WO2019042016A1 - 一种核酸探针以及一种核酸测序方法 - Google Patents

一种核酸探针以及一种核酸测序方法 Download PDF

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WO2019042016A1
WO2019042016A1 PCT/CN2018/094902 CN2018094902W WO2019042016A1 WO 2019042016 A1 WO2019042016 A1 WO 2019042016A1 CN 2018094902 W CN2018094902 W CN 2018094902W WO 2019042016 A1 WO2019042016 A1 WO 2019042016A1
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nucleic acid
base
acid probe
sequencing
probe
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刘二凯
章文蔚
陈奥
徐崇钧
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MGI Tech Co Ltd
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Priority to US16/643,161 priority patent/US11993813B2/en
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    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6869—Methods for sequencing
    • C12Q1/6874—Methods for sequencing involving nucleic acid arrays, e.g. sequencing by hybridisation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • 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
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6811—Selection methods for production or design of target specific oligonucleotides or binding molecules
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6853—Nucleic acid amplification reactions using modified primers or templates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6853—Nucleic acid amplification reactions using modified primers or templates
    • C12Q1/6855—Ligating adaptors

Definitions

  • the invention belongs to the field of gene sequencing, and relates to a nucleic acid probe and a nucleic acid sequencing method.
  • the nucleic acid probe is a DNA sequencing probe.
  • the nucleic acid sequencing method is a nucleic acid sequencing method for sequencing while ligating.
  • the ligation sequencing technology was developed by Complete Genomics and ABI, and is presented in different forms.
  • the basic principle is to attach a fluorescently modified DNA probe through a ligase.
  • the DNA probe is a specific base at certain positions to identify the sequence to be tested, and the remaining positions are random sequences to form a complementary strand with other positions to be detected.
  • the unligated probe is removed, and then the probe signal on the link is optically detected to determine the sequence at that position.
  • Complete Genomics uses probes labeled with 4 different bases with different fluorophores.
  • the first position of the 5' end of the first set of probes is 4 different bases, and the subsequent bases are random sequences so that they can bind to the random sequence to be tested, and after ligase is ligated to the primer sequence, the detection is performed. Fluorescence obtains base information at this position.
  • the added sequence is removed, and a new sequencing primer is added for hybridization and ligation of the second set of probes.
  • the second position of the second set of probes is 4 different bases at the 5th position, and the other positions are random sequences. After that, the fluorescent molecule at the second position is detected to determine the sequence of the second position.
  • the base sequences at positions 3-8 are determined separately. A total of 8 sets of probes were used to determine the first eight base sequences, followed by the addition of longer new sequencing primers for the next 8 bases of sequencing.
  • ABI's ligation sequencing method is called Sequencing by Oligonucleotide Ligation and Detection (SOLiD). Sixteen different probes are used. The first two of each set of probes are fixed sequences, the last three bases are random sequences, and the last three are three.
  • the base is a universal base, and the method of connecting the random base to the universal base (5th and 6th) is OPS, and the above sequence can be sorted from the 5' end or the 3' end. As shown in the following Formula I, if sorted from the 5' end, the 3' end of the random base is an oxygen atom, and the 5' end of the first universal base is a sulfur atom. The ordering from the 3' end is the same, the 5' end of the third random base is an oxygen atom, and the 3' end of the first universal base is a sulfur atom.
  • Sequencing was carried out from the 3' end to the 5' end, and sequencing primers were added, and the sequencing probes were used for ligation detection.
  • the template was complementary to the primers, and the primers were attached to the magnetic beads to directly photograph. Excision with silver ions (silver ions can specifically bind to sulfur, thereby cutting off the SP bond, P is followed by hydrogen and oxygen ions, and S is connected to silver ions), so that all common bases are cut off, leaving the 5' end The lower phosphate group is attached to the next 5 positions after the next cycle. After repeated iterations, the sequencing strand was removed and the new primers were re-hybridized.
  • the new primers were one more base than before, and after re-circulation multiple times, the primers of the sequencing strand hybridization were removed. Repeating this multiple times allows the entire sequence to be joined, and the sequence information for each position is determined by cross-correlation of the signals.
  • the DNA ligation reaction sequencing method has the following shortcomings: (1) the number or variety of probes leads to higher cost; (2) the SOLiD method adds 5 bases at a time, and needs to be renewed after one cycle or several cycles. Loading sequencing primers increases sequencing cost and time cost; (3) the excision method is not friendly to the sequencing system.
  • a biochemical reaction in sequencing many enzymes are very sensitive to transition metals.
  • the use of silver ions is prone to residue, and it is difficult to avoid the influence of subsequent experiments.
  • the reaction buffer may have chloride ions, and silver ions are easily generated. Precipitation; in addition, silver ions can also form mismatches with the T bases to form T-Ag-T structures.
  • the inventors have skillfully designed a nucleic acid probe through intensive research and creative labor, and on this basis, a method for nucleic acid sequencing using the nucleic acid probe is obtained.
  • the inventors have surprisingly found that the new sequencing method uses only four sets of probes, which significantly reduces the number of probes and can effectively reduce the cost; the probe designed by the present invention only increases the sequencing chain by one base after each cycle. Therefore, as sequencing continues to expand, there is no need to replace new sequencing primers, which also reduces costs.
  • the excision of the excess portion of the probe of the present invention uses a novel rapid excision method, which reduces damage to the sequencing system and is advantageous for improving the read length of the sequencing. The following invention is thus provided:
  • nucleic acid probe comprising a first portion, a second portion, a linker and a detectable label, wherein:
  • the base of the first part is A, T, U, C or G,
  • the base of the second part is a random base and/or a universal base, and is at least 3 bases.
  • the first portion and the second portion are connected by a linker, and the connection between the first portion and the connector can be cut off,
  • the detectable label is attached to the second portion or the linker.
  • Nucleic acid probes are also referred to simply as probes.
  • first part and the second part are referred to only for the sake of clarity and do not have the meaning of order.
  • the first part is a sequencing base.
  • the first portion is at the 5' or 3'end; preferably, the first portion is at the 5' end.
  • the detectable label and/or the chemical group can be designed at the 5' end; conversely, if the first base at the 5' end is The sequencing base is then detectable and/or the chemical group is at the 3' end.
  • the base of the second portion is a random base.
  • the base of the second portion is a universal base.
  • the base of the second portion is a combination of a random base and a universal base.
  • the base of the second portion is 3-15 bases, preferably 5-12 bases, more preferably 5-10 bases (eg 5, 6, 7, 8, 9 or 10 bases), particularly preferably 6-9 bases.
  • the preferred probe length allows for the stability of the hybridization of the probe to the template, as well as the cost of the synthetic probe.
  • the end of the second portion is blocked, meaning that there is no free 3'-OH at the end of the second portion, and thus it is impossible to form a new 3',5'-phosphodiester bond, that is, it is impossible to connect other probes. needle.
  • the 3' terminal OH or 5' terminal phosphate group of the first moiety is exposed when the linkage between the first moiety and the linker is cleaved.
  • the second portion and the connector are cut away when the connection between the first portion and the connector is severed.
  • the detectable label is attached to the second portion
  • the detectable label is attached to the 3'-OH at the end of the second portion;
  • the detectable label is attached to the 3'-OH at the end of the second moiety via a phosphate linkage.
  • the detectable label is a fluorophore, preferably at least one selected from the group consisting of cy3, cy5, Texas Red, 6-FAMTM, AF532, AF647, and AF688;
  • the fluorophore is attached to the 3'-OH at the end of the second moiety; preferably, the fluorophore is attached to the 3'-OH at the end of the second moiety via a phosphate linkage. At this point the probe is blocked.
