CN1434286A - immobilized nucleic acid probe for non-labeling detection - Google Patents

Abstract

The invention discloses a solid-phase nucleic acid probe which can be used for non-labeled detection, which comprises a solid carrier, on which an oligonucleotide containing a fluorescent group, a fluorescent quenching group and a specific nucleic acid recognition sequence fragment is immobilized acid probe, the relative physical position of the fluorescent group and the fluorescent quencher group in the probe is the position that can quench the fluorescence; when the target gene is hybridized with the specific nucleic acid recognition sequence fragment in the oligonucleotide probe, through A biochemical reaction that separates the fluorophore and the fluorescence quencher on the oligonucleotide probe, thereby allowing the nucleic acid probe to emit fluorescence. The present invention utilizes the physical space position to overcome the problem that the selection of fluorescent dye types in existing detectors is limited to the excitation wavelength range; avoids the existing technical difficulties of multiple PCR fluorescent labeling target sequences; and realizes high-throughput sample throughput, non-toxic Automated detection of markers. The time required for detection is greatly reduced; it has the potential to realize micro-full analysis.

Description

Immobilized nucleic acid probes for label-free detection

1. Technical field:

The invention relates to a solid-phase nucleic acid probe, in particular to a solid-phase nucleic acid probe which can be used for non-label detection.

2. Background technology

With the deepening of genome research, it will become possible to understand the differences in life, the rules of disease occurrence and development, and the interaction between drugs and living organisms at the genetic level. The high-throughput detection and analysis technology of nucleic acid sequence information will become one of the core technologies in the field of life sciences such as medicine. Recently, gene chip technology has attracted more and more attention. However, there are still some problems in gene chip technology, which affect its application in biology and clinical medicine. 1) The detection process is complicated. The extracted sample DNA needs to be amplified and incorporated with fluorescent markers. In this process, cross-contamination between samples is easy to occur, which affects the reliability of detection. 2) The incorporation of fluorescent markers is not only complicated but also costly to use, which is not conducive to the popularization and application of gene chips in clinical diagnosis. People need to develop high-throughput, accurate, low-cost, and label-free genetic information detection methods. At present, unlabeled detection technologies mainly include some probes, such as TaqMan probes and molecular beacon probes, which are used in fluorescent quantitative PCR reactions. The biggest disadvantage of these technologies in liquid phase environments is their low-throughput capabilities, which cannot be used simultaneously. Detect multiple target sequences.

3. Contents of the invention

1. Technical issues:

The invention provides a solid-phase nucleic acid probe capable of high-throughput detection of a gene to be tested and low in use cost, which can be used for non-marker detection.

2. Technical solution:

A solid-phase nucleic acid probe that can be used for non-labeled detection, comprising a solid support 1, on which an oligonucleus containing a fluorescent group 2, a fluorescent quenching group 3 and a specific nucleic acid recognition sequence fragment 4 is immobilized Nucleic acid probe, the relative physical position of the fluorescent group 2 and the fluorescent quencher 3 in the probe is the position that can quench the fluorescence; when the target gene hybridizes with the specific nucleic acid recognition sequence fragment in the oligonucleotide probe Finally, through a biochemical reaction, the fluorescent group on the oligonucleotide probe is separated from the fluorescent quenching group, so that the nucleic acid probe emits fluorescence.

