WO2023025218A1 - 一种用于检测单碱基突变的引物组和基因芯片方法 - Google Patents

一种用于检测单碱基突变的引物组和基因芯片方法 Download PDF

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WO2023025218A1
WO2023025218A1 PCT/CN2022/114653 CN2022114653W WO2023025218A1 WO 2023025218 A1 WO2023025218 A1 WO 2023025218A1 CN 2022114653 W CN2022114653 W CN 2022114653W WO 2023025218 A1 WO2023025218 A1 WO 2023025218A1
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nucleic acid
primer
chip
primer set
acid sequence
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French (fr)
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胡飞驰
王琪
吴政宪
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南京金斯瑞生物科技有限公司
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    • 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
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism
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    • 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
    • C12Q1/6858—Allele-specific amplification
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    • 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
    • C12Q1/686—Polymerase chain reaction [PCR]
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes

Definitions

  • the invention belongs to the field of biological detection, and in particular relates to a primer set and a gene chip method for detecting single base mutations.
  • Gene chip because of its high-throughput, fast and simple features, is a new development direction in the field of in vitro diagnostics. application prospects.
  • the in-depth development of gene chip diagnostic applications will effectively strengthen the diagnosis and treatment of diseases and protect human health.
  • some challenges are also faced in the process of its development, such as the single detection method (direct in situ hybridization) and the lack of delicate design, which limits the sensitivity of detection.
  • SNP nucleic acid monotyping
  • Another method is based on the in situ hybridization of the chip, and realizes the immobilization of large molecular weight by means of circular chain reaction, so as to realize highly sensitive detection (see Biosensors and Bioelectronics 49 (2013) 472-477), and its problem
  • the reason is that excessive in-situ reaction will bring more adsorption and pollution, which will easily cause the detection baseline to rise or false positive results.
  • the purpose of the present invention is to overcome the defects of low detection sensitivity, or high detection cost and operational complexity in the single-base mutation detection method in the prior art, and provide a method that can accurately, quickly and low-cost detect single-base mutations (or A method known as single nucleotide mutation, single nucleotide typing, single nucleotide polymorphism, SNP).
  • the present invention develops a new detection method for unbalanced PCR combined with chip capture.
  • the invention further combines unbalanced PCR with a high-throughput and high-sensitivity electrochemical chip through ingenious primer design, and realizes simultaneous quantitative detection of mutation sites of multiple target sequences.
  • the present invention provides a primer set for detecting single base mutations in nucleic acid sequences, characterized in that, the primer set comprises the following primers:
  • One or more recognition primers sequentially include in the 5' to 3' direction: a) a nucleotide sequence that can specifically bind to the detection tool; b) a gap that can prevent further amplification by PCR arm molecule; c) a nucleotide sequence complementary to a stretch of continuous nucleotides in the nucleic acid sequence to be detected, the 5' end of the continuous nucleotides starting from the first nucleoside downstream of the mutation detection site acid, and d) a single nucleotide selected from any of A, T, C, and G, and
  • An amplification primer wherein the 5' end of the amplification primer is modified with a group that can bind to a signal response molecule, and the amplification primer is capable of amplifying the amplification obtained by amplifying the nucleic acid sequence to be detected using the recognition primer. increase product.
  • nucleic acid sequence may be a double-stranded or single-stranded nucleic acid, such as double-stranded DNA, single-stranded DNA or RNA.
  • single base mutation refers to a mutation resulting from the substitution of a single base in a nucleic acid sequence.
  • the primer set used for single nucleotide mutation detection of the present invention adopts the design of two primers with different amplification functions (usually different lengths), one of which is called the recognition primer, and the other is called the amplification primer. primers.
  • the recognition primers include short-chain primers that are specifically complementary to the nucleic acid sequence to be detected
  • the amplification primers include short-chain primers that can be combined with a signal response Molecular groups of long-chain primers.
  • the nucleotide length of the specific complementarity between the recognition primer and the sequence to be detected can be 1 to 19 nucleotides less than that of the amplification primer, especially is 3 to 15 (eg, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15) nucleotides.
  • the nucleotide length of the specific complementarity between the recognition primer and the sequence to be detected (target sequence) can be 2 to 8 nucleotides less than that of the amplification primer, for example, 2 or 3 nucleotides less. , 4, 5, 6, 7 or 8 nucleotides.
  • the total nucleotide length of parts c) and d) of the recognition primer may be between 11 and 16 nucleotides (eg 11, 12, 13, 14, 15 or 16 nucleotides) and the nucleotide length of the amplification primer can be between 15 and 30 nucleotides (for example, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 , 28, 29 or 30 nucleotides).
  • the total nucleotide length of parts c) and d) of the recognition primer is 12 to 15 nucleotides.
  • the total length of the nucleotides of the lengths c) and d) of the recognition primer is 12 nucleotides.
  • the length of the amplification primer is 16 to 20 nucleotides.
  • the length of the amplification primer is 20 nucleotides.
  • the term “recognition primer” refers to a primer comprising a probe specific complementary binding sequence, a spacer molecule and a sequence specific complementary binding to a target sequence, wherein the 3' terminal nucleotide of the primer can be Choose any one of A, T, C, and G according to the detection purpose.
  • the term “short-chain primer” refers to a primer comprising a sequence specifically complementary to a target sequence, and the 3' terminal nucleotide of the primer can be selected from any one of A, T, C, and G according to the purpose of detection.
  • the recognition primers include short-chain primers, and the sequence of the short-chain primers is a key sequence for recognizing the target sequence.
  • amplification primer refers to a long-chain primer that includes a group that can bind to a signal-responsive molecule, for example, the modification at the 5' end of the long-chain primer can bind to a signal-responsive molecule group.
  • long-chain primer refers to a primer capable of amplifying an amplification product obtained by amplifying the target sequence using the recognition primer or the short-chain primer.
  • PCR using the primers of the present invention is also called unbalanced PCR.
  • the short-chain primer and the target nucleic acid sequence The binding force is greatly weakened and it is difficult to hybridize, resulting in no amplification product; and when the nucleotide at the 3' end of the short-chain primer is correctly paired with the single-base mutation site of the target nucleic acid sequence, it can be amplified at an appropriate annealing temperature.
  • Double-stranded amplification products are obtained, and the products amplified by long-chain primers and short-chain primers have strong hybridization power. Under this condition, the amplification products of short-chain primers can be combined and copied normally. After multiple PCR cycles, amplified , the correct specific amplification products generated by the long and short primers are exponentially amplified.
  • each short-chain primer will first modify the spacer molecule, and then connect to the nucleotide sequence that can specifically bind to the detection tool, wherein the role of the spacer molecule is to prevent further PCR amplification and ensure the recognition
  • the single-chain state of part a) of the primer; and the 5' end of the long-chain primer will be modified with a group that can bind to a signal response molecule.
  • the detection tool can be any detection tool that can detect nucleotide sequences common in the art, for example, the detection tool can be a chip modified with a gene probe, an electrochemical sensor modified with a gene probe Detection electrodes or fluorescent detection nanomaterials modified with gene probes.
  • the chip modified with the gene probe can be obtained by a common method in the art, and the chip can be, for example, a metal chip, a semiconductor chip, a glass chip, a paper chip, etc.; the electrochemical detection electrode modified with the gene probe can be used in the electrode, For example, it is obtained by linking oligonucleotide fragments with sulfhydryl groups on platinum electrodes or directly growing and synthesizing on chips; the fluorescent detection nanomaterials modified with gene probes can be metal nanomaterials, silicon, silicon dioxide, and carbon nanomaterials, which can be obtained by The silylating agent acts on the surface of the nanometer material to generate amino groups and link the oligonucleotide fragments modified with carboxyl groups.
