阅读说明：本技术 一种荧光探针 (Fluorescent probe ) 是由 唐卓 陈刚毅 董娟 于 2019-11-18 设计创作，主要内容包括：本发明属于分子生物学技术领域,提供一种可以实现靶分子定性和实时荧光定量检测的特异性核酸探针。探针的5’和3’端分别标记淬灭基团和荧光基团,与靶序列互补之后在3’末端形成至少1个碱基错配,并能在3’末端错配核苷酸剪切酶作用下被剪切而释放荧光信号。将探针3’末端最后一个碱基设计在单核苷酸多态性位点上,可实现单核苷酸多态性的检测。探针还可结合核酸扩增策略对靶核酸进行更为灵敏的检测。探针设计简单,可广泛适用于所有可引入3’末端错配核苷酸剪切酶的核酸检测体系。(The invention belongs to the technical field of molecular biology, and provides a specific nucleic acid probe capable of realizing qualitative and real-time fluorescent quantitative detection of target molecules. The 5 'end and the 3' end of the probe are respectively marked with a quenching group and a fluorescent group, at least 1 base mismatch is formed at the 3 'end after the probe is complementary with a target sequence, and the probe can be sheared under the action of 3' end mismatch nucleotide shear enzyme to release a fluorescent signal. The last base at the 3' end of the probe is designed on the single nucleotide polymorphism site, so that the detection of the single nucleotide polymorphism can be realized. The probe may also be combined with a nucleic acid amplification strategy to provide more sensitive detection of the target nucleic acid. The probe has simple design and can be widely applied to all nucleic acid detection systems capable of introducing 3' end mismatch nucleotide splicing enzymes.)

1. A fluorescent probe, characterized by: the probe is an oligonucleotide sequence with a 5 'end labeled with a quenching group and a 3' end labeled with a fluorescent group, and the probe sequence is not completely complementary with a sequence with a certain length on the target nucleic acid; the incomplete complementarity is specifically at least a mismatch of at least 1 base between the 3' end of the probe and the target sequence.

2. The fluorescent probe according to claim 1, characterized in that: when the probe is complete, the fluorescence emitted by the fluorescent group at the 3 'end is absorbed by the quenching group at the 5' end.

3. The fluorescent probe according to claim 1, characterized in that: the 5 ' end sequence of the probe except the 3 ' end mismatched base can be completely complementary with the target molecule, and a small amount of base mismatch can be introduced into the 5 ' end sequence on the premise of ensuring the stable combination of the probe and the target molecule.

4. The fluorescent probe according to claim 1, characterized in that: can be used for detecting a target nucleic acid sequence, when the probe specifically recognizes the target sequence and hybridizes with the target sequence, the 3 'end mismatch nucleotide shear enzyme can shear and release the 3' end nucleotide of the probe, and fluorescence is released; when the probe does not recognize the target sequence, the probe keeps a complete single-chain structure and is not sheared by 3' end mismatch nucleotide shear enzyme, fluorescence emitted by the fluorescent group is quenched by the quenching group, and no fluorescent signal is generated; and reporting the information of the target nucleic acid according to the fluorescent signal.

5. The fluorescent probe according to claim 1, characterized in that: can be used for detecting single nucleotide polymorphism, and the last nucleotide at the 3' end of the designed probe exactly corresponds to the single nucleotide polymorphism site of a target nucleic acid sequence; if the designed probe is perfectly complementary with the normal sequence and forms 3 'terminal base mismatch with the mutant sequence, when the normal sequence is detected, the probe is completely complementary with the normal sequence, the probe can not be sheared by 3' terminal mismatch nucleotide shear enzyme, and no fluorescent signal is generated; when the mutant sequence is detected, the probe has a base mismatch at the 3 ' end after hybridizing with the mutant sequence, and the mismatch nucleotide at the 3 ' end of the probe can be recognized and sheared by the mismatch nucleotide shearing enzyme at the 3 ' end, and the fluorescent signal is released. Similarly, if the probe is perfectly complementary to the mutated sequence and forms a 3' terminal base mismatch with the normal sequence, then a fluorescent signal will be generated when the normal sequence is detected, and no fluorescent signal will be generated when the mutated sequence is detected; the genotype of the single nucleotide polymorphism can be judged according to the difference of the fluorescence signals.

6. The fluorescent probe according to claim 1, characterized in that: detection of a target nucleic acid sequence may be performed in conjunction with a primer-based nucleic acid amplification strategy.

7. The fluorescent probe according to claim 1, characterized in that: single nucleotide polymorphism detection can be performed in conjunction with primer-based nucleic acid amplification strategies.