3. Technical effect:

The solid-phase nucleic acid probe proposed by the present invention includes a nucleic acid sequence that can recognize each other with the target gene to be tested, and a fluorescent group and a fluorescent quenching group that are physically close to each other. Under normal conditions, the fluorescence quenching group can effectively quench the fluorescence of the adjacent fluorophore, and the probe does not emit fluorescence. However, after the detected target gene is identified with the probe, under the action of biologically active enzymes, the fluorescent quenching group is removed from the probe to make the probe emit fluorescence, thereby realizing the non-labeled detection of the tested nucleic acid sequence. The probes that can be aimed at different measured nucleic acid sequences are integrated together to form a microarray chip. The present invention has the following advantages: a. The probes can be directly fixed on a solid carrier in different forms to form a high-density microarray chip, Using spatial resolution to distinguish different detection sites, so as to achieve high-throughput parallel detection. It avoids the problem that the selection of fluorescent dyes in the existing quantitative PCR instruments is limited to the excitation wavelength range; b. Since the excision of the fluorescent quenching group in the nucleic acid probe is achieved by a biochemical reaction, for probes that do not hybridize with the target molecule Needle, the fluorescent molecular group is quenched by the quenching molecular group, so the fluorescence background of the whole chip is very low, and the detection sensitivity is high; c. Overcoming the technical difficulties of the existing multiple PCR fluorescent labeling target sequence, real-time , High-throughput non-labeled detection, at the same time, there is no PCR process, which greatly reduces the time required for detection, and also reduces the difficulty and cost of chip use; d. No need to fluorescently label the target gene, which can greatly simplify the operation The process saves the cost of use and can perform quantitative detection of target genes; e. By building a microfluidic chip, the automation of the entire detection process can be realized.

4. Description of drawings:

Figure 1 is a schematic diagram of three structures of the solid-phase nucleic acid probe proposed by the present invention. Fig. 1 is the solid-phase nucleic acid probe of fluorescent group and quenching group on two complementary chains, Fig. 1 (B) the solid-phase nucleic acid probe of fluorescent group and quenching group on the same complementary chain Needles, Figure 1 (C) Immobilized nucleic acid probes prepared by hybridization by direct method.

Fig. 2 is a working schematic diagram of the solid-phase nucleic acid probe described in Fig. 1 (A) of the present invention in the presence of exonuclease I.

Fig. 3 is a working schematic diagram of the first solid-phase nucleic acid probe proposed by the present invention in the presence of 5'-nuclease.

Fig. 4 is a working schematic diagram of the third solid-phase nucleic acid probe proposed by the present invention in the presence of 5'-nuclease.

Fig. 5 is a working schematic diagram of the second solid-phase nucleic acid probe proposed by the present invention in the presence of an endonuclease.

Fig. 6 is a working schematic diagram of the second solid-phase nucleic acid probe proposed by the present invention in the presence of 5'-nuclease.

Fig. 7 is a working schematic diagram of the thiobase-modified solid-phase nucleic acid probe proposed by the present invention in the presence of 5'-nuclease.

5. Specific implementation methods:

Embodiment one

The solid-phase nucleic acid probe proposed by the present invention includes two protruding ends formed by hybrid double strands of a fluorescent chromophoric group and a fluorescent quenching group, and is fixed on a solid substrate through an arm molecule in the middle of the double strands. The target sequence is capable of forming a complete duplex when complementary to a specific sequence on one overhang of the probe. Under the action of E.coliexonucleaseI, the other single-strand overhang together with the quencher molecule is excised, and the fluorescent chromophore can emit a fluorescent signal under excitation. In the presence of non-specific target sequences, the target sequence cannot hybridize to the specific sequence on the probe. E.coli exonuclease I cannot recognize and excise the overhang of the other single strand, so that the double strand remains intact, the fluorescent signal is still quenched, and no fluorescent signal is released (Figure 2).

Designed nine specific solid-phase nucleic acid probes, prepared microarray chips by contact spotting method, and successfully detected 4, 18, 12, 17, 19, 44 of the human dystrophin gene (dystrophin gene). , 45, 48, 51 exon deletion.

Embodiment two

Apply the microarray chip prepared in Example 1, add the biological sample solution containing the target gene, and DNA polymerase (Taq polymerase), buffer, Mg ²⁺ or Mn ²⁺ , dNTP, etc., and cover with a hydrophobized cover After slides or in situ hybridization membranes, place them in a PCR instrument to react under appropriate reaction conditions such as temperature and time. After the reaction, use the excitation wavelength of the instrument to periodically collect the fluorescence signal of the chip, wash the slide with MilliQH ₂ O, dry it with N ₂ and use a fluorescence scanner to give the intensity of the fluorescence signal.

Fluorescence quantitative PCR program, such as 95°C 10min; 95°C 30sec, 60°C 1min, 72°C 30sec; 40cycles. The fluorescence signal of the chip is detected once at the end of each extension reaction (Figure 3), and the increase curve of the fluorescence signal is analyzed to obtain the amount of the target gene in the sample.

