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Stem-ring type oligonucleotide probe

An oligonucleotide and probe technology, applied in the field of nucleotide structure, can solve the problems of low thermodynamic metastability and high design difficulty

Inactive Publication Date: 2008-06-18
THE FIRST AFFILIATED HOSPITAL OF THIRD MILITARY MEDICAL UNIVERSITY OF PLA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, since the initial state of the target molecule recognition region of this stem-loop oligonucleotide is a single-stranded form, the specificity of its binding to the target molecule needs to be further improved; in addition, because the stem sequence is shorter, As a result, its thermodynamic metastability is low, so the design is very difficult, and there are certain special requirements for the sequence composition of the target molecule, which limits the scope of application to a certain extent

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0078] Example 1. Monomer probes with cohesive ends

[0079] A monomeric oligonucleotide probe P with a stem-loop structure, the probe is composed of an oligonucleotide chain, and its initial state has a loop formed by sequence c, and complementary sequences b and b* combined to form The stem part and the sticky end a are composed of a fluorescent group at the b* end, and a quencher group is marked at the junction of b and a, and the recognition region is composed of the stem b and the sticky end a. The target molecule T is formed by connecting the sequences b* and a*, and is completely complementary to the stem sequence b and the sticky end a of the probe P. When there is no target molecule T in the reaction system, the probe P maintains the initial state, the fluorescence signal of the fluorophore is absorbed by the adjacent quenching group, and there is no detectable fluorescence signal in the system; when there is a target molecule in the reaction system When the molecule...

Embodiment 2

[0080] Example 2. Monomer probes without sticky ends

[0081] A monomeric oligonucleotide probe P with a stem-loop structure, the probe is composed of an oligonucleotide chain, and its initial state has a loop formed by sequence c, and complementary sequences b and b* combined to form The end of the stem is a blunt end, and the two ends are respectively labeled with a fluorescent group and a quencher group. The recognition region is composed of the stem sequence b and the loop sequence c. The target molecule T is formed by connecting the sequences b* and c*, and is completely complementary to the stem sequence b and the loop sequence c of the probe P. When there is no target molecule T in the reaction system, the probe P maintains the initial state, the fluorescence signal of the fluorophore is absorbed by the adjacent quenching group, and there is no detectable fluorescence signal in the system; when there is a target molecule in the reaction system When the molecule is T, t...

Embodiment 3

[0082] Example 3. Two-body probes with cohesive ends

[0083] A double oligonucleotide probe P1·P2 with a stem-loop structure, the probe is composed of two oligonucleotide chains complementary to each other, wherein the oligonucleotide chain P1 consists of the stem sequence b and The sticky end sequence a is connected; the oligonucleotide chain P2 is composed of the sequence b1*, c, b2*, wherein, b1* and b2* are complementary to the two ends of b of P1 respectively, and b1* and b2* are in the whole It is completely complementary to b, and c forms the ring part of the double-body probe; a fluorescent group is labeled at the end of P2, and a quencher group is labeled at the symmetry between the ends of P1 and P2 (that is, at the junction of b and a). The region consists of a stem b and a sticky end a. The target molecule T is formed by connecting the sequences b* and a*, and is completely complementary to the stem sequence b and the sticky end a of the probe P1·P2. When there ...

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Abstract

The invention provides a novel stem-loop oligonucleotide probe which is provided with the initial status of a stem-loop secondary structure and consists of a loop part and a stem part. The stem part is a hairpin structure which is formed from two complementary base sequences that are combined through hydrogen bond. An identification region which is combined with the specificity of a target molecule is mainly composed of the stem part sequence of the probe. When no target molecule exists, the identification region exists as the hairpin structure of the initial status. When the target molecule exists, the hairpin structure of the stem part disintegrates when the target molecule is combined with the specificity of the identification region, and the probe or the target molecule can produce signal differences that can be detected. The identification region of the invention probe specificity combined or complementary hybrid target molecule mainly or completely bases on the oligonucleotide sequence of the stem part structure. And the oligonucleotide sequence which competes with the target molecule exists in the probe molecule. Therefore, the probe of the invention is provided with higher specificity.

Description

technical field [0001] The present invention relates to a nucleotide structure, in particular to the structure of a nucleotide as a probe. Background technique [0002] Stem-loop oligonucleotides are one of the common tools in the field of molecular biology and biotechnology. Compared with linear oligonucleotides, stem-loop oligonucleotides have higher specificity in hybridization or binding to target molecules, and different forms of stem-loop oligonucleotides can be flexibly designed according to different application requirements Acids, such as molecular beacons (molecular beacons), inhibitory polymerase chain reaction (polymerase chain reaction, PCR), LUX (light upon extension) primers, etc. At present, stem-loop oligonucleotides have been used in various molecular biology techniques in different forms, mainly including single nucleotide polymorphism detection, gene mutation analysis, real-time fluorescent quantitative PCR and biochips, etc., fully demonstrated And emb...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
Inventor 黄庆府伟灵
Owner THE FIRST AFFILIATED HOSPITAL OF THIRD MILITARY MEDICAL UNIVERSITY OF PLA
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