8. The fluorescent probe according to claim 6 or 7, characterized in that: the probes are designed for sequences between primer binding regions on the target nucleic acid.

9. The fluorescent probe according to claim 1, characterized in that: the length of the probe is determined according to the reaction temperature and the GC content of the probe, and preferably, the Tm value of the probe is 2 to 3 ℃ higher than the reaction temperature.

10. The 3' -end mismatch nucleotidase according to claim 4 or 5, which is characterized in that: it may be a DNA polymerase having 3 '-5' exonuclease activity, preferably a high fidelity DNA polymerase (Pfu DNA polymerase).

Technical Field

The invention relates to a nucleic acid probe technology, in particular to a nucleic acid probe capable of realizing real-time fluorescence quantification and specific detection of target molecules.

Background

The nucleic acid probe is a nucleic acid sequence with known sequence, detection mark and complementary target gene sequence. Nucleic acid probes are usually labeled with a certain chemical group, and when the nucleic acid probes are combined with target genes through molecular hybridization, hybridization signals are generated, so that nucleic acid sequence signals are converted into optical or electrical signals which are easy to read and analyze, and the target sequences are detected from a large number of unrelated nucleic acid sequences, so that the nucleic acid probes become powerful analytical tools commonly used in the field of gene detection. The earliest nucleic acid probes were usually labeled with a radioisotope for labeling and signal reading, such as Northern and Southern hybridization using a radiolabeled oligonucleotide probe to RNA or DNA on a membrane, respectively, and concentration of the site of the sequence of interest detected by a phospho-screen scanner. However, the isotope has a series of problems of short half-life period, difficult operation, environmental pollution, influence on human health and the like. Consequently, nucleic acid probes have been developed that typically employ nonradioactive labels such as fluorescent molecules, biotin, and enzymes.

The most widely used non-radioactive labeled nucleic acid probe is the fluorescent labeled nucleic acid probe, and the fluorescent labeled nucleic acid probe can be directly used for nucleic acid positioning (such as fluorescent in situ hybridization) in tissues and cells and can also be used for realizing the specific detection of a target sequence by a method of releasing a fluorescent signal through the shearing action of enzyme in the amplification process. At present, the fluorescent probe used in the amplification reaction is mainly a TaqMan probe, and the use of the TaqMan probe greatly improves the specificity and the accuracy of the PCR method. However, the TaqMan probe depends on 5 '-3' exonuclease activity of Taq enzyme, and is therefore not suitable for amplification reactions using polymerase having no 5 '-3' exonuclease activity, such as most of the existing isothermal amplification reactions (SDA, RPA, LAMP, RCA, CPA, HAD, EXPAR, NASBA, MDA). Therefore, the development of a nucleic acid probe which can be used for variable temperature reactions such as PCR and the like and has wider applicability in isothermal amplification reactions is of great significance to the field of gene detection.

Disclosure of Invention

The invention combines the fluorescence resonance energy transfer principle and the enzyme which can recognize the base mismatch at the 3' end and cut to release the mismatch nucleotide, provides an oligonucleotide probe which is easy to prepare, highly stable and highly specific, can be complementarily hybridized and cut with a target sequence, generates accurate optical and electrical signals readable by an instrument, and finally realizes the real-time fluorescence quantification and the specificity detection of the target nucleic acid.

The technical scheme of the invention is as follows: a fluorescent probe, the 5 'end of the probe is marked with a quenching group, the 3' end is marked with a fluorescent group, and the sequence of the probe is designed to be incompletely complementary with a sequence with a certain length on a target nucleic acid; incomplete complementarity is defined as at least a mismatch of at least 1 base at the 3' end of the probe to the target sequence.

When the structure of the fluorescent probe is complete, the fluorescence emitted by the fluorescent group at the 3 'end and the absorbed light of the quenching group at the 5' end meet the requirement of fluorescence resonance energy transfer, the fluorescence emitted by the fluorescent group at the 3 'end can be absorbed by the quenching group at the 5' end, and the probe does not generate fluorescence; when the probe is broken, the quenching group at the 5 'end is separated from the fluorescent group at the 3' end, and the condition for generating fluorescence resonance energy transfer is lost, and the probe generates fluorescence.

According to the fluorescent probe, the 5 ' end sequence of the probe except the 3 ' end mismatched base can be completely complementary with the target molecule, and a small amount of base mismatch can be introduced into the 5 ' end sequence on the premise of ensuring the stable combination of the probe and the target molecule.