Embodiment three

Another solid-phase nucleic acid probe proposed by the present invention includes a fluorescent chromophoric group and a fluorescent quenching group, a specific nucleic acid sequence region, a non-specific spacer region and an arm molecule region. The probes are connected with the solid support to form a microarray chip. Probes were fixed with appropriate methods, washed with 0.1% SDS for 5 min, and then blocked with sodium borohydride, then washed with MilliQ H ₂ O, and dried with N ₂ . Store in the dark at 4°C for later use or use directly.

For the G/C mutation at position 92 of the uid gene in E.coli O157:H7, three corresponding probes were designed, including a negative control, and a specific restriction endonuclease site was included in the reporter molecule and the quencher molecule point. When the wild-type target sequence exists, the endonuclease cannot recognize it, and no enzyme cleavage reaction occurs; when the mutant target sequence exists, the endonuclease recognizes and binds to the double strand, and an enzyme cleavage reaction occurs to cut off the quencher molecule from the reporter molecule , the fluorescent signal is released (Figure 5). Design probes for virulence factors in E.coli O157:H7 such as rfbE, flicH7, hlyA, etc. When these corresponding target sequences exist, under the action of the 5'-3' exonuclease activity of Taq polymerase, The quencher molecule is cleaved and the fluorescent signal is released (Figure 6).

Embodiment Four

The solid-phase nucleic acid probe proposed by the present invention includes a fluorescent chromophoric group and a fluorescent quenching group, which can be fixed on microspheres or microbeads (microparticles or microbeads) to form a suspended microarray. Nucleic acid probes labeled with different fluorescent chromophores can be immobilized in combination on each microsphere or microbead, and the instant detection of multiple sites in a single reaction tube can be completed through specific excitation wavelength combination coding. Design specific probes for virulence factors in E.coliO157:H7 such as rfbE, flicH7, hlyA, etc. When these corresponding target sequences exist, under the action of 5' exonuclease activity, along with the corresponding nucleic acid chain As the displacement reaction proceeds, the quencher molecule is excised and the fluorescent signal is released. Analysis of the detected fluorescent signal can give information about the presence of the corresponding target sequence.

Embodiment five

The solid-phase nucleic acid probe proposed by the present invention is composed of two oligonucleotide chains with complementary partial sequences, one of which includes a fluorescent chromophoric group and a fluorescent quenching group, and is arranged between the two groups. A thio base to prevent enzyme cleavage. Due to the small physical distance between the fluorescent chromophore and the fluorescent quencher, the molecule has no fluorescent emission. The other strand consists of chemical groups (such as amino groups) that can be immobilized on the substrate and DNA segments that can specifically hybridize to the target gene and the first strand. The target sequence can be complementary to the specific sequence of the probe to form a double strand, and it will be extended under the action of 5'nuclease, and the other single-stranded DNA bases hybridized on the probe will be excised one by one. When the quencher molecule is excised, Fluorescent chromophores can emit fluorescent signals upon excitation. When the 5'nuclease is extended to the thio base on the hybrid strand, the biochemical reaction is stopped. In the presence of non-specific target sequences, the target sequence cannot hybridize with the specific sequence on the probe. The 5'nuclease cannot recognize and excise the overhang of the other single strand, so that the double strand remains intact, the fluorescent signal is still quenched, and no fluorescent signal is released (Figure 7).

Designed nine specific solid-phase nucleic acid probes, prepared microarray chips by contact spotting method, and successfully detected 4, 18, 12, 17, 19, 44 of the human dystrophin gene (dystrophin gene). , 45, 48, 51 exon deletion.