  • the spacer molecule can be any molecule that can prevent further amplification of the sequence amplified by the short-chain primer, for example, the spacer molecule can be a molecule with a length not less than 5 carbon-carbon bonds, carbon-oxygen , carbon-nitrogen bonds, etc., and are not molecules of deoxyribonucleotides.
  • the spacer molecule may be a non-deoxyribonucleotide whose main chain molecule length is not less than 5 carbon-carbon bonds.
  • the spacer molecule can be a hydrocarbon chain molecule whose main chain molecular length is not less than 5 carbon-carbon bonds, for example, the main chain molecular length can be 6 carbon atoms, 9 carbon atoms or 12 carbon atoms Hydrocarbon chain molecules, such as C6, C9 or C12, etc.
  • the group that can bind to the signal response molecule can be selected according to the experience and actual needs of those skilled in the art, and any chemical molecule or biomolecular group that can react with the signal response molecule in the aqueous phase at room temperature can be used.
  • the bindable signal response molecule can be biotin, phenylboronic acid, mannose, antigen, antibody and the like.
  • the signal-responsive molecule and the combinable signal-responsive molecule refer to any pair of molecules that can be linked or bonded in a chemical reaction, wherein the signal-responsive molecule contains an electrochemical substance or a chromogenic substance.
  • the signal response molecule can be streptavidin linked with electrochemical substances such as horseradish peroxidase and alkaline phosphatase, or linked with chromogenic substances such as fluorescein, fluorescent protein or acridine compound , sugar molecules with adjacent hydroxyl groups, concanavalin, antibodies, antigens, etc.
  • a modified group on the amplification primer that can bind to a signal-responsive molecule, such as biotin binds to a signal-responsive molecule, such as horseradish peroxidase-labeled streptavidin, and the reaction substrate is added. , such as TMB, electrochemical signal detection can be performed after the reaction is terminated.
  • the part a) in the single-chain state that can specifically bind to the detection tool and the group that can bind to the signal response molecule will be introduced into the double-stranded product, wherein a) in the single-chain state
  • Some specific binding detection tools such as probe chips, can be immobilized on the chip; groups that can bind to signal-responsive molecules can be combined with signal-responsive molecules that are connected with electrochemical substances or chromogenic substances to generate detection signals, which is convenient for subsequent Chip capture and detection.
  • the primer set of the present invention may contain one or more recognition primers according to the needs of the detection purpose, for example, when the detection method of the present invention is used to detect whether the nucleotide at the expected single base mutation site of the target nucleic acid sequence is In the case of mutation, a recognition primer whose base at the 3' end is complementary to the base at the expected mutation site of the unmutated wild-type nucleic acid sequence can be used for detection, that is, a recognition primer.
  • the detection method of the present invention when used to determine what kind of base is the base mutation at the single base mutation site of the target nucleic acid sequence, the bases with the 3' end can be used as A, T, C and G One or more (eg, two, three, or four) recognition primers of any one of these are used for detection.
  • part d) of the recognition primer is the same as the The non-mutated nucleotide or the nucleotide complementary to the expected mutated nucleotide at the expected single base mutation site of the nucleic acid sequence to be detected.
  • the unbalanced PCR reaction amplifies the target nucleic acid sequence while identifying the single nucleotide (base) mutation site, and the amplified product is convenient for subsequent chip capture and detection, the detection sensitivity is realized.
  • the high-throughput lattice and fast reading speed of the chip can realize simultaneous and rapid detection of multiple target nucleic acid sequences. It can be seen that the method of the present invention can have the characteristics of high throughput, high sensitivity, and rapid detection.
  • the present invention also provides a gene chip method for detecting single base mutations in nucleic acid sequences, characterized in that the method comprises:
  • the gene chip method for detecting single base mutations in nucleic acid sequences is also called unbalanced PCR chip method, which mainly includes three parts: unbalanced PCR part, purification part and chip capture inspection part.
  • Fig. 1 exemplarily shows the flowchart of the unbalanced PCR chip method of the present invention.
  • the unbalanced PCR part involves using the primer set of the present invention to amplify the target nucleic acid sequence through unbalanced PCR, wherein the characteristics of the primer set used are the same as those of the previous part, in order to avoid unnecessary redundancy, the The characteristics of the primer set will not be described in detail.
  • the amplification reaction (ie, the polymerase chain reaction) of the method of the present invention can be performed in an amplification reaction mixture.
  • the mixture comprises the reagents required to perform a primer extension reaction or nucleic acid amplification, non-limiting examples of such reagents include primers, polymerases, buffers, cofactors (e.g. divalent or monovalent cations), nucleotides (e.g. dNTP).
  • the polymerase chain reaction is performed using DNA polymerase.
  • the DNA polymerase can be a common DNA polymerase known in the art.
  • the DNA polymerase is a high-fidelity polymerase.
  • the DNA polymerase is selected from: hot start Taq polymerase (E00049, GenScript Biotechnology Co., Ltd.), TaqNova Stoffel DNA polymerase (RP810, BLIRT), HiFi-KAPA polymerase 2X (KK2601 , Roche), Hemo KlenTaq polymerase (M0332S, NEB) etc.
  • the DNA polymerase is HiFi-KAPA polymerase.
  • the polymerase chain reaction may include a pre-denaturation step, a cyclic amplification step and a final extension step, and each cycle in the cyclic amplification step may include denaturation, annealing and extension steps.
  • the cyclic amplification step is performed for 18-30 cycles, such as 20 cycles.
  • the condition of each cycle in the cycle amplification step is 98°C, 10s; 45-52°C, 15-30s; 72°C, 15s.
  • the annealing temperature is 44 to 52°C, such as 44°C, 45°C, 46°C, 47°C, 48°C, 49°C, 50°C, 51°C or 52°C, preferably 45-50°C . In a specific embodiment, the annealing temperature is 45°C. In another specific embodiment, the annealing temperature is 50°C.
  • the purification process of the present invention can be carried out by using column purification or magnetic beads, the purpose of which is to remove unreacted primers and enzymes and obtain a relatively single target double-stranded DNA product.
  • the purified PCR product can be added to 50uL of PBST buffer, and then the mixture is added to the surface of the chip and reacted at 45°C for 40 minutes.
  • Various probe sequences that can hybridize with the PCR product have been synthesized in advance on the chip. Mainly hybridize to the specific end sequences of different primers, because the long-chain primers are modified with sites that can bind to electrochemical signal molecules, and then through a reaction at room temperature for 20 minutes, the signal molecules are connected to the top of the double strands, and the chip is placed on the In the chip reader ECD, the signal of each point can be read.
  • the information about the mutation of the target sequence can be known, for example, including information such as the amount of the mutant sequence, the type of the mutation, and the location of the mutation.
  • the present invention also provides a kit for detecting a single base mutation in a nucleic acid sequence, which comprises the primer set as described above.
  • the kit may also include a DNA polymerase, preferably a high-fidelity polymerase.
  • the present invention provides the use of the above primer set or the kit for detecting single base mutations in nucleic acid sequences.
  • the above-mentioned primer set or the kit is used in the detection of single base mutation of nucleic acid sequence by gene chip method.
  • Fig. 1 exemplarily shows the flowchart of the unbalanced PCR chip method of the present invention.
  • Figure 2 shows the gel electrophoresis of the unbalanced PCR amplification of the wild-type and mutant (single-base mutation) of the target sequence, in which lane 1 is the mutant target sequence, lane 2 is the wild-type sequence, and lane 3 is the Water replaces the blank control of the target sequence, and the leftmost and rightmost lanes are molecular weight markers (molecular weights from top to bottom are: 3000, 2000, 1500, 1000, 700, 500, 250, 100bp).