The fluorescent probe can detect a segment of nucleic acid sequence and Single Nucleotide Polymorphism (SNP) in a segment of nucleic acid sequence.

The fluorescent probe of the invention detects a target nucleic acid sequence: when the probe specifically recognizes and hybridizes with the target sequence, an enzyme capable of recognizing the base mismatch at the 3 ' end and cleaving to release the mismatched nucleotide (hereinafter referred to as "3 ' end mismatched nucleotide cleaving enzyme") can cleave and release the nucleotide at the 3 ' end of the probe, thereby separating the fluorescent group labeled at the 3 ' end of the probe from the quencher group at the 5 ' end and releasing fluorescence; when the probe does not recognize the target sequence, the probe keeps a complete single-chain structure and is not sheared by 3' end mismatch nucleotide shear enzyme, fluorescence emitted by the fluorescent group is quenched by the quenching group, and no fluorescent signal is generated; and reporting the information of the target nucleic acid according to the fluorescent signal.

The fluorescent probe of the invention detects the single nucleotide polymorphism: the last nucleotide at the 3' end of the probe is designed to correspond exactly to a single nucleotide polymorphic site in the target nucleic acid sequence. If the designed probe is perfectly complementary with the normal sequence and forms 3 'terminal base mismatch with the mutant sequence, when the normal sequence is detected, the probe is completely complementary with the normal sequence, the probe can not be sheared by 3' terminal mismatch nucleotide shear enzyme, and no fluorescent signal is generated; when the mutant sequence is detected, the probe has a base mismatch at the 3 ' end after hybridizing with the mutant sequence, and the mismatch nucleotide at the 3 ' end of the probe can be recognized and sheared by the mismatch nucleotide shearing enzyme at the 3 ' end, and the fluorescent signal is released. Similarly, if the probe is perfectly complementary to the mutated sequence and forms a 3' terminal base mismatch with the normal sequence, then a fluorescent signal will be generated when the normal sequence is detected and no fluorescent signal will be generated when the mutated sequence is detected. The genotype of the single nucleotide polymorphism can be judged according to the difference of the fluorescence signals.

The fluorescent probe can be combined with a nucleic acid amplification technology to carry out more sensitive detection on target nucleic acid information.

The fluorescent probe provided by the invention is combined with a nucleic acid amplification strategy based on a primer to detect a target nucleic acid sequence: probes of the invention are designed for sequences between primer binding regions on a target nucleic acid and introduced into an amplification system. When the sample does not contain the target nucleic acid, no primer-initiated amplification product is formed, the probe keeps a single-stranded structure, is not sheared by 3' end mismatch nucleotide shear enzyme, and does not release a fluorescent signal; if the primer is subjected to non-specific amplification, an intermediate product generated by amplification does not contain a probe complementary sequence, the probe keeps a single-chain structure, cannot be sheared by 3' end mismatch nucleotide shear enzyme, does not generate a fluorescent signal, and can effectively control false positive amplification; when the target nucleic acid is contained in the sample, the probe specifically recognizes and is complementary to the primer-based amplification product, the 3 'terminal mismatch nucleotide is recognized and cleaved by the 3' terminal mismatch nucleotidelapsin, the fluorescent group labeled on the 3 'terminal nucleotide is separated from the quencher group at the 5' terminal, and a fluorescent signal is released to report the target nucleic acid information.

The probe of the invention combines a nucleic acid amplification strategy based on a primer to carry out single nucleotide polymorphism detection: the probes of the invention are designed with respect to the sequence between the primer binding regions on the target nucleic acid such that the last nucleotide at the 3' end of the probe corresponds exactly to the single nucleotide polymorphic site of the target nucleic acid sequence. If the designed probe is perfectly complementary with the normal sequence and forms 3 'terminal base mismatch with the mutant sequence, when the normal sequence is detected, the probe is completely complementary with an amplification product of the normal sequence, at the moment, the probe cannot be sheared by 3' terminal mismatch nucleotide shear enzyme, the probe is kept complete, fluorescence emitted by a fluorescent group is quenched by a quenching group, and no fluorescent signal is generated; when the mutant sequence is detected, the probe has a base mismatch at the 3 ' end after hybridizing with the amplified product of the mutant sequence, and the mismatch nucleotide at the 3 ' end of the probe can be recognized and cleaved by 3 ' -mismatch nucleotide cleaving enzyme, so that the fluorescent group labeled on the 3 ' end nucleotide is separated from the quenching group at the 5 ' end, and fluorescence is released. Similarly, if the probe is perfectly complementary to the mutated sequence and forms a 3' terminal base mismatch with the normal sequence, then a fluorescent signal will be generated when the normal sequence is detected and no fluorescent signal will be generated when the mutated sequence is detected. Thus, the genotype of the single nucleotide polymorphism can be judged based on the presence or absence of the fluorescent signal.