Embodiment six

The present invention is a solid-phase nucleic acid probe that can be used for non-labeled detection, comprising a solid support 1 on which a fluorescent group 2, a fluorescent quenching group 3 and a specific nucleic acid recognition sequence fragment 4 are immobilized. The oligonucleotide probe, the relative physical position of the fluorescent group 2 and the fluorescent quenching group 3 in the probe is the position that can quench the fluorescence, when the target gene and the specific nucleic acid in the oligonucleotide probe recognize After the sequence fragments are hybridized, the fluorescent group on the oligonucleotide probe is separated from the fluorescence quenching group through a biochemical reaction, so that the nucleic acid probe emits fluorescence. In this embodiment, the specific nucleic acid recognizes the sequence fragment 4 , fluorescent group 2, fluorescent quenching group 3 and the site for immobilizing the nucleic acid probe are located on the same oligonucleotide probe, the specific nucleic acid recognition sequence fragment is close to the 5' end of the probe, and the fluorescent The group is close to the 3' end of the oligonucleotide probe, and there is an endonucleic acid site 5 between the fluorescent group 2 and the fluorescent quenching group 3, and the oligonucleotide probes are distributed on the solid support in a microarray 1. In this embodiment, the solid carrier of the probes with different specific nucleic acid recognition sequence fragments is placed in the same solution to prepare a suspension microarray.

Embodiment seven

The present invention is a solid-phase nucleic acid probe that can be used for non-labeled detection, including a solid support 1, which is characterized in that the solid support 1 contains a fluorescent group 2, a fluorescent quenching group 3 and a specific nucleic acid recognition The oligonucleotide probe of sequence fragment 4, the relative physical position of the fluorescent group 2 and the fluorescent quenching group 3 in the probe is a position capable of quenching the fluorescence, when the target gene is specific to the oligonucleotide probe After the hybridization of the specific nucleic acid recognition sequence fragments, the fluorescent group on the oligonucleotide probe is separated from the fluorescent quenching group through a biochemical reaction, so that the nucleic acid probe emits fluorescence. In this embodiment, the fluorescent group 2 , the fluorescent quenching group 3 is on the complementary strand of the oligonucleotide probe, and the complementary strand of the oligonucleotide probe is combined on the oligonucleotide probe through a molecular recognition reaction, and the fluorescent quenching group and the fluorescent There is a modification 6 between the bases of the group to prevent the action of the enzyme. In this embodiment, the solid carrier of the probes with different specific nucleic acid recognition sequence fragments is placed in the same solution to prepare a suspension microarray.

The preparation method adopted in the present invention is as follows: (a) The chemically synthesized labeled fluorescent oligonucleotide probe is connected to the solid phase carrier through covalent bonding or physical adsorption. The probe includes one or more fluorescent molecular groups and one or more quencher molecular groups, both of which are kept at a physical distance of 10-100 Å. Due to the effect of energy transfer or electron cloud overlap, the fluorescent molecular groups The fluorescent signal is quenched by the quenching molecular group; (b) the immobilized labeled probe can be in various forms such as single-strand or double-strand, and can constitute a microarray chip; (c) when it is in the system and the probe After the complementary target sequence is hybridized with the complementary sequence on the probe, the quenching molecular group is cut off from the probe molecule through biochemical reactions, such as the enzymatic cleavage of structure-specific biological enzymes, or the excision activity of DNA polymerase (d) Due to the change of the physical distance between the fluorescent molecular group and the quenching molecular group, a fluorescent signal can be excited, and the fluorescent signal can be obtained through the fluorescence detector, and then the gene information in the sample can be obtained. The preparation method adopted in the present invention is as follows: (a) The chemically synthesized labeled fluorescent oligonucleotide probe is connected to the solid phase carrier through covalent bonding or physical adsorption. The probe includes one or more fluorescent molecular groups and one or more quencher molecular groups, both of which are kept at a physical distance of 10-100 Å. Due to the effect of energy transfer or electron cloud overlap, the fluorescent molecular groups The fluorescent signal is quenched by the quenching molecular group; (b) the immobilized labeled probe can be in various forms such as single-strand or double-strand, and can constitute a microarray chip; (c) when it is in the system and the probe After the complementary target sequence is hybridized with the complementary sequence on the probe, the quenching molecular group is cut off from the probe molecule through biochemical reactions, such as the enzymatic cleavage of structure-specific biological enzymes, or the excision activity of DNA polymerase (d) Due to the change of the physical distance between the fluorescent molecular group and the quenching molecular group, a fluorescent signal can be excited, and the fluorescent signal can be obtained through the fluorescence detector, and then the gene information in the sample can be obtained.