  • Figure 3 shows the gel electrophoresis images of PCR amplification of the mutant target sequence by primer p-1-c9 and primer p-1.
  • lane 1 is the gel map of the target sequence after PCR with primers p-1-c9 modified with spacer molecules and complementary to the chip probe
  • lane 2 is without molecular modification of the spacer and without complementarity with the chip probe
  • lane 3 is the molecular weight marker (the molecular weight from top to bottom is: 3000, 2000, 1500, 1000, 700, 500, 250, 100bp).
  • Figure 4 shows the results of chip hybridization detection of PCR products
  • a is the electrochemical imaging image obtained by the chip reader ECD detection of the four products after hybridization and capture on the chip surface
  • b is the measured target lattice on the chip Electrochemical signal collection
  • the abscissa is the corresponding lattice probe
  • the ordinate is the average value of the net added value of the signal.
  • Figure 5 shows the low-concentration detection results of unbalanced PCR combined with chip hybridization.
  • a is the gel image of the gradient concentration target sequence after the first step of unbalanced PCR
  • b is the electrochemical imaging image obtained by hybridizing the purified PCR product to the chip and putting it into the chip reader ECD
  • c is the image b
  • the abscissa is the PCR product of different concentrations
  • the ordinate is the average value of the net value-added of the signal.
  • Embodiment 1 Unbalanced PCR primers carry out SNP detection (specificity detection)
  • the design of unbalanced PCR primers is shown in Table 1 below, wherein the recognition primer p-1-c9 includes three parts: the 5' end is a sequence (CAGCTAGAGCTCCAGT, 16nt, SEQ ID NO: 7) that can specifically complement the chip probe; The middle part is a specially designed modified spacer molecule C9, whose function is to block the further amplification of PCR and ensure that the 5' end part is in a single-stranded state, so that it can further hybridize with the chip probe; the 3' end part can be combined with the target sequence Part of the PCR short-chain primer that specifically binds. Design the long-chain primer p-2, in which the 5' end is modified with biotin, a group that can bind to signal response molecules.
  • Both wild-type and mutant nucleotide sequence template amounts were 1 nmol ⁇ L -1 , and PCR reactions were performed.
  • the specific reaction system and reaction conditions are as follows.
  • the PCR reaction was carried out on a Biometra T1 thermocycler.
  • the reaction system was as follows: primer p-1-c9 (10 ⁇ mol L -1 ) 1 ⁇ L, primer p-2 (10 ⁇ mol L - 1 ) 1 ⁇ L, template DNA (1nmol ⁇ L -1 ) 1 ⁇ L, 2X HiFi-KAPA polymerase (KK2601, Roche) 10 ⁇ L, the rest was made up to 20 ⁇ L with ddH 2 O.
  • the reaction conditions were: pre-denaturation at 98°C for 30s; denaturation at 98°C for 10s, annealing at 50°C for 30s, extension at 72°C for 15s, and after 20 cycles, extension at 72°C for 5min.
  • the target sequence to be detected is a mutant target sequence
  • the recognition primer p-1-c9 which includes three parts: the 5' end is a sequence (CAGCTAGAGCTCCAGT, 16nt) that can specifically complement the chip probe; the middle part is a specially designed modified spacer molecule C9, whose role is to Block the further amplification of PCR to ensure that the 5' end part is in a single-stranded state and can further hybridize with the chip probe; the 3' end part can specifically bind to the target sequence and is the part of the PCR short-chain primer.
  • the 5' end is a sequence (CAGCTAGAGCTCCAGT, 16nt) that can specifically complement the chip probe
  • the middle part is a specially designed modified spacer molecule C9, whose role is to Block the further amplification of PCR to ensure that the 5' end part is in a single-stranded state and can further hybridize with the chip probe; the 3' end part can specifically bind to the target sequence and is the part of the PCR short-chain primer.
  • the template concentration of the synthesized mutant target sequence was 1 nmol ⁇ L -1
  • the PCR reaction system of lane 1 was 20 ⁇ L, including 1 ⁇ L of primers p-1-c9 (10 ⁇ mol ⁇ L -1 ), primer p-2 (10 ⁇ mol ⁇ L -1 ) 1 ⁇ L, template DNA (1 nmol ⁇ L ⁇ 1 ) 1 ⁇ L, 2X HiFi-KAPA polymerase (KK2601, Roche) 10 ⁇ L.
  • the PCR reaction system in lane 2 includes 1 ⁇ L of primer p-1 (10 ⁇ mol L -1 ), 1 ⁇ L of primer p-2 (10 ⁇ mol L -1 ), 1 ⁇ L of template DNA (1 nmol L -1 ), 2X HiFi-KAPA polymerase (KK2601, Roche) 10 ⁇ L, the rest was made up to 20 ⁇ L with ddH 2 O.
  • the reaction conditions of PCR were: pre-denaturation at 98°C for 30s; denaturation at 98°C for 10s, annealing at 50°C for 30s, extension at 72°C for 15s, and after 20 cycles, extension at 72°C for 5min.
  • agarose gel electrophoresis analysis was carried out: 2 ⁇ L of the above corresponding PCR products were mixed with 0.5 ⁇ L plasmid dye (Goldview, Beijing Saibaisheng) respectively, and the two mixtures were added to 2.5% agarose gel ( Invitrogen) was used for gel electrophoresis in the well corresponding to the lane, and the gel image was collected (DYY-8C type, Beijing Liuyi Biotechnology; 120V, 20min).
  • Embodiment 3 PCR product purification
  • the three PCR products obtained in Example 1 were purified by magnetic beads (smart beads, Yisheng Biology), and the following operations were performed according to the instructions provided by the manufacturer: the magnetic beads were taken out from the refrigerator, equilibrated at room temperature for at least 30 minutes, vortexed Vortex or invert the beads thoroughly to ensure thorough mixing. Take the same volume (1.0 ⁇ , ⁇ represents the volume of the DNA sample to be purified) of Hieff Put Smarter DNA Clean Beads into the DNA solution (PCR product EP tube) and incubate at room temperature for 5 minutes. Centrifuge the PCR tube briefly and place it in a magnetic stand to separate the magnetic beads and liquid. After the solution is clear (about 5 minutes), carefully remove the supernatant.
  • the magnetic beads were taken out from the refrigerator, equilibrated at room temperature for at least 30 minutes, vortexed Vortex or invert the beads thoroughly to ensure thorough mixing.
  • Hieff Put Smarter DNA Clean Beads into the DNA solution (PCR product EP tube) and in
  • the concentrations of the three purified products were detected with Thermo Scientific TM NanoDrop TM One Microvolume UV-Vis Spectrophotometers, and the concentrations of the above-mentioned purified products were measured as shown in Table 3.
  • the concentration of sample 1 (the PCR product of the mutant target sequence) was much greater than that of the sample 2 and 3 (PCR products of the wild-type sequence and the blank control, respectively), prove that the unbalanced PCR based on primers of different lengths can effectively identify and amplify the single-base mutation of the mutant target sequence, and successfully purify it.
  • Primer p-2 is modified with a site (biotin) that can bind to an electrochemical signal molecule. Therefore, the amplified PCR product also has this binding site.
  • the signal molecule SA-HRP , horseradish peroxidase-labeled streptavidin
  • SA-HRP horseradish peroxidase-labeled streptavidin
  • Figure 4b is the measured electrochemical signal collection of the target lattice on the chip.
  • the abscissa is the corresponding lattice probe, and the ordinate is the average value of the net added value of the signal. It can be found that only the first part of the target lattice probe There is an obvious signal increase, and there is almost no obvious signal in other parts of the lattice probe, which corresponds to the imaging figure 4a and is consistent with the theory. Through the specific signal in Figure 4b, the purpose of quantitative detection of SNP can also be achieved.
