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DNA fluorescent capillary biosensor and preparing process thereof

A biosensor and DNA probe technology, applied in the fields of biochemistry and molecular biology analysis, can solve the problems of small DNA probes, complex detection instruments, weak fluorescence signals, etc. economic effect

Inactive Publication Date: 2006-08-30
SICHUAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the disadvantages of optical fiber DNA sensors are that the bare part at one end is easily broken, the immobilized amount of DNA probe and the amount of bound target DNA are small, the generated fluorescent signal is weak, the detection sensitivity is low, the stability is poor, and the detection equipment is complicated and expensive.

Method used

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  • DNA fluorescent capillary biosensor and preparing process thereof
  • DNA fluorescent capillary biosensor and preparing process thereof

Examples

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Effect test

Embodiment 1

[0031] In this example, the synthesized 100 μmol / L oligonucleotide sequence 5'-GAAA CCTG TTTG TTGG ATAC-3' was used as a DNA probe, which was immobilized on the inner surface of a capillary according to the above-mentioned method to prepare a DNA-FCBS.

[0032] During the measurement, 10 μL of 33 μmol / L target nucleotide sequence 5′-GTATCC AACAAACA GGTTTC-3′ solution was inhaled with DNA-FCBS, and allowed to hybridize at 45°C for 90 minutes; EB) staining; wash out the free EB staining solution in DNA-FCBS with 0.2% SDS and diethyl ester pyrocarbonic acid (DEPC) water; use RF-5000 fluorescence spectrometer to measure at excitation wavelength 526nm, emission wavelength 584nm, the result obtained Yes: the average value of fluorescence intensity before and after hybridization is 74.01±14.12, the statistical analysis result is t=4.1106, p<0.05, and there is a significant difference before and after hybridization.

Embodiment 2

[0034] In this example, 10 μL of 1 μmol / L Cy5-labeled target nucleotide sequence 5′-GTATCCAACAAACA GGTTTC-3 solution was sucked into the DNA-FCBS of the present invention, and hybridized at 45° C. for 90 minutes; washed out with 0.2% SDS and DEPC water Free target nucleotides in DNA-FCBS; measured with RF-5000 fluorescence photometer at excitation wavelength 646nm, emission wavelength 664nm, the results are shown in Table 1; the average value of fluorescence intensity before and after hybridization is 61.66 ± 18.22, statistics The analysis result was t=6.7684, 0.005<P<0.01, and there was a highly significant difference before and after hybridization.

[0035] DNA-FCBS serial number

Embodiment 3

[0037] In this example, under the experimental conditions that the hybridization time is 90min and the hybridization temperature is 45°C, a series of Cy5-labeled target nucleotide solutions with different concentrations are inhaled with the DNA-FCBS of the present invention, and the RF-5000 fluorescence photometer is used to detect 646nm, measured at the emission wavelength of 664nm, the results are as follows figure 2 shown. The Cy5-labeled target nucleotide concentration has a good linear relationship with the fluorescence intensity in the range of 0.1-1 μmol / L: y=139.73x+39.613, the correlation coefficient is 0.9985, and the detection limit is 1.7 pmol.

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Abstract

This invention discloses a DNA fluorescence capillary biologic sensor (DNA-FCBS) and its preparation method, it belongs to biochemical field. DNA-FCBS is made by using crosslinking agent fixing DNA probe in capillary tube, it includes capillary tube, poly lysine and DNA probe. It can realize the classification of DNA and quantitative determination. DNA sample solution with butt is inhaled by DNA-FCBS for hybridization and coloration reaction, and then the butt DNA is quantitative determined in fluorescence device according to the fluorescence strength. Different butt nucleotides can be determined by changing DNA-FCBS inner probe type, multiple probes are fixed in DNA-FCBS to determine multiple butt nucleotides once time. The DNA-FCBS also can be made into all kinds of DNA fluorescence capillary biologic testing cassette. The reproducibility of this invention is good, operation is simple, and trace quick determination can be realized, so gene detection can be popular and practical.

Description

technical field [0001] The invention relates to a DNA fluorescent capillary biosensor (DNA-FCBS) for DNA fluorescent capillary analysis (DNA-FCA) and a preparation method thereof, belonging to the field of biochemical and molecular biological analysis. The invention is applicable to gene detection of biological samples such as medicine, sanitation, agriculture and forestry. Background technique [0002] At present, DNA detection methods mainly use DNA chips and DNA sensors. The essence of DNA chip analysis is to arrange a series of addressable recognition molecules in an orderly array on the surface of a small substrate. According to the principle of base complementarity, use a sophisticated scanner or CCD imaging technology to scan the target DNA. to identify. The characteristic of DNA chip is that it can measure multiple target DNA at one time, but there is the possibility of non-specific hybridization, which takes a long time, high production...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
Inventor 李永生高秀峰
Owner SICHUAN UNIV
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