  • 5' is a sequencing base
  • 3' is a fluorescent group
  • 3' is a sequencing base
  • 5' is a fluorescent group
  • the linkage between the first moiety and the linker can be cleaved in a different manner such that the 3' terminal OH or 5' terminal phosphate group that can be attached is exposed for the next cycle of sequencing reactions.
  • the linker does not contain a sulfur atom; preferably, the linker is selected from the group consisting of the following formula IV-formula IX:
  • R 1 is selected from the group consisting of H, OH, C 1 -C 6 alkyl, C 2 -C 6 alkenyl and C 2 -C 6 alkynyl;
  • R 2 is selected from H, OH, F, Cl and Br.
  • linkages represented by the groups of the above formula IV-formula IX are abbreviated as AP sites, azide, azide, allyl, cyanovinyl and inosine sites, respectively.
  • the excision methods were Endonuclease IV, organic phosphine, organic phosphide, palladium catalytic reagent, organic phosphide and hAAG enzyme + endonuclease IV enzyme.
  • the ligation base and the second base (N1) are linked by a circular deoxyribose or deoxyribose derivative.
  • ligation methods please refer to Xipeng Liu, Jianhua Liu. The mechanism of base excision repair in Chlamydiophila pneumoniae.DNA Repair 4 (2005) 1295–1305.
  • the above formula X is a structural formula of the 5'-end sequencing base, wherein R 1 is selected from the group consisting of H, OH, C 1 -C 6 alkyl, C 2 -C 6 alkenyl and C 2 -C 6 alkynyl; H, OH, F, Cl and Br.
  • a method of attaching a chemical group to a sequencing base in Formula XI or Formula XII can be referred to the prior art, for example, U.S. Patent No. 8,084,590 B2.
  • the attachment of a chemical group in formula XI or formula XII to a sequencing base can be cleaved by an organophosphine (e.g., THPP, TCEP).
  • organophosphine e.g., THPP, TCEP
  • the attachment of the chemical group to the sequencing base in formula XIII can be cleaved by the PdCl 2 + sulfonated triphenylphosphine complex.
  • the excision operating conditions may be, for example, 20 mM Tris-HCl 10 mM (NH 4 ) 2 SO 4 10 mM KCl 2 mM MgSO 4 0.1% X-100 pH 8.8, 1 mM Na 2 PdCl 4 , 5 mM P (PhSO 3 Na) 3] was reacted for 5 min at 60 ° C.
  • the method of linking the chemical group to the sequencing base in the formula XIV can be referred to the prior art, for example, PNAS, 2006.103, 13635-19640.
  • the method of linking the chemical group to the sequencing base in the formula XV can be referred to the prior art, or can be linked by a commercial company such as (Biotech (Shanghai) Co., Ltd.).
  • Excision methods 50 mM KOOCCH 3 , 20 mM Tris-Acetate, 10 mM Mg(OOCCH 3 ) 2 , 1 mM DTT, 100 ⁇ g/mL BSA, 2 U/mL hAAG enzyme + 40 U/ml endonuclease IV enzyme.
  • nucleic acid probe combination comprising four sets of probes, wherein:
  • the first set of nucleic acid probes (abbreviated as A probe or A probe): comprising the nucleic acid probe according to any one of the invention, wherein the base of the first part is A;
  • T probe comprising the nucleic acid probe according to any one of the invention, wherein the base of the first part is T or U;
  • C probe or C probe comprising the nucleic acid probe according to any one of the invention, wherein the base of the first part is C;
  • a fourth set of nucleic acid probes comprising: the nucleic acid probe according to any one of the invention, wherein the base of the first part is G;
  • detectable markers in the four sets of nucleic acid probes are different;
  • the four sets of nucleic acid probes are mixed or not mixed;
  • the first set of nucleic acid probes is equal to the number of moles of the fourth set of nucleic acid probes
  • the second set of nucleic acid probes is equal to the number of moles of the third set of nucleic acid probes
  • the sum of the moles of the first set of nucleic acid probes and the fourth set of nucleic acid probes is less than or equal to the sum of the moles of the second set of nucleic acid probes and the third set of nucleic acid probes.
  • the A probe and the G probe are easier to connect.
  • the sum of the moles of the A probe and the G probe is less than or equal to the sum of the number of moles of the T probe and the C probe, which is advantageous for the connection. Balance and efficiency.
  • the molar ratio of the A probe: T probe: C probe: G probe is (0.5-2): (2-5): (2-5): (0.5-2);
  • the A probe T probe: C probe: G probe molar ratio is (0.8-1.5): (2-5): (2-5): (0.8-1.5);
  • the A probe: T probe: C probe: G probe molar ratio is (0.8-1.5): (3-5): (3-5): (0.8-1.5);
  • the molar ratio of the A probe: T probe: C probe: G probe is (0.5-2): (3-5): (3-5): (0.5-2); more preferably 1 :4:4:1.
  • T probe C probe: G probe molar ratio facilitates the balance and efficiency of the linkage.
  • Another aspect of the invention relates to a ligating solution comprising the nucleic acid probe of any one of the invention, or a nucleic acid probe combination of the invention, and a DNA ligase.
  • the ligation solution wherein the DNA ligase is one or more selected from the group consisting of T4 DNA ligase, T7 DNA ligase, and T3 DNA ligase.
  • the ligation solution, wherein the nucleic acid probe has a concentration of from 0.1 ⁇ M to 5 ⁇ M, preferably 1 ⁇ M.
  • the ligation solution wherein the concentration of the DNA ligase is from 0.01 ⁇ M to 2 ⁇ M or from 0.1 ⁇ g/ml to 20 ⁇ g/ml, preferably 0.5 ⁇ M.
  • the ligation solution further comprises the following components:
  • the balance of the connecting liquid is water.
  • a further aspect of the present invention relates to a kit comprising the nucleic acid probe of any of the present invention, or comprising the nucleic acid probe combination of the present invention, or comprising the ligation liquid of the present invention;
  • a reagent capable of cleaving the connection between the first portion and the linker, a buffer for dissolving the nucleic acid probe, and a sequencing primer;
  • the reagent in the kit does not contain silver ions.
  • the kit wherein the DNA ligase is one or more selected from the group consisting of T4 DNA ligase, T7 DNA ligase, and T3 DNA ligase.
  • the kit wherein the reagent capable of cleaving the first portion and the second portion is an endonuclease (eg, endonuclease IV or endonuclease V), an organophosphonide (eg, THPP or TCEP) or a combination of PdCl 2 and a sulfonated triphenylphosphine complex.
  • endonuclease eg, endonuclease IV or endonuclease V
  • an organophosphonide eg, THPP or TCEP
  • PdCl 2 eg, THPP or TCEP
  • a further aspect of the invention relates to a nucleic acid sequencing method comprising the steps of:
  • the method further comprises the steps of:
  • the nucleic acid molecule to be tested in step (1) is attached to a solid support.
  • the solid support includes, but is not limited to, a chip, a flow cell, a magnetic bead, and the like.
  • a linker sequence is immobilized on the solid support, and the linker sequence is capable of binding to the nucleic acid molecule to be tested.
  • the linker sequence is joined to the linker sequence of the constructed library by base pairing.
  • the method of constructing the DNA library can be a method known to those skilled in the art.
  • the nucleic acid sequencing method further comprises the step of amplifying the nucleic acid molecule to be detected attached to the solid support.
  • the purpose of amplification is to obtain a sufficient number of samples to amplify the signal intensity of the base to achieve the signal requirements for sequencing.
  • the amplified product is still attached to the solid phase to form a localized enrichment.