  • the target sequence to be detected is a (mutant) target sequence
  • the concentration of the synthesized mutant target sequence was 1nmol ⁇ L -1 , and the mutant target sequence was diluted to 1pM, 100fM, 10fM, 1fM respectively by gradient dilution with TE buffer as templates for amplification, and then PCR amplification was performed respectively.
  • Amplification system 1 ⁇ L of primer p-1-c9 (10 ⁇ mol L -1 ), 1 ⁇ L of primer p-2 (10 ⁇ mol L -1 ), 1 ⁇ L of different concentrations of templates, 10 ⁇ L of 2X HiFi-KAPA polymerase (KK2601, Roche) , and make up the rest to 20 ⁇ L with ddH 2 O.
  • reaction conditions were: pre-denaturation at 98°C for 30s; denaturation at 98°C for 10s, annealing at 50°C for 30s, extension at 72°C for 15s, and after 20 cycles, extension at 72°C for 5min.
  • Figure 5b is the electrochemical imaging image obtained after the purified PCR product is hybridized to the chip and placed in the chip reader ECD.
  • the experimental operation is as described in the chip hybridization detection part of Example 4, wherein the electrochemical signal can also be replaced
  • the analysis was performed with a higher amount of SA-poly-HRP molecules to further increase the sensitivity. It can be seen that the four areas on the chip from top to bottom were hybridized with different concentrations of PCR products, and the corresponding display points were gradually brightened, and the gray value gradually increased, while the gray value of the control array points did not increase significantly, indicating that the chip The target object was detected accurately, and it was consistent with the theory.
  • Figure 5c is the specific signal value corresponding to Figure 5b. The measured electrochemical signal value of the chip increases.
  • the abscissa is the PCR product of different concentrations, and the ordinate is the average value of the net value of the signal. As the concentration increases, the value of the electrochemical signal increases in turn. increase, and there is no significant change in the point signal of the control array, which corresponds to imaging Figure 5b, and is consistent with the theory, indicating that this method is feasible and can achieve ultra-low concentration ( ⁇ 1fM) target sequence detection.

Abstract

提供了一种用于检测单碱基突变的引物组和基因芯片方法，其中所述基因芯片方法包括：提供包含待检测的核酸序列的样品；使用引物组通过聚合酶链反应扩增所述样品中的所述待检测的核酸序列，纯化所述聚合酶链反应后的扩增产物，以除去未反应的引物和酶；以及将纯化后的扩增产物与芯片探针杂交，并使用芯片阅读器检测所述待检测的核酸序列的突变信息。该方法在识别碱基突变位点的同时将目标核酸序列进行扩增，不仅实现了检测的增敏，而且基于芯片的高通量点阵，读取速度快等特点，可实现多目标核酸序列的同时快速检测。

Description

一种用于检测单碱基突变的引物组和基因芯片方法
相关申请的交叉引用
本申请要求于2021年8月25日提交的申请号为202110980219.2的中国专利申请的优先权，其全部内容通过引入并入本文。
技术领域
本发明属于生物检测领域，具体地，涉及一种用于检测单碱基突变的引物组和基因芯片方法。
背景技术
基因芯片，因其具备高通量、快速、简便的特点，是体外诊断领域的新发展方向，在遗传病检测、疾病筛查、疾病分型、病原体检测、个性化用药等方面均呈现出广阔的应用前景。深入开发基因芯片诊断应用将有力加强疾病诊疗，为人类健康保驾护航。然而，其发展的过程中也面临一些挑战，如检测方法过于单一(直接原位杂交)，缺乏精巧设计，这便限制了检测的灵敏度。特别是表现在核酸单分型(SNP)检测方面，因其既要保证可以准确识别突变位点，同时要满足足够高的检测灵敏度，这便是传统的原位杂交很难得实现的。
为此，本领域产生了一些联用或者增敏的方法，例如，全球知名的基因芯片公司Illumina开发的Golden-gate方法，其中将滚环放大法与原位杂交相结合，从而可以有效促进检测灵敏度，但这也同时提高了检测成本与操作复杂度(参见ILLUMINAGOLDENGATEDNA甲基化芯片的KL-FCM聚类分析，生物信息学.2014(000)002)。另有方法是在芯片原位杂交基础之上，借助循环链反应来实现大分子量的固载，以此来实现高灵敏的检测(参见Biosensors and Bioelectronics 49(2013)472-477)，而其问题在于过量的原位反应，会带来较多的吸附和污染，从而容易造成检测基线升高或者假阳性结果。还有方法借助纳米材料或者新合成信号响应的灵敏分子来结合于芯片上，以此实现高灵敏检测目的(参见Sensors  and Actuators B:Chemical,272,53-59；Journal of the American Chemical Society,138(42),13975-13984)，其缺点在于纳米材料或新分子合成技术难度高，尺寸或纯度不好控制，导致方法检测重现性降低。
