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Method for detecting deletion mutation of nucleic acid molecule

A technology for nucleic acid molecules and deletion mutations, which is applied in biochemical equipment and methods, microbiological determination/inspection, etc., and can solve the problems of PCR product contamination, multiple operation steps, false positives, etc.

Inactive Publication Date: 2015-05-27
XIAMEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This technique is widely used clinically, but electrophoresis analysis after PCR is required, and there are many operation steps, and it is easy to cause contamination of PCR products, resulting in false positives.
The MLPA (Multiplex ligation-dependent Probe amplification) technology developed in recent years can simultaneously detect the deletion and duplication of multiple nucleic acid fragments in one reaction, and has also been widely recognized in clinical practice. However, its detection cost is high, the operation is cumbersome, and special equipment is required, so it is not suitable for routine clinical use.

Method used

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  • Method for detecting deletion mutation of nucleic acid molecule
  • Method for detecting deletion mutation of nucleic acid molecule
  • Method for detecting deletion mutation of nucleic acid molecule

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0124] Example 1: The self-quenching fluorescent probe melting curve analysis method is used for the detection of short-segment (tens of bases) gene deletions.

[0125] In this example, taking the detection of the 16 bp deletion c.176-191del16 in the gap junction protein-encoding gene (GJB2 gene) (NCBI sequence number NC_000013.11) as an example, a fluorescent probe P1 and a pair of primers F1 and R1, the double-stranded hybrid formed by the hybridization of the fluorescent probe with the gene fragment before and after the deletion can form different melting points (there are 4 bases at the 3′ end of the probe that are complementary to the wild-type nucleic acid molecule, but not complementary to the nucleic acid molecule with the deletion mutation) , based on which the genotype of the sample can be determined. The probe and primer sequences used are:

[0126] P1:5′-HEX-CAGCAACACCCTGCAGCCAG GCTG -BHQ1-3' (SEQ ID NO: 1), where the underlined part indicates the 4 bases with d...

Embodiment 2

[0132] Example 2: The self-quenching fluorescent probe melting curve analysis method is used for the detection of gene deletions with medium fragment length (hundreds of bases).

[0133] This example takes the β-globin gene (HBB gene, NCBI reference sequence number is NG_000007.3) 619bp gene deletion Del619 (deletion position is g.71609-72227) as an example, and designs a fluorescent probe for this type of gene deletion P2 and primer sets F2, R2 and R3, the double-stranded hybrids formed by the hybridization of the fluorescent probes with the gene fragments before and after the deletion can form different melting points (6 bases at the 3′ end of the probe are complementary to the wild-type nucleic acid molecule, and the presence of The nucleic acid molecule of the deletion mutation is not complementary), based on which the genotype of the sample can be judged. The probe and primer sequences used are:

[0134] P2: 5′-ROX-CCGCATATAAATATTTCTGCATATAAATTGTAAC TGATGT GG-BHQ2-3' (...

Embodiment 3

[0141] Example 3: The melting curve analysis method of self-quenching fluorescent probe is used for large gene deletion.

[0142] In this example, the Southeast Asian deletion type (-- SEA ) (deletion position is g.26264-45564) as an example, design a fluorescent probe P3 and primer set F3, R4 and R5 for this type of gene deletion, and the double-stranded hybrid formed by hybridization between the fluorescent probe and the gene fragment before and after the deletion can be Different melting points are formed (4 bases at the 3' end of the probe are complementary to the wild-type nucleic acid molecule, and not complementary to the nucleic acid molecule with deletion mutation), based on which the genotype of the sample can be judged.

[0143] The probe and primer sequences used are:

[0144] P3:5′-FAM-CGCCTTGGGGAGGTTC TAGC -BHQ1-3' (SEQ ID NO: 8), where the underlined part indicates the 4 bases with differences in matching;

[0145] F3: 5'-CCGTCCGACTCAAGGAC-3' (SEQ ID NO: 9);...

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Abstract

The invention relates to a method for detecting deletion mutation of a nucleic acid molecule, and particularly relates to a method for detecting deletion mutation of a nucleic acid molecule through a probe melting curve, and a reaction system and kit for detecting deletion mutation of a nucleic acid molecule. The method provided by the invention is accurate and stable, rapid, simple and convenient, free of PCR after-treatment and capable of overcoming defects that the operation is complex, long time is consumed, pollution is easily caused, the detecting flux cannot meet the conventional demand and the like in the prior art, so that the method has a favorable application prospect.

Description

technical field [0001] The invention relates to a method for detecting deletion mutations of nucleic acid molecules, in particular to a method for detecting deletion mutations of nucleic acid molecules through probe melting curves, as well as a reaction system and a kit for detecting deletion mutations of nucleic acid molecules. Background technique [0002] Deletion mutation refers to a mutation in which a part of a chromosome or DNA or RNA sequence is lost, which can cause the loss of genetic material. Deletion mutations can be deletions of varying numbers of nucleotides, ranging from just a single base deletion to deletions of entire chromosomal segments. Deletion mutation in a narrow sense refers to the deletion of a longer fragment of a nucleic acid molecule, excluding the deletion of a single base or a few bases. Deletion mutations often cause the loss of genetic material, often directly lead to changes in the expression and expression levels of encoded proteins, resu...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
Inventor 黄秋英李庆阁
Owner XIAMEN UNIV
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