  • the means of amplification may be polymerase chain reaction (PCR), for example by emulsion PCR (Emulsion PCR) or bridge PCR.
  • PCR polymerase chain reaction
  • the emulsion PCR can be referred to, for example, an emulsion PCR operation in ABI's Solid sequencing method, or an emulsion PCR operation in the Roche 454 sequencing method.
  • the bridge PCR can be referred to the bridge PCR operation in Illumina's Solexa sequencing method.
  • the nucleic acid probe of the present invention or the nucleic acid probe of the present invention is ligated to a sequencing primer by using the ligation liquid of the present invention.
  • the ligation of the probe in step (1) is the same as the other ligation sequencing, which is a universal T4 DNA ligase ligation reaction.
  • the reagent used for elution may be a reagent known to those skilled in the art, such as 5X SSC + 0.05% tween 20.
  • the reagent used for elution may be a reagent known to those skilled in the art, such as 5X SSC + 0.05% tween 20.
  • step (5) after the first portion of the nucleic acid probe is cleaved to the linker, the 3' end OH is exposed, and the next cycle ligation can be performed (as shown in FIG. 1). ).
  • step (6) the above steps (1) to (5) are usually repeated; in the case where one round of sequencing has been completed, only the aforementioned steps (1) to (4) may be repeated.
  • the nucleic acid molecule to be tested may be a single-stranded or double-stranded DNA molecule, or a single-stranded or double-stranded RNA molecule.
  • the DNA molecule can be a DNA molecule derived from an animal, plant or microorganism.
  • the DNA molecule is in the form of a DNA library, which may be a DNA library constructed using a library construction kit.
  • Nx means having x N bases, and x may be a positive integer such as 3, 4, 5, 6, 7, 8, 9, 14, 14, and the like.
  • random base means that the position is 4 bases each of 25%.
  • universal base means that the base can form a base pairing structure with any of the four AGTCs.
  • a 5-nitroguanidine ring for example, a 5-nitroguanidine ring, a 2-nitropyrrole ring, and the like.
  • sampling base means that the base at this position is a fixed base, for example, if the base at this position is T, the probe is responsible for detecting base A.
  • C 1 -C 6 alkyl means a straight or branched alkyl group having 1 to 6 carbon atoms, such as methyl, ethyl, propyl, isopropyl, n-butyl. , sec-butyl, tert-butyl, pentyl, 2-pentyl, isopentyl, neopentyl, hexyl, 2-hexyl, 3-hexyl, etc.; C 1 -C 4 alkyl and C 1 -C 3 alkane
  • a preferred alkyl group is a C 1 -C 4 alkyl group, and a more preferred alkyl group is a C 1 -C 3 alkyl group.
  • C 2 -C 6 alkenyl refers to an alkenyl group having 2 to 6 carbon atoms and at least one double bond, and includes ethenyl, propenyl, 1-but-3-enyl, 1-pent-3- Alkenyl, 1-hex-5-enyl, and the like; C 3 -C 5 alkenyl can also be similarly understood. Preferred is a C 3 -C 5 alkenyl group.
  • C 2 -C 6 alkynyl refers to a hydrocarbon group having 2 to 6 carbon atoms and at least one triple bond, and includes ethynyl, propynyl, butynyl, pentyn-2-yl, and the like; C 3 The -C 5 alkynyl group can also be similarly understood. Preferred is a C 3 -C 5 alkynyl group.
  • the probe or probe combination or sequencing method of the present invention can significantly reduce the number or type of probes in sequencing, thereby significantly reducing the cost.
  • the present invention also employs a resection method that is friendly to the sequencing reaction system.
  • the present invention is advantageous for increasing the read length of sequencing.
  • Figure 1 Schematic diagram of the removal of probes containing AP sites by the action of endonuclease IV.
  • nucleic acid probes used in the following examples can be synthesized according to methods known in the art, and if not specified, commercial companies such as Heiya Medical Technology (Shanghai) Co., Ltd. or Bio-Bio Engineering (Shanghai) Co., Ltd. can be commissioned. Generation for synthesis.
  • the instrument is based on the BGISEQ-500 platform.
  • other sequencing platforms such as the hiseq platform of illumina, etc.
  • the selected modification dye is close to the dye absorption emission wavelength used by the BGISEQ-500 reagent, and thus can be well detected by the BGISEQ-500 optical system.
  • reagents used in this experiment were identical to BGISEQ-500, including the photo buffering reagent, elution buffer 2 used in this experiment.
  • the partial reagent used in this experiment is different from BGISEQ-500, including: replacing the probe polymerization solution in BGISEQ-500 with a ligation solution containing "probe of the present embodiment, enzyme and buffer", using the excision buffer of the experiment.
  • the solution replaced the excision buffer of BGISEQ-500.
  • the experimental sample is genomic DNA of E. coli, which is a standard sample carried by BGISEQ-500.
  • MGIEasy TM DNA library preparation kit (Shenzhen Hua Taizhi Ltd. made) to a standard strain of E. coli DNA extraction was prepared starting library for the sequencing, loaded onto a sequencing chip.
  • Group 1 (A probe): The first part is the sequencing base A, and the second part is 6 random bases.
  • T probe The first part is the sequencing base is T, and the second part is 6 random bases.
  • Group 3 (C probe): The first part is the sequencing base is C, and the second part is 6 random bases.
  • Group 4 The first part is the sequencing base G, and the second part is 6 random bases.
  • the concentration of the probe in the ligation solution was 1 ⁇ M, wherein the molar ratio A probe:T probe:C probe:G probe was about 1:4:4:1.
  • the concentration of DNA ligase in the ligation solution was 0.5 ⁇ M.
  • reaction solution containing the above four probes, T4 DNA ligase and buffer was added by means of an instrument, and the reaction was ligated at 25 ° C for 30 minutes;
  • the photographing buffer is added for image collection (photographing); the base information of each DNA nanosphere site is analyzed by software;
  • Group 1 (A probe): The first part is the sequencing base A, and the second part is 3 random bases + 3 universal bases.
  • T probe The first part is the sequencing base T, and the second part is 3 random bases + 3 universal bases.
  • Group 3 (C probe): The first part is the sequencing base C, and the second part is 3 random bases + 3 universal bases.
  • Group 4 (G probe): The first part is the sequencing base G, and the second part is 3 random bases + 3 universal bases.
  • Group 1 (A probe): The first part is the sequencing base A, and the second part is 6 random bases.
  • T probe The first part is the sequencing base is T, and the second part is 6 random bases.
  • Group 3 (C probe): The first part is the sequencing base is C, and the second part is 6 random bases.
  • Group 4 The first part is the sequencing base G, and the second part is 6 random bases.
  • the concentration of the probe in the ligation solution was 1 ⁇ M, wherein the molar ratio A probe:T probe:C probe:G probe was about 1:4:4:1.
  • the concentration of T4 DNA ligase in the ligation solution was 0.5 ⁇ M.
  • reaction solution containing the above four probes, T4 DNA ligase and buffer was added by means of an instrument, and the reaction was ligated at 25 ° C for 30 minutes;
  • the photographing buffer is added for image collection (photographing); the base information of each DNA nanosphere site is analyzed by software;
  • Cycle number (CycleNumber) 30 Photograph area (number of areas) 1632 All readings (TotalReads) 352.89M Alignment (MappedReads) 293.01M a Q30 77.5% Lag phase (Lag) 0.78% Leading phase (Runon) 0.39% Effective reading ratio (ESR) 80.73% Comparison rate (MappingRate) 83.3% b error rate 1.83%
  • Q30 indicates that the probability of base error detection is 0.1%, that is, the accuracy rate is 99.9%; Q30 is 77.5% means that the accuracy of 77.5% base call is 99.9%.
