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Magnetic particle and single base extension based SNP automatic detection method

An automated detection and single-base extension technology, applied in biochemical equipment and methods, microbial measurement/testing, fluorescence/phosphorescence, etc., can solve the problems that hinder the in-depth development of post-genome research, the accuracy of the results is not enough, and the lack of practicability, etc. problems, to achieve the effects of easy automation, high typing signal strength, and high ratio of positive and mismatching signals

Inactive Publication Date: 2009-06-03
SOUTHEAST UNIV
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  • Summary
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  • Claims
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AI Technical Summary

Problems solved by technology

But in general, the existing methods are generally limited by sample purification and concentration, complicated operation, and label detection technology. The number of SNPs and the number of samples that can be detected at the same time are very limited. Some methods have high analysis costs or poor results. Inaccurate enough and lacking in real utility
These technical and methodological bottlenecks hinder the in-depth development of post-genome research

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] 1. In this example, we take the detection of the M235T SNP site of the AGT gene as an example, and the designed extension primers are labeled with biotin at the 5' end. The specific primer sequence is: Biotin-(N) 15 -GGTGTC CACACTGGCTCCC.

[0026] 4. Single-base extension reaction Each sample was carried out in two separate PCR tubes, each tube contained 20 μL of reaction system, and each tube contained 20 μM corresponding TARMA-ddNTP, TARMA-ddATP was added to the wild-type reaction tube, TARMA- Add ddGTP to the mutant reaction tube, and the remaining reaction components include 10mM Tris-HCl (pH8.3), 50mM KCl, 2.0mM MgCl2, 1.5U of Taq enzyme, 100ng genomic DNA and 24pmol biotin-labeled extension primer.

[0027]5. The specific procedure of the extension reaction is as follows: first, keep warm at 95°C for 5 minutes, and then perform 35 cycles of variable temperature thermal cycles, specifically including 95°C for 30 seconds, 58°C for 30 seconds, and 72°C for 30 second...

Embodiment 2

[0031] 1. Synthesize Fe according to the method of Shen et al. (Chemistry Letters Vol.33, No.11, 1468-1469, 2004) 2 o 3 magnetic particles.

[0032] 2. The magnetic particles are soaked in 5% (volume ratio) APTES ethanol solution to modify the surface with NH 2 After washing with ethanol and deionized water, the magnetic particles are soaked in 5% (volume ratio) glutaraldehyde aqueous solution to modify the surface with aldehyde groups. Finally, the avidin diluted with PB buffer was reacted with the aldehyde-modified magnetic particles to prepare the avidin-modified magnetic particles.

[0033] 3. In this example, we take the detection of the M235T SNP site of the AGT gene as an example, and the designed extension primer is labeled with biotin at the 5' end. The specific primer sequence is: Biotin-(N) 15 -GGTGTC CACACTGGCTCCC.

[0034] 4. According to the method of Liu et al. (Journal of Nanoscience and Nanotechnology, 2008, 8: 405-409), the whole genome amplification pro...

Embodiment 3

[0040] 1. According to the method of Wang et al. (J. Nanosci. Nanotechnol. 2008, 8, 1797), the magnetic nanoparticles coated with silica were synthesized.

[0041] 2. The magnetic particles are soaked in 5% (volume ratio) APTES ethanol solution to modify the surface with NH 2 After washing with ethanol and deionized water, the magnetic particles are soaked in 5% (volume ratio) glutaraldehyde aqueous solution to modify the surface with aldehyde groups. Finally, the avidin diluted with PB buffer was reacted with the aldehyde-modified magnetic particles to prepare the avidin-modified magnetic particles.

[0042] 3. In this example, we take the detection of the M235T SNP site of the AGT gene as an example, and the designed extension primer is a 5' terminal biomarker. The specific primer sequence is: biotin-(N) 15 -GGTGTCCACACTGGCTCCC.

[0043] 4. According to the method of Staessenu et al. (Journal of Hypertension. 17 (1): 9-17, 1999), the PCR product of the M235T site of the A...

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Abstract

The invention discloses a novel single nucleotide polymorphism detection method based on magnetic particles and single base extension technology. The detection method is characterized in that through extension primer of a detected locus marked with biotin, specific ddNTP marked with fluorescence is extended to the 3' end of the primer under the action of Taq enzyme, so as to amplify the genotype signal of the template through thermal cycle reaction including denaturation, annealing and extension. Then the extension primer is fixed to the surface of magnetic particles modified with avidin to detect the subtype signal of the specimen. The detection method has the advantages of simplicity, accuracy, low cost, high flux and high sensitivity, can realize automatic operation and has higher practicability than the traditional method.

Description

1. Technical field [0001] The invention belongs to the technical field of gene detection, in particular to an automatic detection method for single nucleotide polymorphism using magnetic particles and single base extension technology. 2. Background technology [0002] The study of DNA sequence variation associated with genetic phenotype has become one of the research topics in the post-genome era. Single nucleotide polymorphism (Single Nucleotide Polymorphism, SNP) is the most common single base difference in the human genome. It occurs frequently in the genome. It is estimated that it occurs once every 300-1000 bases, and the distribution density is very large. Microsatellite repeat sequences, etc., thus becoming the most commonly used third-generation genetic markers. In different populations, there are differences in the frequency distribution of SNPs, and these differences can represent genetic differences among a certain race or population. Therefore, the study of SNP...

Claims

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Application Information

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IPC IPC(8): C12Q1/68G01N21/64
Inventor 刘洪娜何农跃李松
Owner SOUTHEAST UNIV
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