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Method for realizing single base mispairing identification using nano gold particles and electroelution

A nano-gold particle and electro-elution technology, which is applied in the field of nucleic acid mismatch recognition, can solve the problems of high cost, reduced cDNA hybridization signal changes, complicated probe design and preparation, etc. Effect

Inactive Publication Date: 2010-02-17
HUNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Usually adjust the stringency of hybridization conditions (such as temperature, ionic strength, formamide concentration, etc.) Potential to reduce signal changes caused by cDNA hybridization while affecting DNA
In addition, using some nucleic acid probes with good specificity (such as peptide nucleic acid probes, molecular beacons) instead of ssDNA probes can also improve the ability to identify single base mismatches, but this method has the disadvantages of probe design and preparation. Complicated and expensive

Method used

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  • Method for realizing single base mispairing identification using nano gold particles and electroelution
  • Method for realizing single base mispairing identification using nano gold particles and electroelution
  • Method for realizing single base mispairing identification using nano gold particles and electroelution

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Embodiment 1

[0037] 1. The surface of the gold film needs to be cleaned before modification. First, clean it with acetone, absolute ethanol and secondary water in sequence, then clean it with concentrated sulfuric acid / 30% hydrogen peroxide solution with a volume ratio of 3:1 for 1-2 minutes, and then use two Repeatedly rinse with water, use N 2 Blow dry and ready for finishing. The modification steps of the gold film are as follows: (1) firstly treat with 10mmol / L mercaptopropionic acid / absolute ethanol solution for at least 70min, and then wash repeatedly with 0.01mol / L phosphate buffer (pH=7.3) to remove unadsorbed Mercaptopropionic acid; (2) then react 60min at room temperature with 100mmol / L 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride / N-hydroxysuccinimide, and then use 0.01mol / L phosphate buffer (pH=7.3) was washed repeatedly; (3) Finally, inject 0.57μmol / L amino-modified probe DNA2 (purchased from Yubao Bioengineering Dalian Co., Ltd.) and incubate at room temperatu...

Embodiment 2

[0042] 1. Utilize as step 1 method among the embodiment one that 0.57 μ mol / L amino-modified probe DNA2 is immobilized on the gold film surface of surface plasmon resonance (SPR) sensor, and the sequence of the amino-modified DNA probe is 5'-CGCCTCACAACCAAAAAAA -C 6 h 12 -NH 2 -3', record its SPR spectrum as curve 1, and its resonance wavelength is λ 0 ;

[0043] 2. Then add 33nmol / L single base mismatch target DNA 2 Perform hybridization, the hybridization time is 30min, single base mismatch target DNA 2 The sequence is 5'-GGTTGTGAGGCG A TGCCCAAGCGA-3', after that, repeated washing with 6×SSC (pH 7.0) buffer solution, it was found that the SPR spectrum can be caused to move from curve 1 to the long wave direction, to curve 2, and the resonance wavelength λ of curve 2 1 ; Then add excess thiol DNA modified particle diameter of 13nm gold nanoparticles, react for 30min, the sequence of thiol DNA is 5'-HS-C 6 h 12 -AAAAAATCGCTTGGGCAG-3', wash the gold film repeatedly with...

Embodiment 3

[0047] 1. Same as step 1 in embodiment two.

[0048] 2. Then add 33nmol / L single base mismatch target DNA 3 Perform hybridization, the hybridization time is 30min, single base mismatch target DNA 3 The sequence is 5'-GGTTGTGAGGCG T TGCCCAAGCGA-3', after repeated washing with 6×SSC (pH7.0) buffer solution, it was found that the SPR spectrum could move from curve 1 to long-wave direction and move to curve 2, the resonance wavelength of curve 2 is λ 1 ; Then add excess thiol DNA modified particle diameter of 13nm gold nanoparticles, react for 30min, the sequence of thiol DNA is 5'-HS-C 6 h 12 -AAAAAATCGCTTGGGCAG-3', after repeated washing with 6×SSC (pH 7.0) buffer solution, it is found that the SPR spectrum continues to move towards the long-wave direction, moving to curve 3, and the resonance wavelength of curve 3 is λ 2 , the resonance wavelength shift (λ 2 -λ 1 ) for 4.3nm (see Figure 5 Part I of D).

[0049] 3) Then connect the circuit, adjust the potential of the g...

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Abstract

The nanometer gold particle and electroelution process for identifying single base mispairing includes the following steps: fixing DNA probe, adding target DNA, flushing repeatedly, and detecting theSPR responding signal of target DNA hybridization; adding sulfhydryl DNA modified nanometer gold particle hybridization, flushing repeatedly, and detecting the SPR responding signal after sulfhydryl DNA hybridization; negatively charging the metal film surface for electroelution and detecting the responding signal after electroelution; and judging whether to have single base mispairing DNA of theadded target DNA based on whether the SPR responding signal after electroelution is smaller than the SPR responding signal after sulfhydryl DNA hybridization. The process of the present invention caneliminate the interference of the single base mispairing target DNA and ensure no influence on the cDNA hybridizing signal, and has simple DNA probe design and reuseable sensor chip.

Description

technical field [0001] The invention belongs to a method for recognizing nucleic acid mismatches, in particular to a method for realizing single-base mismatch recognition. Background technique [0002] Single nucleotide polymorphism (SNP) refers to the DNA sequence polymorphism caused by a single nucleotide variation at the genome level. The polymorphism shown by SNP only involves the mutation of a single base, which can be caused by the conversion or transversion of a single base, and the ratio of the two is about 2:1. Single-base mutations can lead to differences between groups and between individuals. Susceptibility to many diseases and individual drug sensitivity are related to single base mutations. The study of single base mutations helps to explain the differences in individual performance, the susceptibility of different groups and individuals to diseases, especially complex diseases, as well as the tolerance to various drugs and the response to environmental facto...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68
Inventor 王柯敏羊小海王青
Owner HUNAN UNIV