  • b refers to the average error rate.
  • the method Q30 of the present invention achieves 77.5%, the error rate is only 1.83%, and the read length can reach at least 30 bases.
  • the cost of the invention is significantly lower than existing ligation sequencing methods due to the reduced number of probes and the need to replace new primers.
  • Example 2 The same procedure as in Example 1 was carried out except that four sets of chemical groups (allyl) were used to reversibly link the probes as follows:
  • Group 1 (A probe): The first part is the sequencing base A, and the second part is 6 random bases.
  • T probe The first part is the sequencing base is T, and the second part is 6 random bases.
  • Group 3 (C probe): The first part is the sequencing base is C, and the second part is 6 random bases.
  • Group 4 The first part is the sequencing base G, and the second part is 6 random bases.
  • Example 8 Sequencing of a chemical group (cyanoethylene) reversibly linked probe (6 random bases)
  • Example 2 The same procedure as in Example 1 was carried out except that four sets of chemical groups (cyanoethylene) were used to reversibly link the probes as follows:
  • Group 1 (A probe): The first part is the sequencing base A, and the second part is 6 random bases.
  • T probe The first part is the sequencing base is T, and the second part is 6 random bases.
  • Group 3 (C probe): The first part is the sequencing base is C, and the second part is 6 random bases.
  • Group 4 The first part is the sequencing base G, and the second part is 6 random bases.
  • Group 1 (A probe): The first part is the sequencing base A, and the second part is 6 random bases.
  • T probe The first part is the sequencing base is T, and the second part is 6 random bases.
  • Group 3 (C probe): The first part is the sequencing base is C, and the second part is 6 random bases.
  • Group 4 The first part is the sequencing base G, and the second part is 6 random bases.

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Abstract

核酸探针以及边连接边测序的核酸测序方法，上述核酸探针为DNA测序探针，其包含第一部分、第二部分、连接体和可检测的标记物，第一部分的碱基为A、T、U、C或G，第二部分的碱基为随机碱基和/或通用碱基，并且至少为3个碱基，第一部分和第二部分通过连接体连接，并且第一部分和连接体之间的连接能够被切断，可检测的标记物连接在第二部分或者连接体上。上述探针或将其组合或者测序方法能够降低测序中的探针数量或种类，从而降低成本。

Description

一种核酸探针以及一种核酸测序方法 技术领域
本发明属于基因测序领域，涉及一种核酸探针以及一种核酸测序方法。具体地，所述核酸探针为DNA测序探针。具体地，所述核酸测序方法为边连接边测序的核酸测序方法。
背景技术
连接法测序技术由Complete Genomics和ABI两家公司分别开发出来，采用不同的形式展现，基本原理都是通过连接酶连接上荧光修饰的DNA探针。该DNA探针在某些位置为特定碱基以识别待测序列，其余位置为随机序列可以和其它待测位置形成互补链。连接反应之后，去掉未连接的探针，之后通过光学手段检测连接上的探针信号而确定该位置的序列。
Complete Genomics采用以不同的荧光基团分别标记4个不同碱基的探针。第一组探针的5’端的第1个位置分别为4个不同的碱基，之后的碱基为随机序列从而可以结合到待测的随机序列上，通过连接酶连接到引物序列之后，检测荧光获得该位置的碱基信息。去掉加上的序列，加入新的测序引物之后进行第二组探针的杂交和连接，第二组探针的5’端第2个位置分别为4个不同的碱基，其它位置为随机序列，在这之后进行检测第2个位置的荧光分子从而确定第2位置的序列。类似地，分别确定第3－8位置的碱基序列。一共使用8组探针，确定前八个碱基序列，之后加入更长的新的测序引物开展后面8个碱基的测序。
ABI的连接测序法被称为Sequencing by Oligonucleotide Ligation and Detection(SOLiD)，采用16组不同的探针，每组探针的前面2个为固定序列，后面三个碱基为随机序列，之后三个碱基为通用碱基，在随机碱基和通用碱基(第5和第6)连接的方法为O-P-S，以上序列既可以从5’端开始排序也可以从3’端开始排序。如下面的式I所示，如果从5’端排序，随机碱基3’端为氧原子，第一个通用碱基5’端为硫原子。从3’端排序则相同，第3个随机碱基的5’端为氧原子，第一个通用碱基的3’端为硫原子。
Figure PCTCN2018094902-appb-000001
测序从3’端向5’端方向，加上测序引物，使用测序探针连接检测，连接反应体系中，模板与引物互补配对，引物连接在磁珠上，直接进行拍照。用银离子切除(银离子可以特异性的和硫元素结合，从而切断S-P键，断裂之后P接氢氧离子，S接上银离子)，如此所有通用碱基被切掉，5’端方向留下磷酸基团，下一个循环连接在5个碱基后面的位置。多次重复之后，去掉测序链重新杂交新的引物，新的引物比之前多一个碱基，再重新循环多次之后，去掉测序链杂交第3次的引物。这样重复多次可以使整个序列连接起来，并通过信号的交叉对比，确定每个位置的序列信息。
DNA连接反应测序法有如下几方面的不足：(1)探针数量或种类多，导致成本较高；(2)SOLiD方法每次加入5个碱基，在一个循环或者几个循环之后要重新加载测序引物，增加测序成本和时间成本；(3)切除方法对测序体系并不友好。作为测序中的生化反应，很多酶对过渡金属非常敏感，另外，使用银离子容易有残留，难以避免对后续实验的影响，例如，反应的缓冲液中可能有氯离子，银离子易于与之生成沉淀；另外，银离子还可以和T碱基形成T-Ag-T结构的错误配对(mismatch)。