由于以上方法均存在一定的缺陷：要么过于单一，限制了检测的灵敏度；要么虽然促进了检测灵敏度，但同时也提高了检测成本与操作复杂度，还有可能减低检测结果的准确度。因此，寻求一种既能够准确快速地检测SNP，同时又低成本、易操作的方法是非常必要的。
发明内容
本发明的目的在于克服现有技术中的单碱基突变检测方法存在检测灵敏度低、或检测成本与操作复杂度高的缺陷，提供一种能够准确快速且低成本地检测单碱基突变(或称为单核苷酸突变、单核苷酸分型、单核苷酸多态性，SNP)的方法。
本发明基于发明人以前的诊断方法开发经验，开发了一种新型不平衡PCR联合芯片捕获的检测方法。本发明进一步通过巧妙的引物设计将不平衡PCR与高通量高灵敏的电化学芯片结合，实现了多靶标序列突变位点的同时定量检测。
为了实现上述目的，在一方面，本发明提供了一种用于检测核酸序列的单碱基突变的引物组，其特征在于，所述引物组包含以下引物：
一种或多种识别引物，其中所述识别引物在5’至3’方向上依次包含：a)一段可与检测工具特异性结合的核苷酸序列；b)可阻止PCR进一步扩增的间隔臂分子；c)与所述待检测核酸序列中的一段连续核苷酸互补的核苷酸序列，所述连续核苷酸的5’端起始于突变检测位点下游的第一个核苷酸，和d)选自A、T、C和G中任一种的单个核苷酸，以及
扩增引物，其中所述扩增引物的5’端修饰有可结合信号响应分子的基团，并且所述扩增引物能够扩增使用所述识别引物扩增所述待检测核酸序列得到的扩增产物。
在本说明书的上下文中，所述“核酸序列”可以是双链或单链核酸，例如双链DNA、单链DNA或RNA。
在本说明书的上下文中，所述“单碱基突变”是指因核酸序列上的单个碱基的替换产生的突变。
本发明的用于单核苷酸突变检测的引物组采用了两种扩增功能不同(通常情况下长度亦不同)的引物的设计，其中一种称为识别引物，另一种称为扩增引物。此外，基于在PCR扩增时引物与模板特异性互补的核苷酸长度的不同，所述识别引物包含与待检测核酸序列特异性互补的短链引物，而扩增引物为包含可结合信号响应分子基团的长链引物。
例如，在本发明的一个实施方式中，所述识别引物与待检测序列(靶序列)的特异性互补的核苷酸长度可以比所述扩增引物的少1至19个核苷酸，特别是3至15个(例如3、4、5、6、7、8、9、10、11、12、13、14或15个)核苷酸。在一些优选实施方式中，所述识别引物与待检测序列(靶序列)的特异性互补的核苷酸长度可以比所述扩增引物的少2至8个核苷酸，例如少2、3、4、5、6、7或8个核苷酸。更具体地，所述识别引物的c)和d)部分的核苷酸总长度可以在11至16个核苷酸之间(例如11、12、13、14、15或16个核苷酸)；并且所述扩增引物的核苷酸长度可以在15至30个核苷酸之间(例如15、16、17、18、19、20、21、22、23、24、25、26、27、28、29或30个核苷酸)。在一些优选实施方式中，所述识别引物的c)和d)部分的核苷酸总长度为12至15个核苷酸。在一个具体实施方式中，所述识别引物的长度c)和d)部分的核苷酸总长度为12个核苷酸。在一个优选实施方式中，所述扩增引物的长度为16至20个核苷酸。在一个具体实施方式中，所述扩增引物的长度为20个核苷酸。
在本说明书的上下文中，术语“识别引物”是指包含探针特异性互补结合序列、间隔臂分子和与靶序列特异性互补结合的序列的引物，其中该引物的3’末端核苷酸可以根据检测目的选择A、T、C、G中任意一个。术语“短链引物”是指包含与靶序列特异性互补结合的序列的引物，该引物3’末端核苷酸可以根据检测目的选择A、T、C、G中任意一个。在本申请中，识别引物包含短链引物，且短链引物序列是发挥识别靶序列功能的关键序列。术语“扩增引物”是指包含可结合信号响应分子的基团的长链引物，例如，在长链引物的5’端修饰上可结合信号响应分子基团。术语“长链引物”是指能够扩增使用所述识别引物或短链引物扩增所述靶序列得到的扩增产物的引物。
基于本发明的引物组的独特设计，采用本发明的引物进行的PCR亦称为不平衡PCR。具体地，当短链引物的3’末端的核苷酸(即d)部分)与待检测(目标)核酸序列的单核苷酸突变位点不能正确配对时，短链引物与目标核酸序列的结合力大大减弱而难于杂交，导致无扩增产物；而当短链引物的3’末端的核苷酸与目标核酸序列的单碱基突变位点正确配对时，在合适的退火温度下可以扩增得到双链扩增产物，长链引物与短链引物扩增出的产物有强的杂交力，在此条件下可正常结合并复制短链引物的扩增产物，通过多个PCR循环放大后，由长链引物和短链引物产生的正确的特异性扩增产物被指数倍地扩增。
进一步地，每条短链引物的5’末端都会先修饰间隔臂分子，再连接上可特异性结合检测工具的核苷酸序列，其中间隔臂分子的作用在于阻止PCR进一步的扩增，保证识别引物的a)部分的单链状态；而长链引物的5’末端会修饰有可结合信号响应分子的基团。
更具体地，所述检测工具可以是本领域中常见的任何可检测核苷酸序列的检测工具，例如，所述检测工具可以为修饰有基因探针的芯片、修饰有基因探针的电化学检测电极或修饰有基因探针的荧光检测纳米材料。修饰有基因探针的芯片可采用本领域常用方法获得，且所述芯片可以例如是金属芯片、半导体芯片、玻璃芯片、纸芯片等；修饰有基因探针的电化学检测电极可采用在电极，如铂电极上通过巯基连接寡核苷酸片段或直接在芯片上生长合成而获得；修饰有基因探针的荧光检测纳米材料可以是金属纳米材料、硅、二氧化硅、碳纳米材料，可通过硅烷化试剂作用纳米材料表面产生氨基与修饰有羧基的寡核苷酸片段连接而获得。
根据本发明，所述间隔臂分子可以为任何可阻止由短链引物扩增的序列进一步扩增的分子，例如，所述间隔臂分子可以为分子长度不小于5个碳碳键、碳氧键、碳氮键等，且不是脱氧核糖核苷酸的分子。在一些实施方式中，所述间隔臂分子可以为主链分子长度不小于5个碳碳键的非脱氧核糖核苷酸。在一些实施方式中，所述间隔臂分子可以为主链分子长度不小于5个碳碳键的烃链分子，例如主链分子长度可以为6个碳原子、9个碳原子或12个碳原子的烃链分子等，即C6、C9或C12等。
更具体地，可结合信号响应分子的基团可以根据本领域技术人员的经验和 实际需要进行选择，满足室温水相中，与信号响应分子发生结合反应的化学分子或生物分子基团均可。例如，所述可结合信号响应分子可以为生物素(biotin)、苯硼酸、甘露糖、抗原、抗体等。信号响应分子与可结合信号响应分子是指任何能发生化学反应连接或结合的一对分子，其中，信号响应分子包含电化学物质或可显色物质。例如，相应地，信号响应分子可以是连接有辣根过氧化物酶、碱性磷酸酶等电化学物质或者连接有荧光素、荧光蛋白或吖啶化合物等可显色物质的链霉亲和素、邻位羟基的糖分子、刀豆素、抗体、抗原等。在一些实施方式中，通过扩增引物上修饰的可结合信号响应分子的基团，如生物素与信号响应分子，如辣根过氧化物酶标记的链霉亲和素结合，加入反应底物，如TMB，终止反应后可以进行电化学信号检测。
进一步地，当如上所述正确配对时，可特异性结合检测工具的单链状态的a)部分和可结合信号响应分子的基团将被引入双链产物中，其中，单链状态的a)部分特异性结合检测工具如探针芯片上，可以固定在芯片上；可结合信号响应分子的基团可以与连接有电化学物质或可显色物质的信号响应分子结合产生检测信号，便于后续的芯片捕获和检测。
