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A rapid and general method for low-abundance mutation detection

A mutation detection, low-abundance technology, applied in the field of rapid and general low-abundance mutation detection, can solve the problems that DNA mutation detection technology cannot have specificity, high sensitivity, versatility, etc., to reduce detection time and cost, The effect of reducing inspection costs

Active Publication Date: 2022-06-07
XIEHE HOSPITAL ATTACHED TO TONGJI MEDICAL COLLEGE HUAZHONG SCI & TECH UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The purpose of the present invention is in order to provide a kind of fast and universal low-abundance mutation detection method, first utilize Holliday cross-chain migration technology to distinguish different nucleic acid sequences, then utilize endonuclease IV and single abasic probe to amplify the signal; this It is a general-purpose amplification system for different nucleic acid sequences, which achieves ultra-high selectivity, fast and low-cost detection of target DNA sequences; solves the problem that existing DNA mutation detection technologies cannot combine specificity, high sensitivity, versatility, and fast low-cost technical issues

Method used

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  • A rapid and general method for low-abundance mutation detection
  • A rapid and general method for low-abundance mutation detection
  • A rapid and general method for low-abundance mutation detection

Examples

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Embodiment 1

[0079] The present embodiment provides a method for applying a rapid and general low-abundance mutation detection method to PTEN130Q(G>A) locus genotyping, and the specific steps are as follows:

[0080] 1) Synthesize a single abasic double-labeled fluorescent probe, the probe and its synthesis method are all prior art;

[0081] 2) Design and synthesize mutant strand-1, wild strand-1, guide strand-1, bridge strand-1, and repressor strand-1 of the PTEN130Q (G>A) mutation site gene (see Table 1 for the specific sequence);

[0082] 3) Use TE buffer to dissolve the mutant chain-1, wild chain-1, guide chain-1, bridge chain-1 in 2), and configure them into liquid;

[0083] 4) Dilute mutant chain-1 with wild chain-1 to prepare a series of mixed samples with a concentration of 200 nm, so that the mutation abundance ranges from 100% to 0.01%;

[0084] 5) Add repressor chain-1 to the mixed sample configured in 4), and the concentration of repressor chain-1 is 400 nm;

[0085] 6) Mix t...

Embodiment 2

[0097] This embodiment provides a fast and general low-abundance mutation detection method applied to the BRCA1 / c.2082C>T locus genotyping method. The specific steps are as follows:

[0098] 1) using the single abasic double-labeled fluorescent probe in Example 1;

[0099] 2) Design and synthesize mutant strand-2, wild strand-2, guide strand-2, bridge strand-2, and repressor strand-2 of the BRCA1 / c.2082C>T site gene (see Table 2 for the specific sequence);

[0100] 3) Use TE buffer to dissolve the mutant chain-2, wild chain-2, guide chain-2 and bridge chain-2 in 2), and configure them into liquid;

[0101] 4) Dilute mutant chain-2 with wild chain-2 to prepare a series of mixed samples with a concentration of 200 nm to make the mutation abundance range from 100% to 0.01%;

[0102] 5) Add repressor chain-2 to the mixed sample configured in 4), and the concentration of repressor chain-2 is 400 nm;

[0103] 6) Mixing the guide chain-2 and bridge chain-2 configured in 3) into a m...

Embodiment 3

[0115] This embodiment clearly provides a method for applying a fast and general low-abundance mutation detection method to EGFRL858 locus genotyping, and the specific steps are as follows:

[0116] 1) using the single abasic double-labeled fluorescent probe in Example 1;

[0117] 2) Design and synthesize mutant strand-3, wild-type strand-3, guide strand-3, bridge strand-3, and repressor strand-3 of the EGFRL858 site gene (see Table 3 for the specific sequence);

[0118] 3) Use TE buffer to dissolve the mutant chain-3, wild chain-3, guide chain-3 and bridge chain-3 in 2), and configure them into liquid;

[0119] 4) Dilute mutant chain-3 with wild chain-3 to prepare a series of mixed samples with a concentration of 200 nm, so that the mutant abundance ranges from 100% to 0.01%;

[0120] 5) Add repressor chain-3 to the mixed sample configured in 4), and the concentration of repressor chain-3 is 400 nm;

[0121] 6) Mixing the guide chain-3 and bridge chain-3 configured in 3) in...

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Abstract

The invention discloses a fast and universal low-abundance mutation detection method. It includes the following steps, step 1: prepare a single abasic double-labeled fluorescent probe; step 2: design the required mutant strand, wild strand, guide strand, bridge strand and repressor strand; step 3: configure the liquid; step 4 : Prepare a mixed sample with a concentration of 500nm; Step 5: Add the repressor strand; Step 6: Mix the guide strand and the bridge strand to form a mixed solution; Step 7: Leave the mixed solution to stand; Step 8: Separate the mixed solution prepared in Step 4 Adding to the mixed solution treated in step 7 to configure a mixed solution; Step 9: Add the probe prepared in Step 1 to the mixed solution in Step 7; Step 10: Add enzymes to the mixed solution in Step 8; Step 11: Add the liquid in step 9 into the detection tube to detect the fluorescence curve; step 12: get the fluorescence curve of the mutation point. The invention has the advantages of high selectivity, rapid and low-cost detection of the target DNA sequence.

Description

technical field [0001] The invention relates to the field of nucleic acid sequence detection, and more particularly to the technical fields of DNA sequence identification, single nucleotide polymorphism (SNP) typing, and multiple detection of low-abundance point mutations. More specifically it is a fast and general low-abundance mutation detection method. Background technique [0002] With the development of genomics, more and more clinical evidence shows that cancer and hereditary diseases are closely related to DNA point mutations. Therefore, detection of point mutations in driver genes is of great significance for the early diagnosis, treatment and prognosis evaluation of cancer. However, mutant DNA is generally present in low abundance in humans. The percentage of mutated DNA in an extracted DNA sample is very low, possibly less than 1% in the early stages of cancer. Therefore, in tissue biopsy or liquid biopsy, the method for detecting DNA point mutation should have ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6827C12Q1/682
CPCC12Q1/6827C12Q1/682
Inventor 汪宏波章伟舒湾赵蓉
Owner XIEHE HOSPITAL ATTACHED TO TONGJI MEDICAL COLLEGE HUAZHONG SCI & TECH UNIV