Method for electrochemically detecting concentration of specific single-stranded DNA based on exonuclease and nucleic acid probe

An exonuclease and nucleic acid probe technology, applied in the field of electrochemical detection of specific single-stranded DNA concentration, can solve problems such as low sensitivity, radioactive contamination, and expensive detection instruments.

Active Publication Date: 2016-08-03
北京聚合美生物科技有限公司
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  • Abstract
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  • Claims
  • Application Information

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Problems solved by technology

Traditional DNA detection methods include enzymatic method, chemical degradation method, and random cloning method. Modern DNA detection methods include fluorescent quantitative PCR, DNA automated sequencing, and colorimetric methods. However, these methods have certain shortcomings, such as enzyma

Method used

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  • Method for electrochemically detecting concentration of specific single-stranded DNA based on exonuclease and nucleic acid probe
  • Method for electrochemically detecting concentration of specific single-stranded DNA based on exonuclease and nucleic acid probe
  • Method for electrochemically detecting concentration of specific single-stranded DNA based on exonuclease and nucleic acid probe

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1. K-ras proto-oncogene fragment as the target DNA sequence to be detected.

[0035] K-ras gene is a proto-oncogene about 35kb long located on chromosome 12, the expression of this gene can regulate the normal growth of cells. Mutations in this gene will lead to abnormal cell growth, excessive cell proliferation and spread to cause cancer. According to statistics, the probability of K-ras gene mutation in patients with pancreatic cancer is as high as 90%-100%. K-ras gene mutations often occur in the early stage of canceration. The 12th codon base of the wild-type K-ras gene is GGT, and it is easy to mutate into GTT, GAT, GCT and other mutant types. GAT mutant type is the most common mutation. The type can be used as a marker to characterize pancreatic cancer. It is of great significance for the early screening of pancreatic cancer to develop a sensitive detection method using the gene fragment where the mutation site is located as the detection target sequence....

Embodiment 2

[0041] Example 2. Specificity Analysis of Electrochemical DNA Sensors

[0042] Still taking the above-mentioned K-ras proto-oncogene fragment as an example, replace the original target DNA with DNA with single-base mismatch and three-base mismatch to participate in the hybridization reaction, and the specific steps are the same as in Example 1.

[0043] The target DNA (TD) sequence is: 5'-TGGAGCTGGTGGCGTAGGCA-3'

[0044] The single base mismatch sequence is: 5'-TGGAGCTG A TGGCGTAGGCA-3'

[0045] The three-base mismatch sequence is: 5'-TGGAGCT CCA GGCGTAGGCA-3'

[0046] Compare the signal response in the presence of target DNA, single-base mismatch DNA, and triple-base mismatch DNA in the presence of three different DNAs. Such as Figure 4 , the sensor can produce a clear comparison of DPV signal intensity in the recognition of single-base mutation and three-base mutation sequence of the target DNA. Single-base (b) and triple-base mismatches (c) produce much lower signal...

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Abstract

The invention relates to a method for electrochemically detecting concentration of specific single-stranded DNA based on exonuclease and a nucleic acid probe and belongs to the technical field of analytical chemistry. The invention designs and synthesizes two types of hairpin-type probes P1 and P2. The method comprises the following steps of: firstly modifying a gold electrode with gold nanoparticles by adopting 1,6-hexanedithiol (HDT), preparing an AuNPs-HDT-Au electrode, and then modifying the probe P1 onto the electrode; taking a specific single stranded target DNA as a to-be-detected object, so that the probes P1 and P2 can coexist when no target DNA exists, and the target DNA can trigger two independent reaction cycles when the target DNA, the probe P2 and the exonuclease ExoIII exist; and finally when heme exists, generating a strong signal under the interaction of a G-tetraplex sequence of the probe P1 on the surface of the electrode and heme, and detecting the target DNA by adopting a differential pulse voltammetry, wherein a peak current signal and the target DNA concentration are correlated in a certain concentration range, so that detection on the target DNA concentration is realized. The method provided by the invention has the advantages of high sensitivity and strong specificity.

Description

technical field [0001] The invention relates to a method for electrochemically detecting the concentration of specific single-stranded DNA based on exonuclease and nucleic acid probe assisted target cycle amplification, which belongs to the technical field of analytical chemistry. Background technique [0002] The detection of specific DNA or RNA sequences (such as susceptibility genes or cancer marker gene sequences related to tumors, diabetes and other diseases) is of great significance in clinical diagnosis, disease prevention and treatment. Traditional DNA detection methods include enzymatic method, chemical degradation method, and random cloning method. Modern DNA detection methods include fluorescent quantitative PCR, DNA automated sequencing, and colorimetric methods. However, these methods have certain shortcomings, such as enzymatic and chemical degradation methods. The operation of the method is cumbersome, may cause radioactive contamination, and the efficiency is...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12Q1/682C12Q1/6825C12Q2521/319C12Q2565/607C12Q2563/137C12Q2527/125
Inventor 周楠迪王淑玲田亚平孙笑凡
Owner 北京聚合美生物科技有限公司
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