Primer-type nucleic acid fluorescent probe subjected to two-way strand displacement

A fluorescent probe and two-way chain technology, applied in the biological field, can solve the problems of increasing the overall difficulty of design, and achieve the effects of low design difficulty, high sensitivity real-time fluorescence detection, and high indication sensitivity

Active Publication Date: 2016-07-27
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Therefore, researchers not only need to design primers for high-efficiency amplification, but also reasonably design fluorescent probes for signal indication, which increases the overall difficulty of design to a certain extent.

Method used

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  • Primer-type nucleic acid fluorescent probe subjected to two-way strand displacement
  • Primer-type nucleic acid fluorescent probe subjected to two-way strand displacement
  • Primer-type nucleic acid fluorescent probe subjected to two-way strand displacement

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Embodiment 1 (probe stability verification)

[0040] In this verification example, two primers containing labeling groups (one labeled as FAM fluorescent group and the other labeled as Dabcyl quenching group, respectively denoted as FAM-primer-1 and Dabcyl-primer-1) were melted Curve analysis was used to judge the stability of the probe in the isothermal reaction system at 60-65°C. The structure and composition of this probe can be found in the appendix figure 1 ,Specific steps are as follows:

[0041] Step 1: Take 5 EP tubes and add 21 μL of reaction buffer to each, labeled as A, B, C, D, E;

[0042] Step 2: Add 2.0 μL of FAM-primer-1 to each of the 5 tubes, and the final concentration of the system is 1.6 μM;

[0043] Step 3: Add 2.0 μL Abcyl-primer-1 to each of the 5 tubes, and the final concentration of the system is 1.6 μM;

[0044] Step 4: Mix the 5 tubes of solution thoroughly and incubate at 37°C for 10 minutes. Then, it was placed in an ABI7900HT real-time...

Embodiment 2

[0055] Embodiment 2 (IMSA mediated by the probe of the present invention)

[0056]The purpose of this example is to verify the real-time detection ability of the probe-mediated IMSA of the present invention for the amplification of target nucleic acid sequences and other non-target nucleic acid sequences at different concentrations, that is, its analytical sensitivity and specificity. The composition of the probe structure in this embodiment is shown in the appendix figure 1 , the mechanism of action of the probe is shown in the appendix figure 2 , and the reaction schematic diagram of IMSA is detailed in patent CN104388581A.

[0057] Specific steps are as follows:

[0058] Step 1: Take an EP tube, add 13 μL of FAM-primer-2 (concentration of 20 μM) and 13 μL of Abcyl-primer-2 (concentration of 20 μM), mix well to make a probe solution, and incubate at 37°C in the dark for about 10 minute.

[0059] Step 2: Take 12 EP tubes and add 20.5 μL of the reaction mixture, labeled 1...

Embodiment 3

[0077] Embodiment 3 (probe of the present invention binds to IMSA mediated by HNB)

[0078] The purpose of this example is to verify that the probe of the present invention combined with HNB can establish real-time fluorescence detection of IMSA and dual fluorescence visualization detection of its amplified products. The composition of the probe structure in this embodiment is shown in the appendix figure 1 , the mechanism of action of the probe is shown in the appendix figure 2 , and the reaction schematic diagram of IMSA is detailed in patent CN104388581A. For the mechanism of dual fluorescence establishment, please refer to the summary of the invention.

[0079] Specific steps are as follows:

[0080] Step 1: Take an EP tube, add 9 μL of FAM-primer-2 (concentration of 20 μM) and 9 μL of Abcyl-primer-2 (concentration of 20 μM), mix well to make a probe solution, and incubate at 37°C in the dark for about 10 minute.

[0081] Step 2: Take 8 EP tubes, add 20.5 μL of react...

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Abstract

The invention provides a primer-type nucleic acid fluorescent probe subjected to two-way strand displacement.The primer-type nucleic acid fluorescent probe is composed of two oligonucleotide strands of which 5'-end sequences are completely complementary, the middle of the primer-type nucleic acid fluorescent probe is a double-strand zone, and the two ends of the primer-type nucleic acid fluorescent probe are provided with single-strand arms respectively.The probe is simple in structure and reasonable in design and can simultaneously serve as an amplification primer and a signal probe in an amplification reaction, chemical modification to the 3' end is not needed, and the process of additionally designing a probe is avoided.According to the probe, the strand displacement activity of nucleotide polymerase is utilized, high-sensitivity real-time fluorescence detection on nucleic acid isothermal amplification such as isothermal multiple-self-matching-initiated amplification can be achieved, visual bifluorescence product detection can be constructed by combining ion indicators such as hydroxynaphthol blue, the potential for constructing single-tube multiple nucleic acid isothermal amplification technology is achieved, and a novel nucleic acid fluorescent probe is provided for diagnosis or detection research of related markers in biology, medicine, chemistry and the like.

Description

technical field [0001] The invention belongs to the field of biotechnology and relates to a primer-type nucleic acid fluorescent probe capable of being replaced by double-stranded strands. Background technique [0002] With the continuous development of chemical synthesis of nucleic acid technology, the use of nucleic acid probes for related research has become one of the commonly used molecular biology techniques in the field of biology and medicine. Nucleic acid fluorescent probes are a form of nucleic acid probes that label specific nucleic acid probes with fluorophores and record changes in fluorescent signals to analyze and detect corresponding target molecules. As the most commonly used signal transduction medium, nucleic acid fluorescent probes have the advantages of high analytical sensitivity, simple detection methods, and less impact on biomolecules. They have been used to detect protein molecules, nucleic acid fragment molecules, small biological molecules, and in...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6844C12Q1/6876C12Q2600/16C12Q2531/119C12Q2537/143C12Q2563/107
Inventor 牟颖丁雄金伟
Owner ZHEJIANG UNIV
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