻璃板下沿对齐，确保制胶玻璃板内侧干净无异物，放入封底槽中，两侧各用两个大弹簧夹夹紧。取20mL 12％变性胶(114g丙烯酰胺、6g甲叉双丙烯酰胺、315g尿素、100mL 10×TBE加超纯水定容至1000mL，混匀。)加入200μL 10％APS和20μL TEMED，温和颠倒混匀后，倒入封底槽中等待凝胶。胶凝固后取60mL 12％变性胶，加入600μL 10％APS和30μL TEMED，混匀后倒入双层玻璃板中，插上梳子，静置等待凝胶，待胶凝固后拔掉梳子和封底槽，将胶板和胶孔清理干净后使用。
2)样品准备：在(1)中获得的偶联产物中不断加入适量尿素粉末，并不断振荡溶解，直至加入的尿素有少许粉末不溶为宜，用涡旋振荡器混匀后，放入沸水中煮沸2min变性。
3)跑胶：将胶板夹在电泳槽上，向电泳槽中倒入1×TBE(10×TBE：108g Tris、56g硼酸、8g EDTA加超纯水定容至1000mL；1×TBE：100mL 10×TBE加超纯水定容至1000mL，混匀)。确保电泳槽不漏液后，用200μL移液器将变性处理后的样品加至样品孔中，开始电泳。电压为300V，电泳时间为2h。
4)切胶：将平面玻璃板剥下，将凝胶剥离后放置于荧光板上，置于紫外分析仪下进行观察。因电泳方向自上而下，目的片段分子量大、含量多，切胶时选择最上方Dual splint oligo条带。切胶时应迅速，避免在紫外灯下照射过长时间，以免引物降解。照胶图片如图4所示。
5)泡胶：将凝胶捣碎后，向盛有凝胶的离心管中加入6倍体积的TE Buffer(pH 8.0)。将离心管置于-80℃冰箱中冷冻10min后，迅速放入90℃水浴锅中浸泡30min，其间不断上下翻转离心管，以确保胶块充分溶解。
6)乙醇沉淀法纯化：将离心管放入离心机中配平后，12000rpm离心3min，小心将上清液转到新离心管中。向上清液中加入1/10体积的3M NaOAc溶液(pH 5.2)和2.5倍体积预先放置于-20℃的无水乙醇，用涡旋振荡器混匀后，置于-20℃冰箱冷藏20min，其后12000rpm离心10min。小心倒掉所有上清液，加入200μL预先放置于-20℃的75％乙醇洗涤，其后12000rpm离心2min。温和地倒掉洗涤液，于室温放置数分钟晾干以除去残余液体。向离心管中加入50μL超纯水，涡旋振荡混匀溶解，获得纯的Dual splint oligo， 使用Qubit ssDNA检测试剂盒(ThermoFisher，Cat#Q10212)在Qubit 4 Fluorometer(ThermoFisher，Cat#Q33230)上测定DNA浓度，浓度为560ng/μL，约为28.5μM。
实施例3
制备DNA纳米球/核酸分子复合物，如图5所示：
(1)以表11为条件配制反应体系：
表11
组分 体积
Dual splint dsDNA文库(6.36ng/μL) 9.5μL
Dual splint oligo(0.28μM) 0.6μL
10*phi29 buffer(退火缓冲液) 2μL
ddH 2O 7.9μL
在PCR仪上，以表12为条件进行反应，获得Dual splint oligo与Dual splint dsDNA的双端退火产物。
表12
Figure PCTCN2022137336-appb-000006
(2)以表13为条件配制反应体系：
表13
双端退火产物 20μL
T4 DNA ligase 1.6μL
10*ligation buffer 2.4μL
反应条件为30℃30min。获得双端退火环化产物。
(3)以表14为条件配制反应体系：
表14
双端退火环化产物 24μL
Make DNB enzyme Ⅰ MIX 20μL
Make DNB enzyme Ⅱ 2μL
反应条件为30℃40min，其后加入10μL stop run buffer(终止液)终止反应。滚环扩增获 得既具有正义链又具有反义链纳米球的DNA纳米球。使用Qubit ssDNA检测试剂盒(ThermoFisher，Cat#Q10212)在Qubit 4 Fluorometer(ThermoFisher，Cat#Q33230)上测定DNA纳米球浓度，浓度为19.3ng/μL。
实施例4
华大智造MGI-SEQ2000平台测序
使用MGISEQ-2000 FCL测序载片，通过手持loader将制备完成的DNA纳米球加载到测序载片上，室温静置30分钟，将载片放置在测序仓内，将MGISEQ-2000标准PE100测序试剂盒放入。使用移除了MDA过程的PE100测序脚本，上机测试，获得序列信息后与大肠杆菌参考基因组比对，结果如表15所示，在不做多重链置换扩增反应的情况下，成功获取了测序文库的双端PE信息，过滤后测序数据Q30达到87.56％，且与参考物种基因组序列比对率可达86.73％，可获得真实的双端序列信息。
表15
测序指标 DNA双链纳米球测序结果
参考物种 大肠杆菌
测序循环数 212
过滤后的读长大小(M) 12.47
可比对到参考基因组的读长大小(M) 10.81
过滤后Q30(％) 87.56
一链测序超前比例(％) 0.08
二链测序超前比例(％) 0.09
一链测序滞后比例(％) 0.13
二链测序滞后比例(％) 0.12
具有双末端信息的序列数目 62229
可比对到参考基因组的序列数目 54651
比对率(％) 86.73
在本说明书的描述中，参考术语“一个实施例”、“一些实施例”、“示例”、“具体示例”、或“一些示例”等的描述意指结合该实施例或示例描述的具体特征、结构、材料或者特点包含于本发明的至少一个实施例或示例中。在本说明书中，对上述术语的示意性表述不必须针对的是相同的实施例或示例。而且，描述的具体特征、结构、材料或者特点可以在任一个或多个实施例或示例中以合适的方式结合。此外，在不相互矛盾的情况下，本领域的技术人员可以将本说明书中描述的不同实施例或示例以及不同实施例或示例的特征进行结合和组合。
尽管上面已经示出和描述了本发明的实施例，可以理解的是，上述实施例是示例性的，不能理解为对本发明的限制，本领域的普通技术人员在本发明的范围内可以对上述实施例进行变化、修改、替换和变型。

