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Method suitable to detect mononucleotide polymorphism in fluorescence polarization technology

A single nucleotide polymorphism, fluorescence polarization technology, applied in fluorescence/phosphorescence, biochemical equipment and methods, polarization influence characteristics, etc., can solve problems such as affecting the accuracy of detection results, cumbersome operation steps, and prone to pollution. , to achieve the effect of low cost, simple result analysis, and less pollution

Inactive Publication Date: 2009-09-23
FOURTH MILITARY MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The present invention is to provide a method suitable for detecting single nucleotide polymorphism in fluorescence polarization technology, so as to overcome the problems of high cost, cumbersome operation steps, easy pollution and affecting the accuracy of detection results in the prior art

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0015] Example 1: In the detection of the C / T single nucleotide polymorphism on the 118th codon of the 4th exon of the ERCC1 gene related to the prediction of platinum drug resistance,

[0016] 1. The reagents used are: MgCl2, 10× reaction buffer, TaqDNA polymerase, dNTPs, DNA primer for the 4th exon of ERCC1 gene, C / T single nucleotide polymorphism on the 118th codon DNA probe composition.

[0017] 2. Amplify the above reagents according to the asymmetric PCR method: carry out the reaction in a 25uL system, add 2.5μL of PCR buffer, and add 1.0U TaqDNA polymerase; the concentrations of dGTP, dCTP, dATP and dTTP are respectively 2.0mmol / L ; Concentration of 0.5uM ERCC1 upstream primer and probe mixture with fluorescent label at the 3' end, concentration of 0.05uM ERCC1 downstream primer.

[0018] The amplification reaction conditions were: denaturation at 94°C for 5 minutes, incubation at 95°C for 1 second, incubation at 60°C for 1 second, incubation at 72°C for 10 seconds, 45...

Embodiment 2

[0019] Example 2: In the detection of the Arg194Trp polymorphism of the XRCC1 gene related to the prediction of platinum drug sensitivity,

[0020] 1. The reagents used are: composed of MgCl2, 10× reaction buffer, TaqDNA polymerase, dNTPs, XRCC1 gene DNA primers, and DNA probes for single nucleotide polymorphisms on the 194th codon.

[0021] 2. Amplify the above reagents according to the asymmetric PCR method: carry out the reaction in a 25uL system, add 2.5μL of PCR buffer, and add 2.0U TaqDNA polymerase; the concentrations of dGTP, dCTP, dATP and dTTP are respectively 2.5mmol / L ; The XRCC1 upstream primer and the 3' end fluorescently labeled probe mixture with a concentration of 2.0uM each, and the XRCC1 downstream primer with a concentration of 0.4uM.

[0022] The amplification reaction conditions were: denaturation at 95°C for 10 minutes, incubation at 95°C for 1 second, incubation at 56°C for 1 second, incubation at 72°C for 10 seconds, 40 cycles, and no incubation at 72°...

Embodiment 3

[0023] Example 3: In the detection of the SUR1 gene polymorphism Ser1369Ala related to the prediction of the curative effect of sulfonylureas,

[0024] 1. The reagents used are: composed of MgCl2, 10× reaction buffer, TaqDNA polymerase, dNTPs, SUR1 gene DNA primers, and DNA probes for single nucleotide polymorphisms on the 1369th codon.

[0025] 2. Amplify the above reagents according to the asymmetric PCR method: carry out the reaction in a 25uL system, add 2.5μL of PCR buffer, and add 1.5U TaqDNA polymerase; the concentrations are 2.0mmol / L of dGTP, dCTP, dATP and dTTP respectively ; The concentration of the SUR1 gene upstream primer and the 3' end fluorescently labeled probe mixture is 2.0uM, and the concentration is 0.4uM of the SUR1 gene downstream primer.

[0026] The amplification reaction conditions are: denaturation at 94°C for 8 minutes, incubation at 72°C for 10 seconds, 35 cycles, and no incubation at 72°C in the last cycle. Then the fluorescence polarization valu...

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Abstract

The invention relates to the technical field of fluorescence polarization, in particular to a method suitable to detect mononucleotide polymorphism in a fluorescence polarization technology. In order to overcome the problems of high cost, complex operation step, easy pollution and detection result influence in the prior art, the technical scheme is achieved as follows: reagents adopted by the method comprise MgCl2, 10* reaction buffer, TapDNA polymerase, dNTPs, target gene DNA primer, and a DNA probe with fluorescence labeling at the 3 end of a target zone; and the reagents are performed with amplified reaction. The invention has the advantages that: firstly, the steps and the operation are simple, the amplified reaction is performed in a hybrid stopped tube by one step without easy pollution and accurate result; secondly, the result analysis is simple and the result is judged by only number comparison so as to be easy to standardize and automate, and the application range is wide; thirdly, the cost is low without any special reagent and fluorescent quenching or microgroove wedding agent.

Description

Technical field: [0001] The invention relates to the technical field of fluorescence polarization detection, in particular to a method suitable for detecting single nucleotide polymorphism in fluorescence polarization technology. Background technique: [0002] Single nucleotide polymorphism (SNP) refers to the presence of different bases at specific nucleic acid positions in the genome of a normal individual of a certain population, which is a difference in human DNA sequence, that is, a single nucleotide change in the genome sequence Polymorphic changes in DNA sequences, human triallelic and tetraallelic SNPs are very rare, so SNPs are also called biallelic markers. Among the 3 billion bases in the human genome, about one SNP occurs every 300 to 500 bases, and there are about 20 million SNPs in the entire human genome. SNPs can occur in both coding genes and non-coding genes, and are sensitive to the phenotype of human individuals (morphology, metabolism, and immune status...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68G01N21/64G01N21/21
Inventor 张菊刘文超张贺龙
Owner FOURTH MILITARY MEDICAL UNIVERSITY
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