另外，本发明的引物组可以根据检测目的的需要包含一种或多种识别引物，例如，当本发明的检测方法用于检测目标核酸序列的预期单碱基突变位点上的核苷酸是否突变时，可以使用3’末端的碱基与未突变的野生型核酸序列的该预期突变位点上的碱基互补的识别引物，即一种识别引物进行检测。又例如，当本发明的检测方法用于确定目标核酸序列的单碱基突变位点上的碱基突变为何种碱基时，可以使用具有3’末端的碱基为A、T、C和G中任一种的一种或多种(例如两种、三种或四种)识别引物进行检测。在一些实施方式中，当本发明的方法用于检测待测核酸序列的预期单碱基突变位点上是否存在突变或者所预期的突变核苷酸，所述识别引物的d)部分为与所述待检测核酸序列的预期单碱基突变位点上的未突变核苷酸或所预期的突变核苷酸互补的核苷酸。
由于不平衡PCR反应在识别单核苷酸(碱基)突变位点的同时将目标核酸序列进行扩增，且扩增产物便于后续的芯片捕获检测，因此实现了检测的增敏。另外，芯片的高通量点阵，读取速度快等特点，可实现多目标核酸序列的同时快速检测，由此可见本发明方法可以具有高通量，高灵敏，快速检测的特点。
在另一方面，本发明还提供了一种用于检测核酸序列的单碱基突变的基因芯片方法，其特征在于，所述方法包括：
1)提供包含待检测的核酸序列的样品；
2)使用如上所述的引物组通过聚合酶链反应扩增所述样品中的所述待检测的核酸序列，
3)纯化所述聚合酶链反应后的扩增产物，以除去未反应的引物和酶；以及
4)将纯化后的扩增产物与芯片探针杂交，并使用芯片阅读器检测所述待检测的核酸序列的突变信息。
根据本发明，所述用于检测核酸序列的单碱基突变的基因芯片方法亦称为不平衡PCR芯片方法，其主要包括三个部分：不平衡PCR部分、纯化部分和芯片捕获检查部分。图1示例性地显示了本发明的不平衡PCR芯片方法的流程图。
不平衡PCR部分
在所述不平衡PCR部分中，其涉及使用本发明的引物组对目标核酸序列通过不平衡PCR进行扩增，其中所采用引物组的特征与前述部分相同，为了避免不必要的冗余，对所述引物组的特征不再赘述。
本发明方法的扩增反应(即所述聚合酶链反应)可以在扩增反应混合物中进行。所述混合物包含完成引物延伸反应或核酸扩增所需的试剂，此类试剂的非限制性实例包括引物、聚合酶、缓冲液、辅因子(例如二价或单价阳离子)、核苷酸(例如dNTP)。
在本发明中，所述聚合酶链反应使用DNA聚合酶进行。所述DNA聚合酶可以是本领域已知的常用DNA聚合酶。在一些实施方式中，所述DNA聚合酶为高保真聚合酶。在一些实施方式中，所述DNA聚合酶选自：热启动Taq聚合酶(E00049，金斯瑞生物科技有限公司)，TaqNova Stoffel DNA聚合酶(RP810，BLIRT)，HiFi-KAPA聚合酶2X(KK2601，Roche)，Hemo KlenTaq聚合酶(M0332S，NEB)等。在一个优选的实施方式中，所述DNA聚合酶为HiFi-KAPA聚合酶。
在本发明的一些实施方式中，所述聚合酶链反应可以包括预变性步骤、循 环扩增步骤和终延伸步骤，所述循环扩增步骤中的各循环可以包括变性、退火和延伸步骤。在一些实施方式中，所述循环扩增步骤进行18-30个循环，例如20个循环。在一些实施方式中，所述循环扩增步骤中每个循环的条件为98℃，10s；45-52℃，15～30s；72℃，15s。在一些实施方式中，所述退火的温度为44至52℃，例如44℃、45℃、46℃、47℃、48℃、49℃、50℃、51℃或52℃，优选45-50℃。在一个具体实施方式中，所述退火温度为45℃。在另一个具体实施方式中，所述退火温度为50℃。
纯化部分
本发明的纯化过程可以采用柱纯化或者磁珠来进行，其目的在于去除未反应的引物和酶，得到较为单一的目标双链DNA产物。以磁珠纯化为例，smart beads(翌圣生物)纯化，按照制造商提供的说明进行以下操作：1)将磁珠由冰箱中取出，在室温下平衡至少30分钟。2)涡旋振荡或充分颠倒磁珠以保证充分混匀。3)取相同体积(1.0×，×表示待纯化DNA样品体积)的Hieff
Figure PCTCN2022114653-appb-000001
Smarter DNA Clean Beads至DNA溶液(PCR产物EP管)中，室温孵育5分钟。4)将PCR管短暂离心并置于磁力架中分离磁珠和液体，待溶液澄清后(约5分钟)，小心移除上清。5)保持PCR管始终置于磁力架中，加入200μL新鲜配制的80％乙醇漂洗磁珠，室温孵育30秒后，小心移除上清。6)重复步骤5，总计漂洗2次。7)保持PCR管始终置于磁力架中，开盖以空气干燥磁珠至刚刚出现龟裂(约5分钟)。8)将PCR管从磁力架中取出，加入21μL ddH 2O，使用移液器轻轻吹打至充分混匀，室温静置5分钟。9)将PCR管短暂离心并置于磁力架中静置，待溶液澄清后(约5分钟)，小心移取20μL上清至新PCR管中，勿触碰磁珠，可得到纯的双链DNA产物。
芯片捕获检查部分
纯化后的PCR产物可以加入PBST缓冲液至50uL，再将此混合液加入到芯片表面，45℃反应40分钟，其中芯片上已提前合成了各种可与PCR产物相杂交结合的探针序列，主要杂交于不同引物的特异性末端序列，因长链引物上修饰有可与电化学信号分子结合的位点，再通过室温20分钟的反应，将信号分子连接于双链顶端，将芯片置于芯片阅读器ECD里，便可读出每一个点的信号。通过对芯片上点阵信号的有无或者强弱分析，便可知道目标序列突变的信息，例 如，包括突变序列的量、突变的类型、突变的位置等信息。
本发明还提供了一种用于检测核酸序列的单碱基突变的试剂盒，其包含根据如上所述的引物组。在一个优选的实施方式中，所述试剂盒中还可以包含DNA聚合酶，优选为高保真聚合酶。
另外，本发明提供了以上所述引物组或所述试剂盒用于检测核酸序列的单碱基突变中的用途。特别是，以上所述引物组或所述试剂盒用于基因芯片方法检测核酸序列的单碱基突变中的用途。
附图说明
附图是用来提供对本发明的进一步理解，并且构成说明书的一部分，与下面的具体实施方式一起用于解释本发明，但并不构成对本发明的限制。在附图中：
图1示例性地显示了本发明的不平衡PCR芯片方法的流程图。
图2显示了对目标序列野生型和突变型(单碱基突变)进行不平衡PCR扩增的凝胶电泳图，其中泳道1是突变型目标序列，泳道2是野生型序列，泳道3是以水替代目标序列的空白对照，最左测和最右侧泳道是分子量标记(从上到下的分子量依次是：3000、2000、1500、1000、700、500、250、100bp)。
图3显示了引物p-1-c9与引物p-1对突变型目标序列PCR扩增的凝胶电泳图。其中泳道1是使用间隔臂分子修饰且存在与芯片探针互补序列的引物p-1-c9对目标序列PCR后的产物胶图，泳道2是无间隔臂分子修饰且不存在与芯片探针互补识别序列的引物p-1对目标序列PCR后的产物胶图，泳道3是分子量标记(从上到下的分子量依次是：3000、2000、1500、1000、700、500、250、100bp)。
图4显示了PCR产物进行芯片杂交检测的结果，a是四种产物在芯片表面杂交捕获后，以芯片阅读器ECD检测所得到的电化学成像图，b是测得的芯片上目标点阵的电化学信号收集，横坐标是对应的点阵探针，纵坐标是信号的净增值的平均值。
图5显示了不平衡PCR联合芯片杂交的低浓度检测结果。a是梯度浓度目标序列经过第一步不平衡PCR后的胶图，b是将纯化后的PCR产物杂交于芯片 后，放入芯片阅读器ECD中所得到的电化学成像图，c是图b对应的具体信号值，测得的芯片电化学信号增值，横坐标是不同浓度PCR产物，纵坐标是信号的净增值的平均值。
具体实施方式
以下对本发明的具体实施方式进行详细说明。应当理解的是，此处所描述的具体实施方式仅用于说明和解释本发明，并不用于限制本发明。
在本文中所披露的范围的端点和任何值都不限于该精确的范围或值，这些范围或值应当理解为包含接近这些范围或值的值。对于数值范围来说，各个范围的端点值之间、各个范围的端点值和单独的点值之间，以及单独的点值之间可以彼此组合而得到一个或多个新的数值范围，这些数值范围应被视为在本文中具体公开。
