阅读说明：本技术 一种新型的自我链置换原理及其多重链置换反应的应用 (Novel self strand displacement principle and application of multiple strand displacement reaction thereof ) 是由 周殿明 于 2020-12-28 设计创作，主要内容包括：本发明提供了一种新型的自我链置换原理及其多重链置换反应的应用,新型自我链置换原理为引物以5’端茎环结构的探针为模板,聚合延伸产生双链DNA,产物存在末端双链杂交结构和解链形成两个茎环结构的平衡,本发明在模板引入损伤碱基,使引物延伸后形成含有错配结构的双链,使平衡向解链方向移动,此时解链形成的茎环结构在聚合酶延伸下发生自我链置换,产生原有模板的再生,和新聚合产物分离,此反应得以循环进行。本发明将该新型自我链置换原理应用于发展全新的多重链置换技术,新的链置换技术突破了原有多重链置换技术中因模板长度限制容易发生置换结束的缺点,真正实现了简便、理论“无终点”、非模板长度依赖的多重链置换技术。(The invention provides a novel self-strand displacement principle and application of a multiple strand displacement reaction thereof, wherein the novel self-strand displacement principle is that a probe of a 5' end stem-loop structure is used as a template for a primer, the primer is polymerized and extended to generate double-stranded DNA, a terminal double-stranded hybridization structure and a product exist, and the product is subjected to the balance of two stem-loop structures formed by de-hybridization. The invention applies the novel self-strand displacement principle to the development of a completely new multiple strand displacement technology, and the new strand displacement technology breaks through the defect that the displacement is easy to finish due to the limitation of template length in the original multiple heavy chain displacement technology, thereby really realizing the simple and convenient multiple strand displacement technology which is theoretically 'endless' and is not dependent on the template length.)

1. A novel self strand displacement principle and its multiple strand displacement reaction are characterized in that: the principle of self-strand displacement is as follows:

the system probe consists of two probes A and B, the 5 'end of the probe A is a stem-loop structure with a damage site, the 3' end is a single-stranded DNA structure, the probe B is a single-stranded DNA which is complementarily matched with the single-stranded region at the 3 'end of the probe A, when the probes A and B are in the same system, the probes A and B can be hybridized with each other, at the moment, if DNA polymerase with strand displacement activity exists in the solution, the 3' end of the probe B can be extended by using the probe A as a template, the damage site is spanned, the stem loop at the 5 'end of the probe A is opened, the probe AB is extended to form double-stranded DNA, after the 3' end of an extension product of the probe B forms a stem loop, the probe A can be separated from the double strand by using the probe A as the template and carrying out polymerization strand displacement reaction, and the probe A is regenerated, and.

2. The novel self strand displacement principle and its multiplex strand displacement reaction according to claim 1, wherein: the principle of the novel multiple strand displacement reaction is as follows:

the self-strand displacement reaction of claim 1, wherein the probe B is designed according to the principle of self-strand displacement reaction, a plurality of repeated sequences capable of hybridizing with the probe B are added at the 3 'end of the probe A, a plurality of probes B can hybridize with the template of the probe A sequentially or simultaneously to perform the strand displacement reaction, when the primer B near the 3' end of the template extends to cover the template and cannot perform the multiple-strand displacement reaction, the design can regenerate the probe A by means of self-strand displacement so as to perform a new round of multiple-strand displacement, the design is the multiple-strand displacement without end point, and the infinite multiple-strand displacement reaction can be completed by the limited template length.

3. The novel self-strand displacement principle and multiplex strand displacement reaction according to claim 1, wherein the self-strand displacement reaction comprises the following steps:

preparing a reaction system containing 1x of polymerization reaction buffer solution, 10-200 nM stem-loop probe, 50-200 nM single-strand primer, 1-10U of Bst Large Fragment and 1-10 uM dNTP, adding water to prepare 20uL, and reacting at 37-65 ℃ for 30min-2 h. The bands were visualized and analyzed using 2-5% agarose gel electrophoresis.

4. The use of the novel self strand displacement principle and its multiplex strand displacement reaction according to any of claims 1-2, wherein: the application steps of the multiple heavy chain replacement reaction are as follows:

preparing a reaction system containing 1x of polymerization reaction buffer solution, 10-200 nM triple primer hybridization hairpin probe and single primer hybridization hairpin probe, 50-200 nM single-strand primer, 1-10U Bst Large Fragment and 1-10 mu M dNTP, adding water to prepare 20 mu L, reacting at 37-65 ℃ for 30min-2h, and performing fluorescence detection.

Technical Field

The invention belongs to the field of biotechnology, and particularly relates to a novel self-strand displacement principle and application of multiple strand displacement reaction thereof.

