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High-sensitivity DNA fluorescence analysis method based on morpholine oligonucleotide functionalized magnetic microspheres

A morpholine oligonucleotide and magnetic microsphere technology, which is applied in the field of highly sensitive DNA fluorescence analysis, can solve the problems of difficult single nucleotide polymorphism detection, high background noise of electrochemical method, low hybridization efficiency, etc. The effect of good linear response, simple method and short analysis time

Inactive Publication Date: 2019-01-18
NANJING UNIV OF SCI & TECH
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At present, the method of using the electroactivity of ferrocene to detect DNA has been widely used. However, the background noise of the electrochemical method is large, the reproducibility is poor, and it is difficult to be used for single nucleotide polymorphism detection. The hybridization efficiency of the DNA hybridization probe used in the method is lower than that of peptide nucleic acid, morpholine oligonucleotide and other probes, and it is not suitable for DNA detection of base mismatches

Method used

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  • High-sensitivity DNA fluorescence analysis method based on morpholine oligonucleotide functionalized magnetic microspheres
  • High-sensitivity DNA fluorescence analysis method based on morpholine oligonucleotide functionalized magnetic microspheres
  • High-sensitivity DNA fluorescence analysis method based on morpholine oligonucleotide functionalized magnetic microspheres

Examples

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Effect test

Embodiment 1

[0029] Example 1: Ultraviolet and fluorescence spectrum characterization of morpholino oligonucleotide functionalized magnetic microspheres.

[0030] Using the highly sensitive DNA fluorescence analysis based on morpholine oligonucleotide functionalized magnetic microspheres of the present invention, suspend 10 μL of 25 mg / mL carboxylated magnetic microspheres in 140 μL of PBS buffer, add 20 μL of 1 μM morpholine Oligonucleotide (sequence is 5'-H 2 N-AAC CAT ACA ACC TAC TAC CTC A-3'), 20 μL of 10 μM NHS and 20 μL of 40 μM EDC, shaken at room temperature for 6 hours, then magnetically separated and washed with PBS buffer for several times, then resuspended in 180 μL of TE buffer Add 20 μL of the target DNA fragment, perform magnetic separation after hybridization and wash with TE buffer for several times, then resuspend in 180 μL of TE buffer, add 20 μL of 0.1 μM biotin-labeled signal probe DNA fragment (sequence: 5'-TCA TGA CGAATC CAT TTT TT-Biotin-3'), after the hybridizatio...

Embodiment 2

[0031] Example 2: Effect of magnesium ion concentration on DNA fluorescence analysis results of the present invention.

[0032] Using the high-sensitivity DNA fluorescence analysis based on morpholino oligonucleotide functionalized magnetic microspheres of the present invention, the operation steps are the same as the above-mentioned Example 1. Wherein, the hybridization time remains excessive, and the concentration of magnesium ions is changed to 0.01, 0.05, 0.1, 0.5, 1, 5, 10mM, and the influence of different magnesium ion concentrations on the DNA fluorescence analysis results of the present invention is analyzed. The analysis results are as attached image 3 As shown in (a), it can be seen from the figure that when the concentration of magnesium ions is 0.01mM, 0.05mM, 0.1mM, and 0.5mM, as the concentration increases, the fluorescence intensity increases significantly, and when the concentration is higher than 1mM, with As the concentration increases, the fluorescence inte...

Embodiment 3

[0033] Example 3: The effect of different hybridization times between target DNA and signal probe DNA fragments on the DNA fluorescence analysis results of the present invention.

[0034] Using the high-sensitivity DNA fluorescence analysis based on morpholino oligonucleotide functionalized magnetic microspheres of the present invention, the operation steps are the same as the above-mentioned Example 1. Wherein, the hybridization time of the morpholino oligonucleotide and the target DNA fragment is kept excessive, and the hybridization time of the target DNA and the signal probe DNA fragment is changed to 40, 50, 60, 70, 80, 90, 100 min, and the target DNA and the signal probe are analyzed. Probe DNA fragment hybridization time is different to the impact of DNA fluorescence analysis result of the present invention, and its analysis result is as attached image 3 As shown in (b), it can be seen from the figure that when the hybridization time is 40, 50, 60, 70, and 80 min, the ...

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Abstract

The invention discloses a high-sensitivity and high-selectivity fluorescent analysis method by introducing morpholine oligonucleotide to detect a specific DNA fragment. The method is as below: using morpholine oligonucleotide covalently attached to the surface of a magnetic microspheres as a capture probe, hybridizing the morpholine oligonucleotide with a long target DNA fragment; hybridizing an un-hybridized segment of the target DNA signal with a signal DNA with terminal modified with biotin; using thecombination effect of streptavidin and biotin, introducing alkaline phosphatase into the terminal of a signal probe DNA; catalyzing L-ascorbic acid 2-phosphate to generate L-ascorbic acid by alkaline phosphatase; and reducing resazurin by L-ascorbic acid to form strong fluorescent resorufin. The fluorescence intensity is positively correlated with the target DNA; through fluorescence analysis, high-sensitivity and high-selectivity detection of DNA fragments is achieved; and the method can be used in the detection of DNA samples in the fields of molecular recognition, clinical diagnosis, environmental monitoring and food safety.

Description

technical field [0001] The invention belongs to the application field of fluorescence analysis, in particular to a highly sensitive DNA fluorescence analysis method based on morpholine oligonucleotide functionalized magnetic microspheres. Background technique [0002] High-sensitivity and high-specificity DNA detection technology has been widely used in disease diagnosis, drug development, food safety, environmental monitoring, etc. In recent years, DNA detection technology tends to be miniaturized and simplified. The improvement of DNA detection technology, the reduction of cost and easier process control are the current development trends. Compared with early DNA detection methods such as colorimetry, gel electrophoresis, and ultraviolet spectroscopy, DNA sensors using fluorescence methods have attracted more and more attention due to their high sensitivity, low cost, and portability. [0003] Document 1 (Li Hongye, Fan Shuguo, Liu Yule, Chen Gang, Wang Huanyu, DNA detect...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N21/64
Inventor 孔金明胡伟文顾淑梅胡琼张学记
Owner NANJING UNIV OF SCI & TECH
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