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High-sensitivity DNA fluorescent analysis method based onmorpholine oligonucleotide functionalized magnetic microballoon

A technology of morpholino oligonucleotides and magnetic microspheres, 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 in electrochemical methods, and low hybridization efficiency. Good linear response, simple method and short analysis time

Inactive Publication Date: 2016-10-19
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 fluorescent analysis method based onmorpholine oligonucleotide functionalized magnetic microballoon
  • High-sensitivity DNA fluorescent analysis method based onmorpholine oligonucleotide functionalized magnetic microballoon
  • High-sensitivity DNA fluorescent analysis method based onmorpholine oligonucleotide functionalized magnetic microballoon

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 25 mg / mL carboxylated magnetic microspheres in 140 μL 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 CGA ATC CAT TTT TT-Biotin-3'), after the hybridization, pe...

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 is kept excessive, and the concentration of magnesium ions is changed to 0.01, 0.05, 0.1, 0.5, 1, 5, 10mM in turn, 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 the As the concentration increases, the fluor...

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 remains 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 target DNA are analyzed. The impact of signal probe DNA fragment hybridization time on DNA fluorescence analysis results of the present invention is different, and its analysis results are 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 m...

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Abstract

The invention discloses a high-sensitivity and high-selectivity fluorescence analysis method for detecting specific DNA fragments by introducing morpholino oligonucleotides. Morpholine oligonucleotides covalently linked to the surface of magnetic microspheres are used as capture probes, and after hybridization with longer target DNA fragments, the unhybridized segments of target DNA are then hybridized with signal DNA with end-modified biotin, using strands The combination of mycoavidin and biotin introduces alkaline phosphatase into the signal probe DNA end, alkaline phosphatase catalyzes L-ascorbic acid 2-phosphate to generate L-ascorbic acid, and then reduces resazurin to form L-ascorbic acid Strong fluorescent resorufin, the fluorescence intensity is correlated with the target DNA, through the method of fluorescence analysis to achieve high sensitivity and high selectivity detection of DNA fragments, it can be used in molecular recognition, clinical diagnosis, environmental monitoring and food safety and other fields detection of DNA samples.

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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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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