Claims (26)

  1. 一种双端环化引物，其特征在于，包括第一核酸单链分子和第二核酸单链分子，所述第一核酸单链分子的5’端与第二核酸单链分子的5’端通过连接子相连。
  2. 根据权利要求1所述的双端环化引物，其特征在于，所述第一核酸单链分子和第二核酸单链分子分别独立的选自DNA或RNA分子。
  3. 根据权利要求1或2所述的双端环化引物，其特征在于，所述连接子包括选自-S-S-、-NH-、-C-S-C-的至少之一。
  4. 根据权力要求1所述的双端环化引物，其特征在于，所述连接子通过支持物连接基团与固体支持物相连，所述支持物连接基团包括选自亲和素或链霉亲和素-生物素基团、氨基-NHS酯或醛基或羟甲基膦或羧基基团、羟基-异氰酸盐基团、丙烯酰胺-硅烷基基团、叠氮-炔烃基团和巯基-马莱酰亚胺或卤代乙酰基或硫代磺酸盐基团的至少之一。
  5. 根据权利要求4所述的双端环化引物，其特征在于，所述固体支持物包括选自：磁珠、凝胶珠、玻璃珠、玻片、纳米金颗粒、多聚物体和芯片的至少之一。
  6. 一种制备权利要求1～5任一项所述的双端环化引物的方法，其特征在于，将第一核酸单链分子和第二核酸单链分子进行连接反应，以便获得所述双端环化引物；
    其中，所述第一核酸单链分子的5’端连接有第一修饰基团，所述第二核酸单链分子的5’端连接有第二修饰基团，所述第一修饰基团与第二修饰基团适于发生连接反应；
    所述第一核酸单链分子的3’端与第二核酸单链分子的3’端不适于发生连接反应；
    所述第一核酸单链分子和第二核酸单链分子之间不能互补配对。
  7. 根据权利要求6所述的方法，其特征在于，所述第一修饰基团与第二修饰基团分别独立的选自化学小分子或生物大分子；
    其中，所述化学小分子之间适于通过共价键结合，所述生物大分子之间适于进行连接反应；
    优选地，所述第一修饰基团与第二修饰基团分别独立的选自二苄基环辛炔(DBCO)、叠氮化物(Azide)、马莱酰亚胺(Maleimide)和巯基(-SH)的至少之一。
  8. 根据权利要求6所述的方法，其特征在于，所述第一修饰基团与第二修饰基团分别为二苄基环辛炔(DBCO)和叠氮化物(Azide)，所述连接反应是在温度为37℃、溶剂为PBS、pH为7.4的条件下进行48小时；
    任选地，所述溶剂进一步包括DMSO；
    任选地，所述DMSO在连接反应体系中的体积分数为10％。
  9. 根据权利要求6所述的方法，其特征在于，进一步包括所述双端环化引物通过第三修饰基团和第四修饰基团与固体支持物相连，所述第三修饰基团与第一修饰基团和第二修饰基团相连，所述第四修饰基团与所述固体支持物相连。
  10. 根据权利要求9所述的方法，其特征在于，所述第三修饰基团和第四修饰基团之间适于形成如下支持物连接基团的至少之一：亲和素或链霉亲和素-生物素基团、氨基-NHS酯或醛基或羟甲基膦或羧基基团、羟基-异氰酸盐基团、丙烯酰胺-硅烷基基团、叠氮-炔烃基团和巯基-马莱酰亚胺或卤代乙酰基或硫代磺酸盐基团。
  11. 根据权利要求9所述的方法，其特征在于，所述固体支持物包括选自磁珠、凝胶珠、玻璃珠、玻片、纳米金颗粒、多聚物体或和芯片的至少之一。
  12. 一种获得环化核酸模板的方法，其特征在于，包括：将双端环化引物与双链核酸模板分子进行环化反应，所述双链核酸模板分子包括正义核酸模板链和反义核酸模板链，所述双端环化引物的两个3’端分别与正义核酸模板链和反义核酸模板链的5’端和3’端的至少部分核酸序列互补配对，以便获得环化核酸模板，所述环化核酸模板具有正义核酸模板链环和反义核酸模板链环；
    其中，所述双端环化引物包括第一核酸单链分子和第二核酸单链分子，所述第一核酸单链分子的5’端与第二核酸单链分子的5’端通过连接子相连。
  13. 根据权利要求12所述的方法，其特征在于，所述双链核酸模板分子的两端连接有接头，所述正义核酸模板链的5’端与所述反义核酸模板链的5’端所连接接头的序列不同。