The length of the fluorescent probe according to the present invention is determined according to the reaction temperature and the GC content of the probe, and generally, the Tm value of the probe is 2-3 ℃ higher than the reaction temperature.

The 3 ' -end mismatch nucleotidase of the present invention may be a DNA polymerase having 3 ' -5 ' exonuclease activity, preferably a high fidelity DNA polymerase (Pfu DNAmerase).

As used herein, the following words/terms have the following meanings unless otherwise specifically indicated.

"DNA": deoxyribonucleic acid is a biological macromolecule with genetic information, is formed by connecting 4 main deoxyribonucleotides through 3 ', 5' -phosphodiester bonds, and is a carrier of the genetic information.

"oligonucleotide": the small molecule nucleic acid is formed by connecting nucleotides through 3 ', 5' -phosphodiester bonds.

"target sequence/target nucleic acid": a nucleic acid molecule to be detected which specifically binds to a probe of the present invention or can be produced by other processes.

"base": bases are the basic building blocks of synthetic nucleosides, nucleotides, and nucleic acids, and are a class of nitrogenous bases.

"nucleotide": a compound is composed of purine base or pyrimidine base, ribose or deoxyribose and phosphate, which are basic composition units of nucleic acid.

"mismatch": refers to the phenomenon of non-complementary base pairing that occurs in a double-stranded nucleic acid molecule, i.e., bases on one strand are not complementary to the corresponding bases on the other strand.

"quencher group": a chemical group which is within a certain distance range from the fluorescent group and can absorb fluorescence emitted by the fluorescent group.

"fluorophore": chemical groups that can emit light at longer wavelengths upon irradiation with laser light of a particular wavelength.

"fluorescence energy resonance transfer": refers to two different groups, if the emission spectrum of one fluorophore (donor) overlaps with the absorption spectrum of the other (acceptor), when the distance between the two fluorophores is appropriate (generally less than

) The phenomenon of fluorescence energy transfer from the donor to the acceptor can be observed.

"Tm value": the increase in absorbance of the double-stranded DNA upon thermal denaturation is a function of temperature, and the temperature at which the absorbance increases by half is the Tm value.

The key point of the probe disclosed by the invention is that the probe combines the fluorescence resonance energy transfer principle and the action of 3 ' end mismatch nucleotide shear enzyme, and is specifically identified with a target sequence, forms nucleotide mismatch at the 3 ' end, and is sheared by the 3 ' end mismatch nucleotide shear enzyme, so that a fluorescence signal is released, and the direct or indirect qualitative or quantitative analysis of a target molecule is realized. The probe is stable, simple and easy to prepare, has wide application range, can be used for various nucleic acid detection systems, and particularly has high practical value for infectious diseases, early cancer detection, targeted drug detection and the like. In the development process of the prior probe technology, the invention mainly embodies the following outstanding advantages:

1. and (4) universality. The oligonucleotide probe of the invention is applicable to all nucleic acid detection systems which can introduce 3' end mismatch nucleotide shear enzymes.

2. And (5) simplicity. The method can be used for detecting the target nucleic acid only by carrying out corresponding sequence design aiming at the target nucleic acid sequences in different samples.

3. And (5) practicability. When the probe is used for nucleic acid detection, the release of a fluorescent signal enables the whole reaction process to be monitored in real time, the qualitative detection of target molecules can be realized, the quantitative detection of the target molecules can also be carried out, and the probe has great practical value in the detection and analysis of real samples.

Drawings

FIG. 1 is a schematic diagram of the design and operation of a fluorescent probe;

FIG. 2 is a schematic diagram of the design and operation of fluorescent probes for single nucleotide polymorphism detection;

FIG. 3 is a schematic diagram of the principle of fluorescent probes applied to loop-mediated amplification.

FIG. 4 is a diagram showing the results of quantitative detection of nucleic acid using the probe of the present invention in a loop-mediated amplification reaction.

FIG. 5 is a diagram showing the principle and result of quantitative detection of nucleic acid in a loop-mediated amplification reaction using the probe of the present invention.

FIG. 6 is a graph showing the results of specific detection using the probe of the present invention in a loop-mediated amplification reaction.

Detailed Description

The invention will be further illustrated by way of example with reference to the accompanying drawings. It will be understood by those skilled in the art that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention.