Single base difference test method based on magnetic separation and solid single base elongation cycle technology

A detection method and single-base technology, which can be used in the determination/inspection of microorganisms, biochemical equipment and methods, material excitation analysis, etc. High signal strength, fast detection effect

Inactive Publication Date: 2009-03-25
SOUTHEAST UNIV
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Problems solved by technology

Especially for tumor samples, because the mutation rate in cancer tissue is often very low, conventional hybridization detection methods are often unable to accurately detect mutations due to false positive signals generated by mismatch hybridization

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] A single base difference detection method based on magnetic separation and solid-phase single base cycle extension technology, the detection steps are: modification of magnetic nanoparticles: functional modification of the surface of magnetic particles without fluorescent background, so that the surface of the magnetic particles has Specific chemical groups or biomacromolecules, the specific chemical groups or biomacromolecules are colloidal gold, avidin or aldehyde groups; modification of designed primers: select important functional SNP sites or mutation sites to be detected, according to Design a single-base circular extension primer for the base sequence of the above site, and the 5' end of the designed primer is functionally modified with a sulfhydryl group, biotin or amino group corresponding to the colloidal gold, avidin or aldehyde group on the surface of the magnetic particle, and the 3' The last base at the end is complementary to the upstream base of the site ...

Embodiment 2

[0032] 1. Apply the method of Jun Lin et al. (Journal of Solid State Chemistry 159, 26-31, 2001) to prepare magnetic particles coated with gold shells, and disperse the particles in 0.1M PB buffer solution with a final concentration of 4mg / M1.

[0033]2. 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 a sulfhydryl group at the 5' end. The specific primer sequence is: SH-(N) 15 -GGTGTCCACACTGGCTCCC.

[0034] 3. 24 pmol sulfhydryl-labeled primers were covalently immobilized on the surface of 80 μg magnetic particles through Au-S bonds.

[0035] 4. Solid-phase cycle extension reaction. Each sample was carried out in two separate PCR tubes. Each tube contained 20 μL of the corresponding TARMA-ddNTP. TARMA-ddATP was added to the wild-type reaction tube, and TARMA- ddGTP was added to the mutant reaction tube, and other reaction components included 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 2.0...

Embodiment 3

[0039] 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.

[0040] 2. The magnetic particles are soaked in 5% (volume concentration) 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 concentration) glutaraldehyde aqueous solution to modify the surface with aldehyde groups.

[0041] 3. 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 the 5' terminal amino group. The specific primer sequence is: NH 2 -(N) 15 -GGTGTCCACACTGGCTCCC.

[0042] 4. 20 pmol amino-labeled primers were covalently immobilized on the surface of 80 μg magnetic particles through the shift effect between amino and aldehyde groups.

[0043] 5. Solid-phase cycle extension reaction Each sample was carried out in two separate PCR tubes...

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Abstract

The invention discloses a new method for detecting single base difference (SBD) based on magnetic separation and a solid-phase single base circular extension (SBCE) technology. The method is characterized in that: magnetic particles are used as carriers for fixing extension primers of locus to be detected; under the action of Taq enzyme, specific fluorescent labeled ddNTP is extended to 3' end of the primers and genotype signals of a template are amplified by thermal cycling reaction which comprises denaturation, annealing and extension. The method is characterized by simplicity, preciseness, low cost, high flux and high sensitivity and automated operation can be realized by the method, therefore, the method is more practical than the traditional methods.

Description

1. Technical field [0001] The invention belongs to the technical field of gene detection, in particular to a single base difference detection method based on magnetic separation and solid-phase single base cycle extension technology. 2. Background technology [0002] Prior Art: The study of DNA sequence variation related to genetic phenotype has become one of the research topics in the post-genome era. Therefore, the study of the most common variation in the human genome—Single Base Difference (SBD), which mainly includes single-base deletions, insertions, substitutions, and single nucleotide polymorphisms, has also become a hotspot. Sex (Single Nucleotide Polymorphism, SNP). 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 in every 300-1000 bases. The distribution density is much higher than that of microsatellite repeat sequences. third-generation genetic markers. In different popu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68G01N21/64
Inventor 李松何农跃刘洪娜
Owner SOUTHEAST UNIV
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