  14. 根据权利要求12所述的方法，其特征在于，所述双链核酸模板分子的5’端具有游离磷酸基团，所述双链核酸模板分子的3’端具有游离羟基基团。
  15. 根据权利要求12所述的方法，其特征在于，进一步包括：对所述环化核酸模板进行滚环扩增反应，获得同时具有多个拷贝的正义链和反义链的核酸分子。
  16. 根据权利要求15所述的方法，其特征在于，进行所述滚环扩增反应之前，将所述环化核酸模板加载在测序芯片上；
    将加载在所述测序芯片上的环化核酸模板进行所述滚环扩增反应，获得同时具有多个拷贝的正义链和反义链的核酸分子。
  17. 根据权利要求12所述的方法，其特征在于，所述双链核酸模板分子是通过如下方式制备获得：
    将核酸双链模板进行3’末端加A处理，所述核酸双链模板有且仅有一个5’末端具有游离磷酸基团，其余末端具有游离羟基基团；
    将3’末端加A处理产物与第一接头进行第一连接处理；
    将第一连接处理产物进行第一5’末端磷酸化处理；
    将第一5’末端磷酸化处理产物与第二接头进行第二连接处理；以及
    将第二连接处理产物进行第二5’末端磷酸化处理，以便获得所述双链核酸模板分子。
  18. 一种环化核酸分子，其特征在于，是通过权利要求12～17任一项所述的方法获得的。
  19. 一种环化核酸分子，其特征在于，包括：权利要求1～5任一项所述的双端环化引物以及正义核酸模板链和反义核酸模板链，所述正义核酸模板链和所述反义核酸模板链反向互补，所述正义核酸模板链和所述反义核酸模板链的两端连接有接头序列，所述正义核酸模板链的5’端和所述反义核酸模板链的5’端的接头序列不同，所述正义核酸模板链的3’端和所述反义核酸模板链的3’端的接头序列不同；
    其中，所述双端环化引物的两个3’端分别与正义核酸模板链或反义核酸模板链的5’端和3’端的至少部分核酸序列互补配对。
  20. 根据权力要求19所述的环化核酸分子，其特征在于，所述双端环化引物以及正义核酸模板链和反义核酸模板链是固定在固体支持物、多聚物或游离在溶液中。
  21. 根据权利要求20所述的环化核酸分子，其特征在于，所述固体支持物包括选自磁珠、凝胶珠、玻璃珠、玻片、纳米金颗粒和芯片的至少之一。
  22. 一种核酸测序芯片，其特征在于，包括：多个核酸样本结合位点和多个固定于所述核酸样本结合位点的核酸分子复合物，其中，所述核酸分子复合物包含正义核酸模板链、反义核酸模板链和双端环化引物，所述双端环化引物与所述正义核酸模板链相连，所述双端环化引物与所述反义核酸模板链相连。
  23. 根据权力要求22所述的测序芯片，其特征在于，所述正义核酸模板链和所述反义核酸模板链为单链环状结构，所述双端环化引物的两个3’端分别与正义核酸模板链或反义核酸模板链的5’端和3’端的至少部分核酸序列互补配对。
  24. 根据权力要求22所述的测序芯片，其特征在于，所述核酸分子复合物包括多个正义核酸链拷贝和多个反义核酸链拷贝。
  25. 根据权利要求24所述的测序芯片，其特征在于，所述多个正义核酸链拷贝和多个反义核酸链拷贝是通过将所述正义核酸模板链、反义核酸模板链进行滚环扩增处理后获得的。
  26. 根据权力要求22所述的测序芯片，其特征在于，所述正义核酸模板链和所述反义核酸模板链的两端连接有接头序列，所述正义核酸模板链的5’端和所述反义核酸模板链的5’端的接头序列不同，所述正义核酸模板链的3’端和所述反义核酸模板链的3’端的接头序 列不同。
PCT/CN2022/137336 2022-12-07 2022-12-07 基于一步法的双核酸纳米球制备方法 WO2024119413A1 (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
PCT/CN2022/137336 WO2024119413A1 (zh) 2022-12-07 2022-12-07 基于一步法的双核酸纳米球制备方法