Background

Most molecular diagnostics are based on simple and reliable molecular amplification reactions, and although Polymerase Chain Reaction (PCR) is the most widely used method, isothermal amplification methods can be easier to handle in many scenarios, including point-of-care diagnostics, due to the avoidance of precise temperature cycling and complex equipment. Isothermal amplification methods encompass a variety of methods, including nucleic acid sequence-based amplification (NASBA), single-primer isothermal amplification (SPIA), isothermal chimeric primer-primed amplification (ICAN), Strand Displacement Amplification (SDA), helicase-dependent amplification (HDA), Recombinase Polymerase Amplification (RPA), isothermal target and signal probe amplification (tpa), Rolling Circle Amplification (RCA), loop-mediated amplification (LAMP), and SmartAmp. In general, these methods have a mechanism of strand invasion and/or primer binding that does not require temperature cycling, and can produce sensitive amplification similar to PCR.

Most of the isothermal nucleic acid amplification methods described above employ a common mechanism, the polymerase-mediated strand displacement mechanism. Polymerase-mediated displacement of nucleic acid strands is a widely used technique in nucleic acid amplification reactions, and is also a simulation and variation of nucleic acid replication processes in organisms. The process mainly comprises two processes of primer extension under the action of polymerase with strand displacement activity and new nucleic acid sequence generated by extension to displace original downstream nucleic acid. The polymerase-mediated strand displacement leads to the unwinding of the original double strand and the generation of a new double strand, and generates an amplification reaction, the displacement reaction replaces the high-temperature unwinding process in the traditional temperature-variable reaction, can be carried out in a wider temperature range, and is simple and convenient to operate. In recent years, nucleic acid strand displacement reactions have been widely used in various fields of molecular biology due to their high specificity and high sensitivity detection characteristics, and have also received great attention in amplification of detection signals and diagnostic biosensing detection.

In order to make nucleic acid amplification reactions more sensitive, polymerase-mediated strand displacement reactions often produce multiple products via amplification of a single template, and the approaches to achieve this goal are broadly divided into three categories:

the first is the strand displacement sequence amplification involving nicking enzymes. A nicking enzyme cleavage site exists on the double strand, and one of the double strands is cleaved by the nicking enzyme to expose the 3' end, followed by extension by polymerase to replace the downstream nucleic acid. The process is carried out circularly, and the same single-stranded DNA is continuously generated, so that the sequence amplification and the signal amplification are realized. Although such exponential strand displacement reactions have high sensitivity, the method introduces nicking enzymes, and the combination of polymerase and nicking enzyme has high background reaction, which limits the practical application of such methods.

The second type is to realize the recycling of the template by designing a template with a special structure. For example, a rolling circle replication RCA reaction based on a circular template, and the 3' end of the primer hybridized on the circle realizes the displacement amplification of the downstream sequence through the extension of a continuous repetitive sequence. The LAMP reaction also aims at realizing sequence amplification based on a special structure template.

The third type is a multiple strand displacement reaction. The multiple heavy chain replacement reaction is to generate multiple amplification products by utilizing the fact that multiple short primers can be hybridized on a template strand, multiple free 3' ends can be extended and reach the subsequent strand replacement reaction. Nevertheless, multiplex strand displacement has the obvious disadvantage that when primer extension near the 3 'end of the template leaves the template cycle incompetent, multiple primers cannot hybridize repeatedly, the reaction is terminated quickly, and if this is to be overcome, it is necessary to extend the 3' end of the template to create a new primer binding site. Cheng et al used a rolling circle amplification method to continuously extend the template strand, and achieved multiple strand displacement reactions by adding primers. Although the extension reaction of this method is not stopped until the substrate is exhausted, in one aspect, the long DNA template itself forms various structures, which is not favorable for the reaction, and in addition, the polymerase itself has a reduced efficiency in extending a particularly long template, and often needs to introduce a special template structure design or a plurality of enzymatic reactions. Therefore, such multiple strand displacement reactions have limited their wide application due to technical difficulties.

Disclosure of Invention

In order to overcome the defects of the prior art, the technical scheme of the invention is realized as follows:

the invention aims to provide a novel self strand displacement principle, a multiple strand displacement reaction principle and application thereof.

The principle of self-strand displacement is as follows:

the system probe consists of two probes A and B, the 5 'end of the probe A is a stem-loop structure with a damage site, the 3' end is a single-stranded DNA structure, the probe B is a single-stranded DNA which is complementarily matched with the single-stranded region at the 3 'end of the probe A, when the probes A and B are in the same system, the probes A and B can be hybridized with each other, at the moment, if DNA polymerase with strand displacement activity exists in the solution, the 3' end of the probe B can be extended by using the probe A as a template, the damage site is spanned, the stem loop at the 5 'end of the probe A is opened, the probe AB is extended to form double-stranded DNA, after the 3' end of an extension product of the probe B forms a stem loop, the probe A can be separated from the double strand by using the probe A as the template and carrying out polymerization strand displacement reaction, and the probe A is regenerated, and.