因此，尚需要开发新的测序方法，特别是新的效率更高、成本更低的边连接边测序的核酸测序方法。
发明内容
本发明人经过深入的研究和创造性的劳动，巧妙地设计了一种核酸探针，并在此基础上得到了利用该核酸探针进行核酸测序的方法。本发明人惊奇地发现，新的测序方法只用4组探针，显著降低了探针的数量从而能够有效地降低成本；本发明所设计 探针在每个循环之后只会增加测序链一个碱基，因此随着测序的不断延伸，不需要更换新的测序引物，也降低了成本。此外，本发明探针多余部分的切除使用新型的快速切除方法，减少了对测序体系的伤害，有利于提高测序的读长。由此提供了下述发明：
本发明的一个方面涉及一种核酸探针，包含第一部分、第二部分、连接体(linker)和可检测的标记物，其中：
第一部分的碱基为A、T、U、C或G，
第二部分的碱基为随机碱基和/或通用碱基，并且至少为3个碱基，
所述第一部分和第二部分通过连接体(linker)连接，并且第一部分和连接体之间的连接能够被切断，
所述可检测的标记物连接在第二部分或者连接体上。
本发明中，如果没有特别说明，
核酸探针也简称为探针。
本发明中，所称第一部分和第二部分仅仅是为了指代清楚，并不具有次序的含义。
本发明中，所述第一部分为测序碱基。
在本发明的一个实施方案中，其中，所述第一部分位于5’端或3’端；优选地，所述第一部分位于5’端。如果探针3’端第一个碱基为测序碱基，则可以把可检测的标记物和/或所述化学基团设计在5’端；反之，如果5’端第一个碱基为测序碱基则可检测的标记物和/或所述化学基团在3’端。
在本发明的一个实施方案中，其中，第二部分的碱基为随机碱基。
在本发明的一个实施方案中，其中，第二部分的碱基为通用碱基。
在本发明的一个实施方案中，其中，第二部分的碱基为随机碱基和通用碱基兼有之。
在本发明的一些实施方案中，其中，所述第二部分的碱基为3－15个碱基，优选为5－12个碱基，更优选为5－10碱基(例如5、6、7、8、9或10个碱基)，特别优选为6－9个碱基。不拘于理论的限制，优选的探针长度考虑到探针与模板杂交的稳定性，以及合成探针的成本。
所述第二部分的末端是阻断的，是指第二部分末端没有游离的3’-OH，因而不能够形成新的3’,5’-磷酸二酯键，即不能够再连接其它探针。
在本发明的一个实施方案中，当第一部分和连接体之间的连接被切断时，第一部 分的3’端OH或者5’端磷酸基团暴露出来。
在本发明的一个实施方案中，当第一部分和连接体之间的连接被切断时，第二部分和连接体被切除。
在本发明的一个实施方案中，优选地，所述可检测的标记物连接在第二部分；
优选地，所述可检测的标记物连接在第二部分末端的3’-OH上；
优选地，所述可检测的标记物通过磷酸酯键连接在第二部分末端的3’-OH上。
在本发明的一些实施方案中，其中，所述可检测的标记物为荧光基团，优选为选自cy3、cy5、Texas Red、6-FAMTM、AF532、AF647和AF688中的至少一种；优选地，所述荧光基团连接在第二部分末端的3’-OH上；优选地，所述荧光基团通过磷酸酯键连接在第二部分末端的3’-OH上。此时探针是阻断的。例如下面的式II或式III所示：
Figure PCTCN2018094902-appb-000002
式II中，5’是测序碱基，3’是荧光基团。
下面的式III中，3’是测序碱基，5’为荧光基团。
Figure PCTCN2018094902-appb-000003
第一部分和连接体之间的连接可以被不同的方式切断，从而使可以连接的3’端OH或者5’端磷酸基团露出来，以进行下一个循环的测序反应。优选地，所述连接体不含有硫原子；优选地，所述连接体选自如下的式IV-式IX所示的基团：
Figure PCTCN2018094902-appb-000004
其中，式IV中，R 1选自H、OH、C 1-C 6烷基、C 2-C 6烯基和C 2-C 6炔基；R 2选自H、OH、F、Cl和Br。
上述式IV-式IX所示的基团所代表的连接方式分别简称为AP位点、叠氮、叠氮、烯丙基、氰乙烯基和inosine位点。切除方式依次分别为Endonuclease IV、有机膦化物、有机膦化物、钯催化试剂、有机膦化物和hAAG酶+endonuclease IV酶。
上述式IV-式IX所示的基团的连接和切除方法可以参考本领域技术人员知悉的方法，也可以参考下面的描述。
(1)AP位点连接方式
Figure PCTCN2018094902-appb-000005
测序碱基和第二个碱基(N1)的连接采用一个环状脱氧核糖或脱氧核糖衍生物连接，具体连接方法可以参考Xipeng Liu,Jianhua Liu.The mechanism of base excision repair in Chlamydiophila pneumoniae.DNA Repair 4(2005)1295–1305。上面的式X为5’端测序碱基的结构式，其中R 1选自H、OH、C 1-C 6烷基、C 2-C 6烯基和C 2-C 6炔基；R2选自H、OH、F、Cl和Br。
(2)两种叠氮连接方式
Figure PCTCN2018094902-appb-000006
式XI或式XII中化学基团与测序碱基的连接方法可以参考现有技术，例如美国专利US8084590B2。
式XI或式XII中化学基团与测序碱基的连接可被有机膦化物(例如THPP，TCEP)切除。切除的条件可以是，例如，100mM TCEP，pH＝7，1M氯化钠，50℃下5分钟。
(3)烯丙基连接方式
Figure PCTCN2018094902-appb-000007
式XIII中化学基团与测序碱基的连接方法可以参考现有技术，例如Jingyue Ju,Dae Hyun Kim,et.al.Four-color DNA sequencing by synthesis using cleavable fluorescent nucleotide reversible terminators.PNAS.2006.103.19635-19640。
式XIII中化学基团与测序碱基的连接可被PdCl 2+磺化三苯基膦配合物切除。切除的操作条件可以是，例如，20mM Tris-HCl 10mM(NH 4) 2SO 4 10mM KCl 2mM MgSO 40.1％
Figure PCTCN2018094902-appb-000008
X-100pH 8.8,1mM Na 2PdCl 4,5mM P(PhSO3Na)3]反应5min 60℃。
(4)氰乙烯基连接方式
Figure PCTCN2018094902-appb-000009
式XIV中化学基团与测序碱基的连接方法可以参考现有技术，例如PNAS，2006.103，19635-19640。
切除方法：100Mm THPP，pH 9，3M NaCl，0.2M tris。
(5)inosine位点连接方式
Figure PCTCN2018094902-appb-000010
Inosine:
Figure PCTCN2018094902-appb-000011
式XV中化学基团与测序碱基的连接方法可以参考现有技术，或者委托商业公司例如(生工生物工程(上海)股份有限公司)连接。
切除方法：50mM KOOCCH 3,20mM Tris-Acetate,10mM Mg(OOCCH 3) 2,1mM DTT，100μg/mL BSA，2U/mL hAAG酶+40U/ml endonuclease IV酶。
本发明的另一方面涉及一种核酸探针组合，其包含4组探针，其中：
第一组核酸探针(简称为A探针或A probe)：包含本发明中任一项所述的核酸探针，其中，第一部分的碱基为A；
第二组核酸探针(简称为T探针或T probe)：包含本发明中任一项所述的核酸探针，其中，第一部分的碱基为T或U；
第三组核酸探针(简称为C探针或C probe)：包含本发明中任一项所述的核酸探针，其中，第一部分的碱基为C；
第四组核酸探针(简称为G探针或G probe)：包含本发明中任一项所述的核酸 探针，其中，第一部分的碱基为G；
