A highly selective DNA fluorescence analysis method based on morpholino oligonucleotide functionalized silicon chip

A morpholino oligonucleotide, a high-selectivity technology, applied in the field of high-selectivity DNA fluorescence analysis, can solve the problems of difficult single nucleotide polymorphism detection, high background noise in electrochemical methods, and low hybridization efficiency. Achieve good linear response, simple method, and short analysis time

Inactive Publication Date: 2018-10-12
NANJING UNIV OF SCI & TECH
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  • Summary
  • 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

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  • A highly selective DNA fluorescence analysis method based on morpholino oligonucleotide functionalized silicon chip
  • A highly selective DNA fluorescence analysis method based on morpholino oligonucleotide functionalized silicon chip
  • A highly selective DNA fluorescence analysis method based on morpholino oligonucleotide functionalized silicon chip

Examples

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Effect test

Embodiment 1

[0046] Example 1: The effect of rhodamine B on the detection results of DNA fluorescence analysis of the present invention under different reaction times.

[0047] Using the highly selective DNA fluorescence analysis based on the morpholine oligonucleotide functionalized silicon-based chip of the present invention, the morpholine oligonucleotide (sequence: 5'-H 2 N-AAC CAT ACA ACC TAC TAC CTC A-3') as capture probe, fully complementary DNA fragment (sequence: 5'-TGA GGT AGT AGG TTG TAT GGTT-3') as target DNA fragment, all operation steps All as described in above-mentioned embodiment 1, wherein, the concentration of morpholine oligonucleotide is 1 μ mol / L, and the concentration of DNA fragment solution to be tested (solvent is 1 * TE buffer) is 0.1nmol / L, hybridization time and rhodan The concentration of rhodamine B remains excessive, and the rhodamine B reaction time is changed to be 10, 15, 20, 25, 30, 35, 40min successively, and the influence of different rhodamine B react...

Embodiment 2

[0048] Example 2: Effects of different rhodamine B concentrations on the detection results of the DNA fluorescence analysis of the present invention.

[0049] Using the highly selective DNA fluorescence analysis based on the morpholine oligonucleotide functionalized silicon-based chip of the present invention, the morpholine oligonucleotide (sequence: 5'-H 2 N-AAC CAT ACA ACC TAC TAC CTC A-3') as capture probe, fully complementary DNA fragment (sequence: 5'-TGA GGT AGT AGG TTG TAT GGTT-3') as target DNA fragment, all operation steps All as described in above-mentioned embodiment 1, wherein, the concentration of morpholine oligonucleotide is 1 μ mol / L, the solution (solvent is 1×TE buffer) concentration of DNA fragment to be tested is 0.1 nmol / L, and hybridization time keeps excessive , change the Rhodamine B concentration to 4, 8, 12, 16, 20, 24, 28 μmol / L in turn, and analyze the influence of different Rhodamine B concentrations on the nucleic acid fluorescence analysis detec...

Embodiment 3

[0050] Example 3: Effects of different hybridization reaction times on the detection results of the DNA fluorescence analysis of the present invention.

[0051] Using the highly selective DNA fluorescence analysis based on the morpholine oligonucleotide functionalized silicon-based chip of the present invention, the morpholine oligonucleotide (sequence: 5'-H 2 N-AAC CAT ACA ACC TAC TAC CTC A-3') as capture probe, fully complementary DNA fragment (sequence: 5'-TGA GGT AGT AGG TTG TAT GGTT-3') as target DNA fragment, all operation steps All as described in above-mentioned embodiment 1, wherein, the concentration of morpholine oligonucleotide is 1 μ mol / L, the solution (solvent is 1×TE buffer) concentration of DNA fragment to be tested is 0.1nmol / L, changes DNA to be tested The hybridization reaction time of the fragment and the capture probe morpholino oligonucleotide is 30, 45, 60, 75, 90, 105, and 120 min in sequence, and the influence of different hybridization reaction times...

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Abstract

The invention discloses a high-sensitivity and high-selectivity fluorescence analysis method for detecting a specific DNA fragment through introducing morpholino oligonucleotides. The morpholino oligonucleotides covalently connected to the surface of a silicon chip are adopted as a capture probe, and are hybridized with the DNA fragment, and rhodamine B is coordinated to a DNA skeleton through using the coordination chemical action of transition metal ions, so the fluorescence analysis method can realize high-sensitivity and high-selectivity detection of the DNA fragment, and can be used to detect DNA samples in the fields of molecular identification, clinic diagnosis, environment monitoring and food safety.

Description

technical field [0001] The invention belongs to the application field of fluorescence analysis, in particular to a highly selective DNA fluorescence analysis method based on a morpholine oligonucleotide functionalized silicon-based chip. Background technique [0002] High-selectivity and high-specificity DNA detection technology has been widely used in molecular recognition, clinical diagnosis, environmental monitoring, food safety, etc. In recent years, DNA detection technology tends to be miniaturized, simplified, low-cost, specific and sensitive , the improvement of detection rate, and easier process control are the current development trends of DNA detection technology. Compared with early DNA detection methods such as gel electrophoresis, ultraviolet spectroscopy, and colorimetry, 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, C...

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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