The principle of the novel multiple strand displacement reaction is as follows:

the probe B designed on the principle of the self-strand displacement reaction is unchanged, a plurality of repeated sequences capable of hybridizing with the probe B are added at the 3 'end of the probe A, a plurality of probes B can hybridize with a probe A template sequentially or simultaneously to generate the strand displacement reaction, when the primer B near the 3' end of the template extends to cover the template and cannot perform the multiple heavy chain displacement reaction, the design can regenerate the probe A in a self-strand displacement mode so as to perform a new round of multiple strand displacement, the design is the multiple strand displacement without an end point, and the infinite multiple strand displacement reaction can be completed by the limited template length.

Further, the self-strand displacement reaction steps are as follows:

preparing a reaction system containing 1x of polymerization reaction buffer solution, 10-200 nM stem-loop probe, 50-200 nM single-strand primer, 1-10U of Bst Large Fragment and 1-10 uM dNTP, adding water to prepare 20uL, and reacting at 37-65 ℃ for 30min-2 h. The bands were visualized and analyzed using 2-5% agarose gel electrophoresis.

Further, the multiple strand displacement reaction steps are as follows:

preparing a reaction system containing 1x of polymerization reaction buffer solution, 10-200 nM triple primer hybridization hairpin probe and single primer hybridization hairpin probe, 50-200 nM single-strand primer, 1-10U Bst Large Fragment and 1-10 mu M dNTP, adding water to prepare 20 mu L, reacting at 37-65 ℃ for 30min-2h, and performing fluorescence detection.

Compared with the prior art, the novel self-strand displacement principle and the application of the multiple strand displacement reaction have the following advantages:

(1) the novel self-strand displacement principle and the application of the multiple strand displacement reaction thereof are based on the novel self-strand displacement principle of damaged base repair mediation, and are applied to the development of a brand new multiple strand displacement technology, the novel strand displacement technology breaks through the defect that the displacement is easy to finish due to the limitation of the template length in the original multiple heavy chain displacement technology, and the simple, convenient and theoretically 'end-point-free' multiple strand displacement technology which is not dependent on the template length is really realized;

(2) the novel self-strand displacement principle and the application of the multiple strand displacement reaction thereof are used as a superior detection platform which is simple and convenient to operate, saves time and labor, can be coupled with various detection targets and signal conversion modes, are widely applied to various fields of molecular biology, and have wide application prospects in amplification of detection signals and diagnosis of biosensing detection.

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate an embodiment of the invention and, together with the description, serve to explain the invention and not to limit the invention. In the drawings:

FIG. 1 is a schematic diagram illustrating the principle of self-strand displacement according to an embodiment of the present invention;

FIG. 2 is a schematic diagram of a multiplex strand displacement reaction according to an embodiment of the present invention;

FIG. 3 is a diagram illustrating the electrophoretic verification of the self-strand displacement principle according to an embodiment of the present invention;

wherein Lane 1: marker;

lane 2: primer T-02, reacting at 37 ℃;

lane 3: TXA + primer T-02 hybridization, and 37 ℃ reaction;

lane 4: TXA + T-02+ Bst, reacting at 37 ℃

Lane 5: TTA + T-02+ Bst, reacting at 37 ℃;

lane 6: TXA + T-02+ Bst, reacting at 60 ℃;

lane 7: TTA + T-02+ Bst, reacting at 60 ℃;

Lane8：Marker。

FIG. 4 is a diagram showing a comparison between a triple strand displacement probe and a single strand displacement probe according to an embodiment of the present invention;

wherein a is a strand displacement effect fluorescence diagram added with a Triple cycle probe Triple;

b is a fluorescence diagram of strand displacement effect of the added single-recycling probe TXA;

and c is a background fluorescence image of the primer T-02.

Detailed Description

The present invention will be described in detail with reference to the following examples and accompanying drawings.

According to the principle of self-strand displacement reaction, the self-strand displacement reaction of the present invention comprises the following steps:

probes A and B were designed according to the principle, and the specific sequences are shown in Table 1.

A reaction system containing 1x of polymerization reaction buffer solution, 60nM stem-loop probe (TTA, TXA), 60nM single-strand primer, 8U Bst Large Fragment and 2.5 μ M dNTP is prepared, water is added to prepare 20 μ L, and reaction is carried out for 2h at 37 or 60 ℃. The bands were observed and analyzed by electrophoresis on a 3% agarose gel, and the band results are shown in FIG. 3.

According to the principle of self strand displacement reaction and the principle of multiple strand displacement, the multiple strand displacement reaction of the invention comprises the following steps:

a reaction system containing 1X polymerization buffer solution, 6nM triple primer probe (triple), single primer probe (TXA), 100nM single-strand primer T-02, 4U Bst Large Fragment, and 2.5. mu.M dNTP was prepared, and 20. mu.L of the reaction system was prepared by adding water and reacted at 60 ℃. Fluorescence data was collected and plotted to compare the performance of each structure.

Table 1 nucleic acid sequences

The above nucleic acid sequences are all synthesized by Biotechnology engineering (Shanghai) GmbH

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.

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