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/CN2022/137336 WO2024119413A1 (zh) 2022-12-07 2022-12-07 基于一步法的双核酸纳米球制备方法

Publications (1)

Publication Number Publication Date
WO2024119413A1 true WO2024119413A1 (zh) 2024-06-13

Family

ID=91378446

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2022/137336 WO2024119413A1 (zh) 2022-12-07 2022-12-07 基于一步法的双核酸纳米球制备方法

Country Status (1)

Country Link
WO (1) WO2024119413A1 (zh)

Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107002291A (zh) * 2014-11-26 2017-08-01 深圳华大基因研究院 一种核酸的双接头单链环状文库的构建方法和试剂
CN107532205A (zh) * 2015-03-31 2018-01-02 伊卢米纳剑桥有限公司 模板的表面连环化
CN110114472A (zh) * 2016-12-21 2019-08-09 深圳华大智造科技有限公司 将线性测序文库转换为环状测序文库的方法
CN110835783A (zh) * 2018-08-17 2020-02-25 深圳华大生命科学研究院 用于长读长高质量测序的核酸文库的构建方法、测序方法及试剂
WO2021178893A2 (en) * 2020-03-06 2021-09-10 Singular Genomics Systems, Inc. Linked paired strand sequencing
WO2021236792A1 (en) * 2020-05-20 2021-11-25 Element Biosciences, Inc. Methods for paired-end sequencing library preparation
CN113736850A (zh) * 2021-08-13 2021-12-03 纳昂达(南京)生物科技有限公司 基于双链环化的文库构建方法及其在测序中的应用
CN113862261A (zh) * 2021-11-15 2021-12-31 纳昂达(南京)生物科技有限公司 一组应用于不同高通量测序平台文库的通用环化陪伴序列、其试剂盒以及环化方法
WO2022087485A1 (en) * 2020-10-22 2022-04-28 Singular Genomics Systems, Inc. Nucleic acid circularization and amplification on a surface