以下由特定的具体实施例说明本发明的实施方式，熟悉此技术的人士可由本说明书所揭露的内容轻易地了解本发明的其他优点及功效，显然，所描述的实施例是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
实施例
实施例1：不平衡PCR引物进行SNP检测(特异性检测)
突变型目标序列
5’-CTTTACTTACTACACCTCAGATATATTTCTTCATGAAGACCTCACAGTAAAAATAGGTGATTTTGGTCTAGCTACAGA AGAAATCTCGATGGAGTGGG(SEQ ID NO:1)
野生型序列
5’-CTTTACTTACTACACCTCAGATATATTTCTTCATGAAGACCTCACAGTAAAAATAGGTGATTTTGGTCTAGCTACAGA TGAAATCTCGATGGAGTGGG(SEQ ID NO:2)
设计不平衡PCR引物见下表1，其中识别引物p-1-c9，包括三部分：5’端为 能与芯片探针特异性互补结合的序列(CAGCTAGAGCTCCAGT，16nt，SEQ ID NO:7)；中间部分为特别设计的修饰间隔臂分子C9，其作用在于阻断PCR进一步扩增，保证5’端部分呈现单链状态，从而能与芯片探针进一步互补杂交；3’端部分能够与靶标序列特异性结合的PCR短链引物部分。设计长链引物p-2，其中5’端修饰有可结合信号响应分子的基团生物素biotin。
表1
Figure PCTCN2022114653-appb-000002
野生型和突变型核苷酸序列模板量均为1nmol·L -1，进行PCR反应。具体反应体系和反应条件如下，PCR反应是在Biometra T1 thermolcycler热循环仪上进行的，反应体系如下：引物p-1-c9(10μmol·L -1)1μL，引物p-2(10μmol·L -1)1μL,模板DNA(1nmol·L -1)1μL,2X HiFi-KAPA聚合酶(KK2601，Roche)10μL，其余用ddH 2O补足至20μL。反应条件为：98℃预变性30s；98℃变性10s，50℃退火30s，72℃延伸15s，20个循环后，72℃延伸5min。将水作为模板，进行相同的PCR反应，作为空白对照。
反应结束后进行琼脂糖凝胶电泳分析：取上述PCR产物各2μL分别与0.5μL质粒大抽染料(Goldview，北京赛百盛)混匀，将混合液加入2.5％琼脂糖凝胶(Invitrogen)孔内，进行凝胶电泳，并收集胶图(DYY-8C型，北京六一生物；120V，20min)。
结果如图2所示，只有突变型目标序列(泳道1)出现明显条带，而野生型序列和空白对照(分别为泳道2和泳道3)没有出现条带，理论上野生型序列和水无法进行反应因而不会显示出条带，这与理论相符合，说明此方法能准确识别单碱基突变。
实施例2：包含间隔臂引物检测单碱基突变的验证
待检测的目标序列为突变型目标序列
5’-CTTTACTTACTACACCTCAGATATATTTCTTCATGAAGACCTCACAGTAAAAATAGGTGATTTTGGTCTAGCTACAGA AGAAATCTCGATGGAGTGGG(SEQ ID NO:1)
设计识别引物p-1-c9，其包括三部分：5’端为能与芯片探针特异性互补结合的序列(CAGCTAGAGCTCCAGT，16nt)；中间部分为特别设计的修饰间隔臂分子C9，其作用在于阻断PCR进一步扩增，保证5’端部分呈现单链状态，能与芯片探针进一步互补杂交；3’端部分能够与靶标序列特异性结合，为PCR短链引物部分。
设计短链引物p-1：仅保留p-1-c9的3’端部分，能够与靶标序列特异性结合，作为PCR的短链引物。
设计长链引物p-2：其中5’端修饰可结合信号响应分子的基团生物素biotin，具体序列见下表2：
表2
引物名称 序列
p-1-c9 CAGCTAGAGCTCCAGT(spacer)ATCGAGATTTCT(SEQ ID NO:3)
p-1 ATCGAGATTTCT(SEQ ID NO:5)
p-2 Biotin-CTTTACTTACTACACCTCAG(SEQ ID NO:4)
合成的突变型目标序列模板浓度均为1nmol·L -1，泳道1的PCR反应体系20μL，包括引物p-1-c9(10μmol·L -1)1μL，引物p-2(10μmol·L -1)1μL,模板DNA(1nmol·L -1)1μL,2X HiFi-KAPA聚合酶(KK2601，Roche)10μL。
泳道2的PCR反应体系包括引物p-1(10μmol·L -1)1μL，引物p-2(10μmol·L -1)1μL,模板DNA(1nmol·L -1)1μL,2X HiFi-KAPA聚合酶(KK2601，Roche)10μL，其余用ddH 2O补足至20μL。PCR的反应条件为：98℃预变性30s；98℃变性10s，50℃退火30s，72℃延伸15s，20个循环后，72℃延伸5min。
反应结束后进行琼脂糖凝胶电泳分析：取上述对应的PCR产物各2μL分别与0.5μL质粒大抽染料(Goldview，北京赛百盛)混匀，将两种混合液加入2.5％琼脂糖凝胶(Invitrogen)对应泳道的孔内，进行凝胶电泳，并收集胶图(DYY-8C 型，北京六一生物；120V，20min)。
结果如图3所示，均能看到明亮的目标条带，且图中泳道1的条带位置略高于泳道2条带这是由于修饰间隔臂与杂交链的引物在PCR后得到的产物分子量稍大于未修饰的引物PCR后得到的产物，这与理论上相符，说明间隔臂及芯片杂交序列成功引入至PCR产物中。
实施例3：PCR产物纯化
将实施例1得到的三种PCR产物进行磁珠(smart beads，翌圣生物)纯化，按照制造商提供的说明进行以下操作：将磁珠由冰箱中取出，在室温下平衡至少30分钟，涡旋振荡或充分颠倒磁珠以保证充分混匀。取相同体积(1.0×，×表示待纯化DNA样品体积)的Hieff
Figure PCTCN2022114653-appb-000003
Smarter DNA Clean Beads至DNA溶液(PCR产物EP管)中，室温孵育5分钟。将PCR管短暂离心并置于磁力架中分离磁珠和液体，溶液澄清后(约5分钟)，小心移除上清。保持PCR管始终置于磁力架中，加入200μL新鲜配制的80％乙醇漂洗磁珠，室温孵育30秒后，小心移除上清。重复步骤5，总计漂洗2次。保持PCR管始终置于磁力架中，开盖以空气干燥磁珠至刚出现龟裂(约5分钟)。将PCR管从磁力架中取出，加入21μL ddH 2O，使用移液器轻轻吹打至充分混匀，室温静置5分钟。将PCR管短暂离心并置于磁力架中静置，溶液澄清后(约5分钟)，小心移取20μL上清至新PCR管中，勿触碰磁珠，得到纯的双链DNA产物。
以Thermo Scientific TM NanoDrop TM One Microvolume UV-Vis Spectrophotometers检测三种纯化产物的浓度，测得上述纯化产物的浓度如表3所示，样品1(突变型目标序列的PCR产物)的浓度远远大于样品2和3(分别为野生型序列和空白对照的PCR产物)，证明基于不同长度引物的不平衡PCR可有效识别并扩增出突变型目标序列的单碱基突变，并成功进行了纯化。
表3
Figure PCTCN2022114653-appb-000004
实施例4：不平衡PCR联合芯片杂交检测PCR产物
向实施例3中三种纯化后的PCR产物中加入PBST缓冲液至50μL，再分别加入至芯片的三个腔体(芯片人为划分为互不干扰的四个区域，本实验中用到上面三个区域)进行杂交，如图4所示，45℃反应40分钟，而芯片上已连接可与PCR产物相杂交结合的探针序列(ACTGGAGCTCTAGCTGTTTTTTTTTT，SEQ ID NO:6)，用于特异性杂交利用引物p-1-c9/p-2进行PCR扩增的产物，其设计形状如图4a中每个区域的三个长方形区域中不同宽度的长条形。芯片点阵上还有其他控制探针，不与纯化的PCR产物杂交，用于检测试验中的干扰信号。