并且所述4组核酸探针中的可检测的标记物各不相同；
所述4组核酸探针混合或者不混合；
优选地，第一组核酸探针与第四组核酸探针的摩尔数相等；
优选地，第二组核酸探针与第三组核酸探针的摩尔数相等；
优选地，第一组核酸探针与第四组核酸探针的摩尔数之和小于或等于第二组核酸探针与第三组核酸探针的摩尔数之和。
不拘于理论的限制，A探针与G探针更容易连接，优选A探针与G探针的摩尔数之和小于或等于T探针与C探针的摩尔数之和，有利于连接的平衡和效率。
优选地，A探针：T探针：C探针：G探针的摩尔比为(0.5-2)：(2-5)：(2-5)：(0.5-2)；
优选地，A探针：T探针：C探针：G探针的摩尔比为(0.8-1.5)：(2-5)：(2-5)：(0.8-1.5)；
优选地，A探针：T探针：C探针：G探针的摩尔比为(0.8-1.5)：(3-5)：(3-5)：(0.8-1.5)；
优选地，A探针：T探针：C探针：G探针的摩尔比为(0.5-2)：(3-5)：(3-5)：(0.5-2)；更优选为1：4：4：1。
不拘于理论的限制，优选的A探针：T探针：C探针：G探针的摩尔比有利于连接的平衡和效率。
本发明的另一方面涉及一种连接应液，其包含本发明中任一项所述的核酸探针，或者包含本发明的核酸探针组合，以及DNA连接酶。
在本发明的一个实施方案中，所述的连接液，其中，所述DNA连接酶为选自T4 DNA连接酶、T7 DNA连接酶和T3 DNA连接酶中的一种或多种。
在本发明的一个实施方案中，所述的连接液，其中，所述核酸探针的浓度为0.1μM－5μM，优选为1μM。
在本发明的一个实施方案中，所述的连接液，其中，所述DNA连接酶的浓度为0.01μM－2μM或者0.1μg/ml－20μg/ml，优选为0.5μM。
在本发明的一个实施方案中，所述的连接液，其还包括如下成分：
50mM CH 3COOK，20mM Tris，10mM Mg(CH 3COO) 2，100μg/ml BSA，1mM ATP， 10％PEG6000；
优选地，所述连接液余量为水。
本发明的再一方面涉及一种试剂盒，其包含本发明中任一项所述的核酸探针，或者包含本发明的核酸探针组合，或者包含本发明的连接液；
优选地，还包含选自能够切断第一部分和连接体之间的连接的试剂、用于溶解核酸探针的缓冲液和测序引物中的一种或多种；
优选地，所述试剂盒中的试剂不含有银离子。
在本发明的一个实施方案中，所述的试剂盒，其中，所述DNA连接酶为选自T4 DNA连接酶、T7 DNA连接酶和T3 DNA连接酶中的一种或多种。
在本发明的一个实施方案中，所述的试剂盒，其中，能够切断第一部分和第二部分连接的试剂为内切酶(例如内切酶IV或内切酶V)、有机膦化物(例如THPP或TCEP)或者PdCl 2与磺化三苯基膦配合物的组合物。
本发明的再一方面涉及一种核酸测序方法，包括下述步骤：
(1)将测序引物杂交至待测核酸分子；
(2)将本发明所述的核酸探针或者本发明的核酸探针组合连接至测序引物；
(3)洗脱未与待测核酸分子结合的核酸探针；
(4)检测结合于待测核酸分子的核酸探针上的可检测的标记物，确定第一部分的碱基信息；
(5)切断核酸探针第一部分与连接体的连接，洗脱核酸探针中除第一部分之外的其余部分；
优选地，还包括下述步骤：
(6)重复前述步骤(2)－(4)或(2)－(5)。
在本发明的一个实施方案中，步骤(1)中所述待测核酸分子连接在固相支持物上。
所述固相支持物包括但不限于：芯片、槽道(Flowcell)、磁珠等。所述固相支持物上固定有接头序列，该接头序列与待测核酸分子能够结合。例如，当待测核酸分子是以DNA文库的形式时，该接头序列与构建文库的接头序列通过碱基配对结合。所述DNA文库的构建方法可以是本领域技术人员知悉的方法。
优选地，所述核酸测序方法还包括将连接在固相支持物上的待测核酸分子进行扩增的步骤。
不拘于理论的限制，扩增的目的是获得足够数量的样本，将碱基的信号强度放大，以达到测序所需的信号要求。扩增产物仍然连接在固相上，形成局部富集。
扩增的手段可以是聚合酶链式反应(PCR)，例如通过乳液PCR(Emulsion PCR)或桥式PCR。所述乳液PCR可以参考，例如，ABI公司的Solid测序方法中的乳液PCR操作，或者参考Roche 454测序方法中的乳液PCR操作。所述桥式PCR可以参考illumina公司的Solexa测序方法中的桥式PCR操作。
在本发明的一个实施方案中，步骤(2)中，通过使用本发明的连接液，将本发明所述的核酸探针或者本发明的核酸探针组合连接至测序引物。步骤(1)中探针的连接和其它连接法测序一样，为通用的T4 DNA连接酶连接反应。
在本发明的一个实施方案中，步骤(3)中，洗脱所用的试剂可以是本领域技术人员知悉的试剂，例如5X SSC+0.05％tween20。
在本发明的一个实施方案中，步骤(5)中，洗脱所用的试剂可以是本领域技术人员知悉的试剂，例如5X SSC+0.05％tween20。
在本发明的一个实施方案中，步骤(5)中，切断核酸探针第一部分与连接体之间的连接之后，暴露出3’端OH，可进行下一循环连接测序(如图1所示)。
对于步骤(6)而言，通常情况下，重复前述步骤(1)－(5)；在已经完成一轮测序的情况下，可以仅重复前述步骤(1)－(4)。
本发明中，所述待测核酸分子可以是单链或双链的DNA分子，或者单链或双链的RNA分子。所述DNA分子可以是来自于动物、植物或者微生物的DNA分子。优选地，所述DNA分子是以DNA文库的形式，所述DNA文库可以是利用文库构建试剂盒构建的DNA文库。
本发明中，Nx是指有x个N碱基，x可以是正整数，例如3、4、5、6、7、8、9……14、15等。
术语“随机碱基”是指该位置为4种碱基各占25％，
术语“通用碱基”是指该碱基可以和AGTC四种中的任何一个形成碱基配对结构。例如，5-硝基吲哚环、2-硝基吡咯环等。
术语“测序碱基”是指该位置的碱基为固定碱基，例如，如果该位置的碱基为T， 则该探针负责检测碱基A。
在本发明中，术语“C 1-C 6烷基”是指具有1-6个碳原子的直链或支链烷基，例如甲基、乙基、丙基、异丙基、正丁基、仲丁基、叔丁基、戊基、2-戊基、异戊基、新戊基、己基、2-己基、3-己基等；C 1-C 4烷基和C 1-C 3烷基也可做类似理解。优选的烷基是C 1-C 4烷基，更优选的烷基是C 1-C 3烷基。
术语“C 2-C 6烯基”是指具有2-6个碳原子以及至少一个双键的烯基，并且包括乙烯基、丙烯基、1-丁-3-烯基、1-戊-3-烯基、1-己-5-烯基等；C 3-C 5烯基也可做类似理解。优选的是C 3-C 5烯基。
术语“C 2-C 6炔基”是指具有2-6个碳原子以及至少一个叁键的烃基，并且包括乙炔基、丙炔基、丁炔基、戊炔-2-基等；C 3-C 5炔基也可做类似理解。优选的是C 3-C 5炔基。
发明的有益效果
本发明具有下述技术效果中的至少一项：
(1)本发明的探针或探针组合或者测序方法能够显著地降低测序中的探针数量或种类，从而显著降低了成本。
(2)由于一次加入一个碱基，不需要重复加引物，也降低了成本。
(3)本发明还采用了对测序反应体系友好的切除方法。
(4)本发明测序准确度高。
(5)本发明有利于提高测序的读长。
附图说明
图1：含有AP位点的探针在内切酶IV作用下被去除的示意图。
具体实施方式
下面将结合实施例对本发明的实施方案进行详细描述，但是本领域技术人员将会理解，下列实施例仅用于说明本发明，而不应视为限定本发明的范围。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。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。
下面实施例所用的核酸探针可以按照本领域已知的方法合成，如果没有特别说 明，可以委托商业上的公司例如合亚医药科技(上海)有限公司或者生工生物工程(上海)股份有限公司代为合成。
实施例1：AP位点可逆连接探针(6个随机碱基)的测序应用
1.仪器和试剂
仪器基于BGISEQ-500平台。理论上，其它测序平台(例如illumina的hiseq平台等)经过适当调整，也可以进行与本实施例相同或类似的实验。
