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A single base mutation detection method

A single-base mutation and target gene technology, applied in biochemical equipment and methods, microbial measurement/testing, etc., can solve problems such as poor performance, and achieve the effects of easy preparation, simple operation, and high repeatability

Active Publication Date: 2020-05-05
BEIJING UNIV OF CHEM TECH
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Problems solved by technology

However, traditional hybrid probes perform poorly in single-base selectivity, which is caused by the thermodynamic barrier of nucleic acid hybridization, that is, the equilibrium constant of the hybridization reaction between the probe and the wild-type and single-base mutants at room temperature near

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] Example 1: Detection of single base A>G mutation in DNA

[0059] Use a signal probe that is 10 bases complementary to the target gene to detect the A>G gene mutation in the target single-stranded DNA.

[0060] Specific steps:

[0061] (1) Magnetic sphere-coupled biotinylated capture probe

[0062] ①Put the solution of magnetic balls coated with avidin on the surface and vortex for 30s to mix the magnetic balls coated with avidin thoroughly. Pipette 100 μl of magnetic bead solution into a 1.5ml centrifuge tube. Place the centrifuge tube on a magnetic separator, let it stand for 2 minutes, and suck off the supernatant.

[0063] ②Remove the centrifuge tube, add 1ml of buffer I (10mM Tris-HCl (pH=7.5), 1mM EDTA, 1M NaCl, 0.1% Tween-20), vortex and mix well, magnetically separate, and remove the supernatant. Repeat this operation once.

[0064] ③ Add 500 μl of buffer I containing 1.6 μM biotinylated capture probe to make the concentration of the magnetic beads 2 mg / ml, ...

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Abstract

The invention relates to a single base mutation detection method combining nucleic acid temporary hybridizing and magnetic separating. The method comprises the following steps: A, designing a capturing probe and a signal probe according to a target gene; B, preparing a magnetic sphere solution from a magnetic sphere coupled with the capturing probe, adding the signal probe to conduct nucleic acid hybridization with a to-be-detected sample; C, conducting magnetic separation after finishing the nucleic acid hybridization, and then re-suspending the magnetic sphere in a low-metal ion solution; D, conducting magnetic separation again, detecting an obtained liquid supernatant, judging whether single base mutation exists in the to-be-detected sample, wherein the capturing probe is completely complementary with a non-mutation part of the target gene; the signal probe is completely complementary with a mutant type gene of the target gene, and forms a single base mismatch with a wild type gene of the target gene. The detection method is fast, and can be used in low-abundance mutation detection.

Description

technical field [0001] The invention belongs to the field of gene mutation detection, and in particular relates to a single base mutation detection method. Background technique [0002] Gene mutation, especially single-base mutation detection technology is of great significance to the early diagnosis and drug strategy of diseases. In the context of precision medicine, gene mutation detection has become an important clinical test indicator. Gene mutation detection technology is divided into sequencing technology and non-sequencing technology. With the development of next generation sequencing technology (Next Generation Sequencing), the cost of sequencing has dropped significantly, enabling many institutions to provide gene sequencing services for patients. However, the next-generation sequencing technology requires a high concentration of nucleic acid and a long analysis time, and is limited by the sequencing depth and the nature of the nucleic acid polymerase itself, maki...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6827
CPCC12Q1/6827C12Q2565/519C12Q2523/308
Inventor 苏昕喻长远王俊秀周旭王磊李泽浩郝丹丹李丽丹
Owner BEIJING UNIV OF CHEM TECH
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