引物p-2上修饰有可与电化学信号分子结合的位点(生物素)，因此，扩增后的PCR产物上也有该结合位点，在室温下反应20分钟，信号分子(SA-HRP，辣根过氧化物酶标记链霉亲和素)连接于PCR产物的双链顶端的结合位点，将芯片置于芯片阅读器ECD里，可读出每一个点的信号。结果如图4所示，三种产物在芯片表面杂交捕获后，以芯片阅读器ECD检测所得到的电化学成像图，对比图4a中三个区域，可以发现加入目标产物的第一部分，只有相匹配的探针灰度值明显增加，而其他阵列点的灰度值与背景相当，说明方法成功检测到目标序列的产物，且无明显干扰信号。芯片的2，3部分是野生型序列及空白(水)的结果，可以发现所有点阵(包括目标点阵与空白点阵)都没有灰度值增加，这说明野生型序列是无法通过设计的引物扩增产生双链产物，与理论相符，这也说明此方法可以正确识别出突变型目标序列的单碱基突变。图4b是测得的芯片上目标点阵的电化学信号收集，横坐标是对应的点阵探针，纵坐标是信号的的净增值的平均值，可以发现只有第一部分的目标点阵探针有明显的信号增量，其他部分点阵探针几乎没有明显信号，这与成像图4a相应，且与理论相符合。通过图4b的具体信号，也可以达到SNP定量检测的目的。
实施例5：对不同低浓度产物进行检测
待检测的目标序列为(突变型)目标序列
5’-CTTTACTTACTACACCTCAGATATATTTCTTCATGAAGACCTCACAGTAAAAATAGGTGATTTTGGTCTAGCTACAGA AGAAATCTCGATGGAGTGGG(SEQ ID NO:1)
合成的突变型目标序列浓度为1nmol·L -1，通过梯度稀释用TE缓冲液将突变型目标序列分别稀释至1pM，100fM，10fM，1fM，作为扩增的模板，然后分 别进行PCR扩增。扩增体系：引物p-1-c9(10μmol·L -1)1μL，引物p-2(10μmol·L -1)1μL,不同浓度的模板1μL,2X HiFi-KAPA聚合酶(KK2601，Roche)10μL，其余用ddH 2O补足至20μL。反应条件为：98℃预变性30s；98℃变性10s，50℃退火30s，72℃延伸15s，20个循环后，72℃延伸5min。
各取2μL PCR产物样品与0.5μL质粒大抽染料混匀，将混合液加入2.5％的琼脂糖凝胶孔内，放在电泳仪器上跑胶，跑胶结束后放于紫外灯下观察。如图5a所示，不同模板量从右到左，1是1fM，2是10fM，3是100fM，4是1pM，5是标尺(从上到下的分子量依次是：3000、2000、1500、1000、700、500、250、100bp)，可以发现随着浓度降低，条带亮度逐渐变暗，说明扩增出的产物逐渐降低，这与理论是相符的。图5b是将纯化后的PCR产物杂交于芯片后，放入芯片阅读器ECD中所得到的电化学成像图，该实验操作如实施例4芯片杂交检测部分所述，其中电化学信号也可替换为具备更高量的SA-poly-HRP分子进行分析，以进一步提高灵敏度。可以看出，芯片上从上到下四个区域分别杂交不同浓度的PCR产物，其所对应的陈列点逐渐加亮，灰度值逐渐增加，而控制阵列点灰度则无明显增加，说明芯片上准确检测到目标物，且与理论相符。图5c是图5b对应的具体信号值，测得的芯片电化学信号增值，横坐标是不同浓度的PCR产物，纵坐标是信号的净增值的平均值，随着浓度增加，电化学信号增值依次增加，且控制阵列点信号无明显变化，这与成像图5b相应，且与理论相符合，说明此方法可行且可以实现超低浓度(<1fM)目标序列检测。
以上详细描述了本发明的优选实施方式，但是，本发明并不限于上述实施方式中的具体细节，在本发明的技术构思范围内，可以对本发明的技术方案进行多种简单变型，这些简单变型均属于本发明的保护范围。
另外需要说明的是，在上述具体实施方式中所描述的各个具体技术特征，在不矛盾的情况下，可以通过任何合适的方式进行组合，为了避免不必要的重复，本发明对各种可能的组合方式不再另行说明。
此外，本发明的各种不同的实施方式之间也可以进行任意组合，只要其不违背本发明的思想，其同样应当视为本发明所公开的内容。

Claims (17)

  1. 一种用于检测核酸序列的单碱基突变的引物组，其特征在于，所述引物组包含以下引物：
    一种或多种识别引物，其中所述识别引物在5’至3’方向上依次包含：a)一段可与检测工具特异性结合的核苷酸序列；b)可阻止PCR进一步扩增的间隔臂分子；c)与所述待检测核酸序列中的一段连续核苷酸互补的核苷酸序列，所述连续核苷酸的5’端起始于突变检测位点下游的第一个核苷酸，和d)选自A、T、C和G中任一种的单个核苷酸，以及
    扩增引物，其中所述扩增引物的5’端修饰有可结合信号响应分子的基团，并且所述扩增引物能够扩增使用所述识别引物扩增所述待检测核酸序列得到的扩增产物。
  2. 根据权利要求1所述的引物组，其中，所述检测工具为修饰有基因探针的芯片、电化学检测电极或荧光检测纳米材料。
  3. 根据权利要求1所述的引物组，其中，所述间隔臂分子为主链分子长度不小于5个碳碳键、碳氧键或碳氮键，且不是脱氧核糖核苷酸的分子，优选为主链分子长度不小于5个碳碳键的烃链分子。
  4. 根据权利要求1所述的引物组，其中，所述可结合信号响应分子的基团为在水相中可与信号响应分子结合的化学分子或生物分子，优选为生物素、苯硼酸、甘露糖、抗原或抗体。
  5. 根据权利要求1-4中任一项所述的引物组，其中，所述识别引物的c)和d)部分的核苷酸总长度比所述扩增引物的核苷酸长度短1至19个核苷酸。
  6. 根据权利要求1-5中任一项所述的引物组，其中，所述识别引物的c)和d)部分的核苷酸总长度在11至16个核苷酸之间。
  7. 根据权利要求1-6中任一项所述的引物组，其中，所述扩增引物的核苷酸长度在15至30个核苷酸之间。
  8. 一种用于检测核酸序列的单碱基突变的基因芯片方法，其特征在于，所述方法包括：
    1)提供包含待检测的核酸序列的样品；
    2)使用根据权利要求1-7中任一项所述的引物组通过聚合酶链反应扩增所述样品中的所述待检测的核酸序列，
    3)纯化所述聚合酶链反应后的扩增产物，以除去未反应的引物和酶；以及
    4)将纯化后的扩增产物与芯片探针杂交，并使用芯片阅读器检测所述待检测的核酸序列的突变信息。
  9. 根据权利要求8所述的方法，其中，所述识别引物的d)部分为与所述待检测核酸序列的预期单碱基突变位点上的未突变核苷酸或所预期的突变核苷酸互补的核苷酸。
  10. 根据权利要求8或9所述的方法，其中，所述聚合酶链反应使用DNA聚合酶进行，所述DNA聚合酶优选为高保真聚合酶。
  11. 根据权利要求10所述的方法，其中，所述DNA聚合酶选自：热启动Taq聚合酶、TaqNova Stoffel DNA聚合酶、HiFi-KAPA聚合酶和Hemo KlenTaq聚合酶。
  12. 根据权利要求8至11中任一项所述的方法，其中所述聚合酶链反应中采用的退火温度为44℃至52℃。
  13. 根据权利要求8所述的方法，其中，所述纯化为柱纯化或磁珠纯化。
  14. 根据权利要求8所述的方法，其中，所述核酸序列的突变信息包括突变序列的量、突变类型和突变位置中的至少一种。
  15. 一种用于检测核酸序列的单碱基突变的试剂盒，其包含根据权利要求1-7中任一项所述的引物组。
  16. 根据权利要求15所述的试剂盒，其还包含DNA聚合酶，优选为高保真聚合酶。
  17. 根据权利要求1-7中任一项所述的引物组、或根据权利要求15或16所述的试剂盒在用于基因芯片方法检测核酸序列的单碱基突变中的用途。
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