另外，为了能够在BGISEQ-500平台上使用，所选用的修饰染料与BGISEQ-500试剂所使用的染料吸收发射波长接近，因此可以很好的被BGISEQ-500光学系统检测。
本实验使用的部分试剂和BGISEQ-500完全一致，包括本实验所使用的拍照缓冲试剂、洗脱缓冲液2。
本实验使用的部分试剂和BGISEQ-500不同，包括：用包含“本实施例的探针、酶和缓冲液”的连接液取代BGISEQ-500中的探针聚合反应液，用本实验的切除缓冲液取代BGISEQ-500的切除缓冲液。
实验样品是大肠杆菌的基因组DNA，其为BGISEQ-500所携带的标准样品。
根据制造商的说明书，采用MGIEasy TMDNA文库制备试剂盒(深圳华大智造科技有限公司)以大肠杆菌标准菌株为原料提取DNA制备用于测序的文库，加载到测序芯片上。
2.探针的设计与合成
4组AP位点可逆连接探针如下(x＝6)：
第1组(A probe)：第一部分即测序碱基为A，第二部分为6个随机碱基。
Figure PCTCN2018094902-appb-000012
第2组(T probe)：第一部分即测序碱基为T，第二部分为6个随机碱基。
Figure PCTCN2018094902-appb-000013
第3组(C probe)：第一部分即测序碱基为C，第二部分为6个随机碱基。
Figure PCTCN2018094902-appb-000014
第4组(G probe)：第一部分即测序碱基为G，第二部分为6个随机碱基。
Figure PCTCN2018094902-appb-000015
以上探针委托生工生物工程(上海)股份有限公司合成。
将以上4组探针和T4 DNA连接酶溶解在如下的缓冲液中：
50mM CH 3COOK，20mM Tris，10mM Mg(CH 3COO) 2，100μg/ml BSA，1mM ATP，10％PEG6000。
得到连接液。
连接液中探针的浓度是1μM，其中摩尔比A probe：T probe：C probe：G probe约为1：4：4：1。
连接液中DNA连接酶的浓度时0.5μM。
3.测序步骤
参照BGISEQ-500说明书完成如下的前期准备工作：文库构建，DNA单链环扩增成DNA纳米球，DNA纳米球装载至BGISEQ-500携带的芯片上和测序引物加载到DNA纳米球上。
利用仪器加入包含上述4种探针、T4 DNA连接酶和缓冲液的连接液，在25℃下 连接反应30分钟；
用洗脱试剂2，洗脱未连接的探针；
然后加入拍照缓冲液进行图像采集(拍照)；利用软件分析每个DNA纳米球位点的碱基信息；
拍照之后加入内切酶IV(New England Biolabs，货号M0304L)和其缓冲液，在37℃下反应5分钟，以切除AP位点，之后加入洗脱试剂2洗脱被切除的部分探针；
可重复加入4组探针进行下一循环的测序。
4.实验结果
结果与BGISEQ-500所携带的标准样品的序列完全一致，说明本发明的测序方法准确。
实施例2：AP位点可逆连接探针(3个随机碱基+3个通用碱基)的测序应用
按照与实施例1相同的方法进行，不同之处在于使用如下的4组探针：
第1组(A probe)：第一部分即测序碱基为A，第二部分为3个随机碱基+3个通用碱基。
Figure PCTCN2018094902-appb-000016
第2组(T probe)：第一部分即测序碱基为T，，第二部分为3个随机碱基+3个通用碱基。
Figure PCTCN2018094902-appb-000017
第3组(C probe)：第一部分即测序碱基为C，，第二部分为3个随机碱基+3个通用碱基。
Figure PCTCN2018094902-appb-000018
第4组(G probe)：第一部分即测序碱基为G，，第二部分为3个随机碱基+3个通用碱基。
Figure PCTCN2018094902-appb-000019
结果与BGISEQ-500所携带的标准样品的序列完全一致，说明本发明的测序方法准确。
实施例3：AP位点可逆连接探针(7个随机碱基)的测序应用
按照与实施例1相同的方法进行，不同之处在于使用的4组AP位点可逆连接探针，其中x＝7。
结果与BGISEQ-500所携带的标准样品的序列完全一致，说明本发明的测序方法准确。
实施例4：AP位点可逆连接探针(8个随机碱基)的测序应用
按照与实施例1相同的方法进行，不同之处在于使用的4组AP位点可逆连接探针，其中x＝8。
结果与BGISEQ-500所携带的标准样品的序列完全一致，说明本发明的测序方法准确。
实施例5：AP位点可逆连接探针(9个随机碱基)的测序应用
按照与实施例1相同的方法进行，不同之处在于使用的4组AP位点可逆连接探 针，其中x＝9。
结果与BGISEQ-500所携带的标准样品的序列完全一致，说明本发明的测序方法准确。
实施例6：化学基团(叠氮)可逆连接探针(6个随机碱基)的测序应用
按照与实施例1相同的方法进行，不同之处在于
1.仪器和试剂
与实施例1相同。
2.探针的设计与合成
使用如下的4组探针，其中x＝6：
第1组(A probe)：第一部分即测序碱基为A，第二部分为6个随机碱基。
Figure PCTCN2018094902-appb-000020
第2组(T probe)：第一部分即测序碱基为T，第二部分为6个随机碱基。
Figure PCTCN2018094902-appb-000021
第3组(C probe)：第一部分即测序碱基为C，第二部分为6个随机碱基。
Figure PCTCN2018094902-appb-000022
第4组(G probe)：第一部分即测序碱基为G，第二部分为6个随机碱基。
Figure PCTCN2018094902-appb-000023
将以上4组探针和T4 DNA连接酶溶解在如下的缓冲液中：
50mM CH 3COOK，20mM Tris，10mM Mg(CH 3COO) 2，100μg/ml BSA，1mM ATP，10％PEG6000。
得到连接液。
连接液中探针的浓度是1μM，其中摩尔比A probe：T probe：C probe：G probe约为1：4：4：1。
连接液中T4 DNA连接酶的浓度时0.5μM。
3.测序步骤
参照BGISEQ-500说明书完成如下的前期准备工作：文库构建，DNA单链环扩增成DNA纳米球，DNA纳米球装载至BGISEQ-500携带的芯片上和测序引物加载到DNA纳米球上。
利用仪器加入包含上述4种探针、T4 DNA连接酶和缓冲液的连接液，在25℃下连接反应30分钟；
用洗脱试剂2，洗脱未连接的探针；
然后加入拍照缓冲液进行图像采集(拍照)；利用软件分析每个DNA纳米球位点的碱基信息；
拍照之后加入切除试剂，切除试剂成份为(10Mm THPP,200Mm tris pH＝9缓冲液，0.5M氯化钠)，切除反应60度3分钟，之后加入洗脱试剂2洗脱被切除的部分探针；
可重复加入4组探针进行下一循环的测序。
4.实验结果
由于大肠杆菌的基因组较小，本发明人在此进行了30个循环的测序，通过BGISEQ-500的测序分析软件进行了结果分析，结果如下面的表1所示。
表1：
参考基因组(Reference) 大肠杆菌
循环数(CycleNumber) 30
拍照区域(区域数目) 1632个
全部读数(TotalReads) 352.89M
比对度数(MappedReads) 293.01M
aQ30 77.5％
滞后相(Lag) 0.78％
超前相(Runon) 0.39％
有效读数比例(ESR) 80.73％
比对率(MappingRate) 83.3％
b错误率 1.83％
a，Q30表示碱基被测错的概率为0.1％，即准确率为99.9％；Q30为77.5％是指77.5％的碱基响应(base call)的准确率达到99.9％。
b，是指平均错误率。
结果显示，利用本发明的方法Q30达到77.5％，错误率只有1.83％，读长至少可达到30个碱基。另外，由于探针的数量减少以及不需要更换新的引物，发明的成本显著低于现有的连接测序法。
实施例7：化学基团(烯丙基)可逆连接探针(6个随机碱基)的测序应用
按照与实施例1相同的方法进行，不同之处在于使用4组化学基团(烯丙基)可逆连接探针，如下：
