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Fluorescence detection method for DNA and kit thereof

A technology for fluorescence detection and kits, applied in fluorescence/phosphorescence, material excitation analysis, etc., to achieve high-efficiency, rapid enrichment and separation, and early diagnosis

Inactive Publication Date: 2009-07-01
SHANGHAI INST OF APPLIED PHYSICS - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The technical problem to be solved by the present invention is to detect the DNA sequence based on the optical amplification of the water-soluble conductive polymer, thereby solving the sensitivity problem of the target (targeted) DNA detection, and to provide a combination of high selectivity and high selectivity. Unique DNA fluorescence detection method and its kit

Method used

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  • Fluorescence detection method for DNA and kit thereof
  • Fluorescence detection method for DNA and kit thereof
  • Fluorescence detection method for DNA and kit thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0064] The materials in the kit are: commercially available streptavidin-coated magnetic beads (Streptavidin coatedMMPs) (purchased from Promega, magnetic beads with a diameter of about 1 μm); the capture probe DNA1 labeled with biotin (Biotin) in a conventional method ;Signal probe——Fluorescein-labeled signal probe DNA2; hybridization solution (750mM NaCl, 150mM Sodium citrate); buffer system containing 50mM NaCl, 10mM Tris-HCl buffer, pH=7.5 or so; CP1; denaturation Solution 50mM NaOH.

[0065] Such as figure 1 As shown, the commercially available streptavidin-coated magnetic beads were washed three times with 0.5×SSC buffer according to their requirements, and then mixed with biotin-labeled oligonucleotide strands (capture probe 1) (1.25 nmol / L capture Probe: 1 mg magnetic beads), light vibration for 20 minutes, using highly specific binding of avidin and biotin (K d =10 -15 M) DNA probes were immobilized on magnetic particles, magnetically separated and the beads were was...

Embodiment 2

[0067] Replace the targeted DNA3 with a single-base mismatched DNA4 and repeat the steps in Example 1, the results are as follows figure 2 shown.

Embodiment 3

[0069] Replace the targeting DNA3 with DNA5 of any sequence, repeat the steps of Example 1, and the results are as follows figure 2 shown.

[0070] Embodiment 1~3 such as figure 2 As shown in Figure A, it can be seen that even if the concentration of completely non-complementary arbitrary sequence DNA is 5000 times that of complementary DNA, there is still no energy transfer signal; when the concentration of pure DNA to be tested is 1nM, there is no single-base mismatch DNA energy transfer signal, while the complementary targeting DNA has a stronger energy transfer signal. It shows that this DNA detection strategy can completely distinguish single base mismatched DNA from random sequence DNA. It can be seen from Figure B that when the pure DNA to be tested is any DNA sequence, even if the concentration is 1000 times that of the complementary chain, no energy transfer signal can be observed, indicating that this DNA detection strategy is not interfered by non-specific DNA s...

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Abstract

The invention discloses a method for fluorescence detection of target DNA in biological samples, which comprises the following steps: 1) magnetically enriching and separating DNA to be detected from biological samples; 2) adding a signal detector labeled with a fluorescent group Needle to hybridize and pair the DNA to be detected after magnetic enrichment and separation with the signal probe, and use a buffer containing 10-100mM sodium chloride or potassium chloride and 10-50mM Tris-hydrochloric acid or phosphate, pH=7.0-8.0 The system washes the single-base mismatched DNA and discards it; 3) Add water-soluble cationic polyfluorene to achieve signal multiplication, use the excitation wavelength of the water-soluble cationic polyfluorene for fluorescence spectrum, and use the fluorescence resonance energy transfer detection method The detection analyzes the DNA to be detected. The invention also discloses a DNA fluorescence detection kit. The invention not only has supersensitivity, but also solves the problem of selectivity, making it of great significance in biological detection, such as early diagnosis and treatment of genetic diseases, such as breast cancer.

Description

technical field [0001] The invention relates to the field of detection of target DNA sequences in biological samples to be tested, in particular to a DNA fluorescence detection method and a kit thereof. Background technique [0002] Magnetism is the basic property of matter. Magnetic materials are ancient and widely used functional materials. Nano-magnetic materials are new magnetic materials that have been gradually produced, developed and expanded since the 1970s and have become the most vigorous and broad application prospects. Superparamagnetic nanoparticles have excellent properties such as small size, large specific surface area, good suspension stability, and magnetically oriented transport and enrichment under the action of an external magnetic field, making them useful in the enrichment and separation of cells and biologically active substances. Very important application prospects, some have entered clinical trials. DNA magnetic nano-separators have extremely impo...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N21/64C12Q1/68
Inventor 樊春海徐慧武海萍李文新
Owner SHANGHAI INST OF APPLIED PHYSICS - CHINESE ACAD OF SCI
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