A method for quantitative analysis of label-free DNA mass spectrometry based on exonuclease III-assisted target cyclic amplification

A technology of exonuclease and target circulation, applied in the field of biological detection

Active Publication Date: 2021-03-26
HUNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] The present invention aims to solve the problem that the current DNA detection method based on Exo III assisted target cyclic amplification and spectral detection technology only judges DNA single nucleotide polymorphisms based on changes in spectral signal intensity and is prone to false positive results, and proposes a Based on Exo III-assisted circular amplification and label-free DNA mass spectrometry quantitative analysis method to achieve sensitive quantitative analysis of DNA in complex samples, and use mass spectrometry to have the ability to identify different residual DNA fragments to clearly identify DNA single nucleotide polymorphisms

Method used

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  • A method for quantitative analysis of label-free DNA mass spectrometry based on exonuclease III-assisted target cyclic amplification
  • A method for quantitative analysis of label-free DNA mass spectrometry based on exonuclease III-assisted target cyclic amplification
  • A method for quantitative analysis of label-free DNA mass spectrometry based on exonuclease III-assisted target cyclic amplification

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Experimental program
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Embodiment 1

[0045] Example 1: Quantitative analysis of target DNA in simulated biological samples

[0046] This embodiment combines the Exo III-assisted target cyclic amplification strategy, mass spectrometry detection and spectral deformation quantitative analysis theory (SSD) to achieve accurate quantitative analysis of target DNA in simulated biological samples.

[0047] Reagents: HPLC grade acetonitrile was purchased from Sweden Opson Company; HPLC grade ammonia water was purchased from Aladdin Reagent Co., Ltd.; Exonuclease III (Exo III) and 10×NEB buffer 1 were purchased from New England Biolabs Company, USA; serum was purchased from Changsha Healthy volunteers in the blood bank center; HPLC-purified target DNA was synthesized by Bao Bioengineering (Dalian) Co., Ltd.; dialysis membrane (1KD, standard grade regenerated cellulose membrane) and other HPLC-purified oligonucleotide strands were purchased from Shenggong Bioengineering (Shanghai) Co., Ltd. The DNA sequence used is as foll...

Embodiment 2

[0071] Example 2: Identification of Base Mismatched DNA Samples

[0072] To test the specificity of the DNA quantitative analysis strategy proposed in the present invention, we designed a base mismatch DNA sequence with the same length as the target DNA (see the reagent section), including a single base mismatch DNA sequence: its mismatched base The positions of the 5th (DNA@5), 9th (DNA@9) and 12th (DNA@12) bases located at the 5′ end of the target DNA sequence, respectively, and the DNA with three base mismatches (DNA@9.10 .11): The mismatched bases are located at the 9th, 10th and 11th bases of the 5' end of the target DNA sequence.

[0073] A sample of base mismatch DNA was prepared in the same manner as in Example 1 for preparing the standard sample. Two samples were prepared at final concentrations of 5 and 15 nM for each base mismatched DNA. Compared with DNA detection methods based on absorption or emission spectroscopy (such as UV-Vis, fluorescence, Raman, etc. spec...

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Abstract

The invention discloses an exonuclease III aided target cycle amplification based unmarked DNA mass spectrum quantitative analysis method. According to the method, exonuclease III aided target cycle amplification strategy, mass spectrum and wave spectrum deformation quantitative theory model are combined together, so as to realize sensitive and accurate quantitative analysis of target DNA in a complicated biologicla sample. Meanwhile, the method overcomes disadvantages of existing methods for identifying single nucleotide polymorphism, and by utilizing the characteristic that mass spectrum hasa capacity of identifying different residual DNA segments, the strategy provided by the invention can clearly identify single nucleotide polymorphism.

Description

technical field [0001] The invention belongs to the field of biological detection. Specifically, it relates to a label-free DNA mass spectrometry quantitative analysis method based on exonuclease III-assisted circular amplification strategy. [0002] technical background [0003] The development of a reliable method for deoxyribonucleic acid (DNA) detection with high sensitivity and good selectivity is of great significance to the development of biological research, clinical diagnosis and biomedicine. Among the existing DNA detection methods, the DNA detection technology based on the exonuclease III (Exo III)-assisted target cyclic amplification strategy has the advantages of high sensitivity, simple operation, isothermal reaction, especially without specific recognition sites, etc. Therefore, it has received extensive attention in the field of biosensing technology. The most suitable substrates for Exo III are double-stranded DNA with flush or recessed 3' ends, stepwise cl...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6813
CPCC12Q1/6813C12Q2565/627C12Q2521/319C12Q2537/165
Inventor 陈增萍石彩霞
Owner HUNAN UNIV
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