Preparation of solid support: The solid support used for immobilizing the solid-phase nucleic acid probe of the present invention can be various forms of solid phase carriers, such as microparticles, microspheres, gels, solid sheets or films, and the like. The surface of the material has active chemical groups, such as amine groups, aldehyde groups, etc., in order to connect with various biomolecules; good optical properties, such as low fluorescence background; and good stability of the material, etc. Glass slides, plastics such as polystyrene, polypropylene, or polycarbonate, etc.

Activation and synthesis of solid-phase supports: use activating reagents to bond active groups on the surface of the carrier through chemical reactions, so as to covalently bond with the corresponding ligands to form affinity carriers with different biological specificities. Immobilize different nucleic acid probes.

Preparation of solid-phase nucleic acid probes: use commercial solid-phase chemical synthesis methods to synthesize designed oligonucleotides containing two or more fluorescent chromophoric groups, fluorescent quenching groups and specific nucleic acid sequences acid probe.

Immobilization of probes: The solid-phase chemically synthesized probes are transferred to the surface of the solid support by machine, manual or electrophoresis, and connected to the solid support under appropriate conditions.

Hybridization and detection: Appropriate ions and buffers are added to the test system, and the target gene and the solid-phase probe of the present invention undergo hybridization reaction, enzyme cleavage reaction or amplification reaction. Adjust the temperature and time of each reaction, detect the fluorescent signal of the corresponding reaction system, analyze the results through computer control and corresponding software, and obtain the detected gene information.

Claims (8)

1, a kind of immobilization nucleic acid probe that can be used for the non-marked detection, comprise solid carrier (1), it is characterized in that going up the oligonucleotide probe that fixed packet contains fluorophor (2), fluorescent quenching group (3) and specific nucleic acid recognition sequence segment (4) at solid carrier (1), the relative physical location of fluorophor in this probe (2) and fluorescent quenching group (3) is for can make the fluorescent quenching position; After specific nucleic acid recognition sequence segment hybridization in target gene and the oligonucleotide probe,, the fluorophor on the oligonucleotide probe is separated with the fluorescent quenching group, thereby make the nucleic acid probe emitting fluorescence by biochemical reaction.

2, the immobilization nucleic acid probe that can be used for the non-marked detection according to claim 1, it is characterized in that specific nucleic acid recognition sequence segment (4), fluorophor (2), fluorescent quenching group (3) and the site that is used for fixing this nucleic acid probe, be positioned on same the oligonucleotide probe.

3, according to claim 1 or the 2 described immobilization nucleic acid probes that can be used for the non-marked detection, it is characterized in that the 5 ' end of specific nucleic acid recognition sequence segment near probe, fluorophor is near 3 ' end of oligonucleotide probe.

4, the immobilization nucleic acid probe that can be used for the non-marked detection according to claim 1 is characterized in that being provided with endonuclease site (5) between fluorophor (2) and fluorescent quenching group (3).

5, the immobilization nucleic acid probe that can be used for the non-marked detection according to claim 1, it is characterized in that fluorophor (2), fluorescent quenching group (3) on the complementary strand of oligonucleotide probe, the complementary strand of oligonucleotide probe passes through the molecular recognition reaction bonded on oligonucleotide probe.

6, the immobilization nucleic acid probe that can be used for the non-marked detection according to claim 5 is characterized in that the modification (6) that stops the enzyme effect is arranged between the base of fluorescent quenching group and fluorophor.

7, according to claim 1,2,3,4, the 5 or 6 described immobilization nucleic acid probes that can be used for the non-marked detection, it is characterized in that oligonucleotide probe is distributed in solid carrier (1) by microarray.

8, according to claim 1,2,3,4, the 5 or 6 described immobilization nucleic acid probes that can be used for the non-marked detection, it is characterized in that the solid carrier of the probe of intrinsic different specific nucleic acid recognition sequence segments is placed in the same solution, be prepared into floated microarray.

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