Detection method for single nucleotide polymorphism

A single nucleotide polymorphism and detection method technology, applied in biochemical equipment and methods, microbial measurement/inspection, etc., can solve the problems of high cost of detection equipment, isotope pollution, etc.

Inactive Publication Date: 2013-05-15
CHENGDU INST OF BIOLOGY CHINESE ACAD OF S
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The current LCR amplification technology is mostly used in combination with isotope labeling or fluorescent modification for the detection of single nucleotide polymorphisms. These methods all have problems such as isotope pollution, high cost of reagents or detection instruments to a certain extent.

Method used

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  • Detection method for single nucleotide polymorphism
  • Detection method for single nucleotide polymorphism
  • Detection method for single nucleotide polymorphism

Examples

Experimental program
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Effect test

Embodiment 1

[0038] Example 1. Detection of sickle cell anemia gene DNAT-1 with group A (Probe L, ProbeL*, Probe R, Probe R*) oligonucleotide probes (mutant DNAT-1 is mutant DNA, normal DNAT-1 is normal DNA).

[0039] For the reaction steps to detect the sickle cell anemia gene, see figure 1 . Group A (Probe L, ProbeL*, Probe R, Probe R*) oligonucleotide probes are designed to detect the sickle cell anemia gene, in which the 3' end of the oligonucleotide probe Probe R is labeled with a porphyrin The DNAzyme sequence with peroxidase activity is used for signal detection; the sequence at the 5' end is a sequence that can form a complementarity with the sequence near the mutation site of the target gene to be detected, and is used for Gap-LCR amplification.

[0040] After adding group A oligonucleotide probes Probe L, ProbeL*, Probe R, Probe R* into the Gap-LCR system, under denaturing conditions, the double strand of the DNA T-1 to be tested and the probe (Probe L, ProbeL* , Probe R, Prob...

Embodiment 2

[0058] Example 2. Using group B (B1-B4) oligonucleotide probes to detect the mutation site 235delC in the neonatal deafness gene.

[0059] For the detection results of the mutation site 235delC in the neonatal deafness gene, see image 3 . Group B (B1-B4) oligonucleotide probes are designed to detect the mutation site 235delC in the neonatal deafness gene, in which the 3' end of the oligonucleotide probe B4 is labeled with a compound that can bind to porphyrin iron, etc. The DNAzyme sequence with peroxidase activity is used for signal detection; the sequence at the 5' end is a sequence that can form a complementary sequence with the sequence near the mutation site of the target gene to be detected, and is used for Gap-LCR amplification.

[0060] After adding group B oligonucleotide probes (B1-B4) into the Gap-LCR system, under denaturing conditions, the double-strand structure of the DNA T-1 to be tested and the double-strand structure of probe 1 are opened; Under the condit...

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Abstract

The invention belongs to the technical field of molecular biology, relates to analysis and detection for single nucleotide polymorphism, and in particular relates to a universal detection method. The detection method comprises the steps of amplifying target DNA (deoxyribonucleic acid) by gap-ligase chain reaction, introducing a DNAzyme sequence into a ligation product, degrading a phosphorylated probe which does not participate in the ligation reaction by Lambda excision enzyme, and releasing DNAzyme by excision enzyme III. The detection method is characterized in that a DNAzyme probe is combined with a Gap-LCR (ligase chain reaction) probe, and is characterized by comprising the following steps of: adding a section of DNAzyme sequence into a phosphorylated probe to be connected in an LCR system, adding an Anti-G sequence into a system (in complementary pairing with a DNAzyme sequence part in the probe) after the Gap-LCR amplification reaction stops, degrading a 5-end phosphorylated DNA probe by the Lambda excision enzyme, and releasing the DNAzyme in the ligation product by the excision enzyme III, thereby qualitatively and quantitatively detecting the SNP (single nucleotide polymorphism) due to the characteristics of the DNAzyme. The method is simple, convenient, practical and economical, and is widely applicable to the detection of various single nucleotide polymorphisms.

Description

technical field [0001] The invention belongs to the technical field of molecular biology and relates to the analysis and detection of single nucleotide polymorphisms, in particular to a universal method for amplifying target DNA by gap-ligase chain reaction, introducing DNAzyme sequences into ligation products, and then A detection method that uses Lambda exonuclease to degrade phosphorylated probes that do not participate in the ligation reaction, and exonuclease III to release DNAzyme. Background technique [0002] Single nucleotide polymorphism (single nucleotide polymorphism, SNP), mainly refers to the DNA sequence polymorphism caused by the variation of a single nucleotide at the genomic level, which is a specific nucleotide position in genomic DNA. Changes such as conversion, transversion, insertion or deletion occur, and the frequency of any allele in the population is not less than 1%. It is the most common type of human heritable variation, accounting for more than...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
Inventor 唐卓周丽
Owner CHENGDU INST OF BIOLOGY CHINESE ACAD OF S
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