Patent Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107002291A (zh) * 2014-11-26 2017-08-01 深圳华大基因研究院 一种核酸的双接头单链环状文库的构建方法和试剂
CN107532205A (zh) * 2015-03-31 2018-01-02 伊卢米纳剑桥有限公司 模板的表面连环化
CN110114472A (zh) * 2016-12-21 2019-08-09 深圳华大智造科技有限公司 将线性测序文库转换为环状测序文库的方法
CN110835783A (zh) * 2018-08-17 2020-02-25 深圳华大生命科学研究院 用于长读长高质量测序的核酸文库的构建方法、测序方法及试剂
WO2021178893A2 (en) * 2020-03-06 2021-09-10 Singular Genomics Systems, Inc. Linked paired strand sequencing
WO2021236792A1 (en) * 2020-05-20 2021-11-25 Element Biosciences, Inc. Methods for paired-end sequencing library preparation
WO2022087485A1 (en) * 2020-10-22 2022-04-28 Singular Genomics Systems, Inc. Nucleic acid circularization and amplification on a surface
CN113736850A (zh) * 2021-08-13 2021-12-03 纳昂达(南京)生物科技有限公司 基于双链环化的文库构建方法及其在测序中的应用
CN113862261A (zh) * 2021-11-15 2021-12-31 纳昂达(南京)生物科技有限公司 一组应用于不同高通量测序平台文库的通用环化陪伴序列、其试剂盒以及环化方法

Similar Documents

Publication Publication Date Title
JP6959378B2 (ja) 酵素不要及び増幅不要の配列決定
JP7097627B2 (ja) 核酸エンコーディングを使用した巨大分子解析
US20210373000A1 (en) Multipart reagents having increased avidity for polymerase binding
EP3388519B1 (en) Vesicular adaptor and uses thereof in nucleic acid library construction and sequencing
WO2021116715A1 (en) Spatial barcoding
JP2019522463A (ja) サンプル中の標的核酸を検出する方法
KR102607124B1 (ko) 핵산 분석을 위한 다가 결합 조성물
CN113005121B (zh) 接头元件、试剂盒及其相关应用
WO2021227129A1 (zh) 一种通用型高通量测序接头及其应用
JP2020513741A (ja) 連結型ライゲーション
US20090318298A1 (en) Methods for Sequencing DNA
CN112771174A (zh) 用于循环微米粒子分析的方法
US20210198731A1 (en) Linked target capture and ligation
Zhao et al. Combining competitive sequestration with nonlinear hybridization chain reaction amplification: an ultra-specific and highly sensitive sensing strategy for single-nucleotide variants
WO2024119413A1 (zh) 基于一步法的双核酸纳米球制备方法
CN112111560B (zh) Dna纳米球及其制备方法和应用
JP7034299B2 (ja) ハイスループットシークエンシングに基づくオリゴヌクレオチド配列不純物の分析方法及び使用
US20220235410A1 (en) Nucleic acid amplification methods
CN116536308A (zh) 测序封闭剂及其应用
CN114761538A (zh) 样品处理条形码化珠组合物、方法、制备和***
US11649452B2 (en) High density sequencing and multiplexed priming
US20230340592A1 (en) Targeted sequencing
Chen et al. Bottlebrush DNA-primed rolling circle amplification for sensitive detection of biomolecules
WO2023092601A1 (zh) Umi分子标签及其应用、接头、接头连接试剂及试剂盒和文库构建方法
US20240035078A1 (en) Methods and compositions for amplifying polynucleotides