第1组(A probe)：第一部分即测序碱基为A，第二部分为6个随机碱基。
Figure PCTCN2018094902-appb-000024
第2组(T probe)：第一部分即测序碱基为T，第二部分为6个随机碱基。
Figure PCTCN2018094902-appb-000025
第3组(C probe)：第一部分即测序碱基为C，第二部分为6个随机碱基。
Figure PCTCN2018094902-appb-000026
第4组(G probe)：第一部分即测序碱基为G，第二部分为6个随机碱基。
Figure PCTCN2018094902-appb-000027
结果与BGISEQ500所携带的标准样品的序列完全一致，说明本发明的测序方法准确。
实施例8：化学基团(氰基乙烯)可逆连接探针(6个随机碱基)的测序应用
按照与实施例1相同的方法进行，不同之处在于使用4组化学基团(氰基乙烯)可逆连接探针，如下：
第1组(A probe)：第一部分即测序碱基为A，第二部分为6个随机碱基。
Figure PCTCN2018094902-appb-000028
第2组(T probe)：第一部分即测序碱基为T，第二部分为6个随机碱基。
Figure PCTCN2018094902-appb-000029
第3组(C probe)：第一部分即测序碱基为C，第二部分为6个随机碱基。
Figure PCTCN2018094902-appb-000030
第4组(G probe)：第一部分即测序碱基为G，第二部分为6个随机碱基。
Figure PCTCN2018094902-appb-000031
结果与BGISEQ-500所携带的标准样品的序列完全一致，说明本发明的测序方法准确。
实施例9：化学基团(Inosine)可逆连接探针(6个随机碱基)的测序应用
按照与实施例1相同的方法进行，不同之处在于使用4组化学基团(Inosine)可逆连接探针，如下：
第1组(A probe)：第一部分即测序碱基为A，第二部分为6个随机碱基。
Figure PCTCN2018094902-appb-000032
第2组(T probe)：第一部分即测序碱基为T，第二部分为6个随机碱基。
Figure PCTCN2018094902-appb-000033
第3组(C probe)：第一部分即测序碱基为C，第二部分为6个随机碱基。
Figure PCTCN2018094902-appb-000034
第4组(G probe)：第一部分即测序碱基为G，第二部分为6个随机碱基。
Figure PCTCN2018094902-appb-000035
结果与BGISEQ-500所携带的标准样品的序列完全一致，说明本发明的测序方法准确。
尽管本发明的具体实施方式已经得到详细的描述，本领域技术人员将会理解。根据已经公开的所有教导，可以对那些细节进行各种修改和替换，这些改变均在本发明的保护范围之内。本发明的全部范围由所附权利要求及其任何等同物给出。

Claims (12)

  1. 一种核酸探针，包含第一部分、第二部分、连接体和可检测的标记物，其中：
    第一部分的碱基为A、T、U、C或G，
    第二部分的碱基为随机碱基和/或通用碱基，并且至少为3个碱基，
    所述第一部分和第二部分通过连接体连接，并且第一部分和连接体之间的连接能够被切断，
    所述可检测的标记物连接在第二部分或者连接体上。
  2. 根据权利要求1所述的核酸探针，其中，所述第一部分位于5’端或3’端。
  3. 根据权利要求1所述的核酸探针，其中，所述第二部分的碱基为3－15个碱基，优选为5－12个碱基，进一步优选为5－10个碱基(例如5、6、7、8、9或10个碱基)，特别优选为6－9个碱基。
  4. 根据权利要求1所述的核酸探针，其中，所述可检测的标记物为荧光基团；优选为选自cy3、cy5、Texas Red、6-FAMTM、AF532、AF647和AF688中的至少一种；
    优选地，所述可检测的标记物连接在第二部分；
    优选地，所述可检测的标记物连接在第二部分末端的3’-OH上；
    优选地，所述可检测的标记物通过磷酸酯键连接在第二部分末端的3’-OH上。
  5. 根据权利要求1所述的核酸探针，其中，所述连接体不含有硫原子；优选地，所述连接体选自如下的式IV-式IX所示的基团：
    Figure PCTCN2018094902-appb-100001
    Figure PCTCN2018094902-appb-100002
    其中，式IV中，R 1选自H、OH、C 1-C 6烷基、C 2-C 6烯基和C 2-C 6炔基；R 2选自H、OH、F、Cl和Br。
  6. 一种核酸探针组合，其包含4组探针，其中：
    第一组核酸探针：包含权利要求1至5中任一权利要求所述的核酸探针，其中，第一部分的碱基为A；
    第二组核酸探针：包含权利要求1至5中任一权利要求所述的核酸探针，其中，第一部分的碱基为T或U；
    第三组核酸探针：包含权利要求1至5中任一权利要求所述的核酸探针，其中，第一部分的碱基为C；
    第四组核酸探针：包含权利要求1至5中任一权利要求所述的核酸探针，其中，第一部分的碱基为G；
    并且所述4组核酸探针中的可检测的标记物各不相同；
    所述4组核酸探针混合或者不混合；
    优选地，第一组核酸探针与第四组核酸探针的摩尔数相等；
    优选地，第二组核酸探针与第三组核酸探针的摩尔数相等；
    优选地，第一组核酸探针与第四组核酸探针的摩尔数之和小于或等于第二组核酸探针与第三组核酸探针的摩尔数之和；
    优选地，第一组核酸探针：第二组核酸探针：第三组核酸探针：第四组核酸探针的摩尔比为(0.5-2)：(3-5)：(3-5)：(0.5-2)；更优选为1：4：4：1。
  7. 一种连接液，其包含权利要求1至5中任一权利要求所述的核酸探针或者包含权利要求6所述的核酸探针组合，以及DNA连接酶。
  8. 根据权利要求7所述的连接液，其特征在于如下的(1)至(5)项中的任意一项或者多项：
    (1)所述DNA连接酶为选自T4DNA连接酶、T7DNA连接酶和T3DNA连接酶中的一种或多种；
    (2)所述核酸探针的浓度为0.1μM－5μM，优选为1μM；
    (3)所述DNA连接酶的浓度为0.01μM－2μM，优选为0.5μM；
    (4)还包括如下成分：
    50mM CH 3COOK，20mM Tris，10mM Mg(CH 3COO) 2，100μg/ml BSA，1mM ATP，10％PEG6000；
    (5)所述连接液余量为水。
  9. 一种试剂盒，其包含权利要求1至5中任一权利要求所述的核酸探针，或者包含权利要求6所述的核酸探针组合，或者包含权利要求7或8所述的连接液；
    优选地，还包含选自能够切断第一部分和连接体之间的连接的试剂、用于溶解核酸探针的缓冲液和测序引物中一种或多种；
    优选地，所述试剂盒中的试剂不含有银离子。
  10. 根据权利要求9所述的试剂盒，其中，能够切断第一部分和连接体之间的连接的试剂为内切酶(例如内切酶IV或内切酶V)、有机膦化物(例如THPP或TCEP)或者PdCl 2与磺化三苯基膦配合物的组合物。
  11. 一种核酸测序方法，包括下述步骤：
    (1)将测序引物杂交至待测核酸分子；
    (2)将权利要求1至5中任一权利要求所述的核酸探针或者权利要求6所述的核酸探针组合连接至测序引物；
    (3)洗脱未与待测核酸分子结合的核酸探针；
    (4)检测结合于待测核酸分子的核酸探针上的可检测的标记物，确定第一部分的碱基信息；
    (5)切断核酸探针第一部分与连接体的连接，洗脱核酸探针中除第一部分之外的其余部分；
    优选地，还包括下述步骤：
    (6)重复前述步骤(2)－(4)或(2)－(5)。
  12. 根据权利要求11所述的测序方法，其中，步骤(2)中，通过使用权利要求7或8所述的连接液，将权利要求1至5中任一权利要求所述的核酸探针或者权利要求6所述的核酸探针组合连接至测序引物。
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