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Low-abundance gene mutation detecting method

A gene and point mutation technology, which is used in the detection of low-abundance gene mutations, can solve the problems of reducing the efficiency of primer hybridization, affecting the competition between primers and repressors, etc.

Active Publication Date: 2017-08-11
XIAMEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, if there is a strong secondary structure in the region near the detected point mutation, the hybridization efficiency of the primer will be significantly reduced, which will directly affect the competition between the primer and the repressor.

Method used

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Examples

Experimental program
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Effect test

Embodiment 1

[0129] Example 1. Investigation of the ability of the self-quenching probe melting curve method that hinders primer extension to detect low-abundance mutations

[0130] The 315th codon of the catalase encoding gene (katG) of Mycobacterium tuberculosis is mutated from serine (Ser, S) to threonine (Thr, T) (AGC→ACC), which will lead to the first-line Resistance to the drug isoniazid (INH). In this example, taking the point mutation (katG S315T(AGC→ACC)) as an example, the ability of the self-quenching probe melting curve method that hinders primer extension to detect low-abundance mutations was investigated.

[0131] 1.1 Synthesis of primers and probes

[0132] In this example, an LNA-modified self-quenching probe and primer set for the katG gene were designed to ensure that the amplicon contained the mutation site. The forward primer is F1, the reverse primer is R1, and the probe is p1. Both the primer and probe sequences were synthesized by Shanghai Sangong, the primers wer...

Embodiment 2

[0160] Example 2. Investigation of the ability of the self-quenching probe melting curve method that hinders primer extension to detect low-abundance mutations

[0161] In this example, taking the point mutation -15C→T in the promoter region of the inhA gene of Mycobacterium tuberculosis as an example, the ability of the self-quenching probe melting curve method that hinders primer extension to detect low-abundance mutations was investigated.

[0162] 2.1 Synthesis of primers and probes

[0163] In this example, an LNA-modified self-quenching probe and primer set targeting the promoter region of the inhA gene were designed to ensure that the amplicon contained the mutation site. The forward primer is F2, the reverse primer is R2, and the probe is p2. Both the primer and probe sequences were synthesized by Shanghai Sangong, the primers were purified by ULTRAPAGE, and the probes were purified by HPLC. The 5' end of the probe is labeled with ROX, and the 3' end is labeled with ...

Embodiment 3

[0190] Example 3. Investigation of the ability of the self-quenching probe melting curve method that hinders primer annealing to detect low-abundance mutations

[0191] In this example, taking the same point mutation (katG S315T (AGC→ACC)) as in Example 1 as an example, the ability of the self-quenching probe melting curve method that hinders primer annealing to detect low-abundance mutations was investigated.

[0192] 3.1 Design of primers and probes

[0193] First, design primer pairs and probes based on its gene sequence to ensure that the amplicon contains the mutation site. The forward primer is F2, and the reverse primer (R1) and self-quenching probe (p1) in this example are the same as those in Example 1. Wherein, the forward primer F2 and the probe have an overlapping sequence, so F2 and the probe can competitively bind to the region to be detected of the nucleic acid sequence to be detected, and when there is no mutation in the region, the probe binds to the target s...

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Abstract

The invention relates to low-abundance gene mutation detection. Particularly, the invention provides a method for detecting whether mutation exists in the nucleic acid molecules of a nucleic acid sample to be detected through self-quenching probe melting curve analysis. Besides, the invention also provides a kit comprising a self-quenching probe and primer pairs, and the kit can be applied to implementing the method.

Description

technical field [0001] The invention relates to the detection of low-abundance gene mutations. In particular, the present invention provides a method for detecting whether there is a mutation in a nucleic acid molecule in a nucleic acid sample to be tested by analyzing the melting curve of a self-quenching probe. In addition, the invention provides kits comprising self-quenching probes and primer sets, which kits can be used to practice the methods of the invention. Background technique [0002] Gene mutation refers to the change of base pair composition or arrangement sequence in the structure of DNA molecules, which is a heritable change and commonly exists in various species in nature. Bacteria will undergo random mutations during the reproduction process, and certain mutations will cause bacteria to develop resistance to drugs. These mutations are called drug resistance mutations. In genetic diagnosis, detect bacterial drug-resistant mutations, especially in samples wi...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12Q1/6827C12Q1/689C12Q2600/106C12Q2600/156C12Q2527/107C12Q2531/113C12Q2563/107
Inventor 李庆阁许晔梁斌
Owner XIAMEN UNIV
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