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DNA molecular machine and nucleic acid detection method

A DNA molecule and detection method technology, applied in biochemical equipment and methods, microbiological determination/inspection, etc., can solve the problems of cumbersome operation, complex sample components, and low target DNA content, so as to reduce processing steps and broaden the scope of application , the effect of high sensitivity

Inactive Publication Date: 2018-12-28
HENAN AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The traditional fluorescence-labeled nucleic acid amplification detection technology requires fluorescent probe labeling, which is cumbersome, time-consuming and expensive, and its sensitivity is not high
Although the fluorescent sensor built on the basis of traditional technology has low background signal and strong stability, however, due to the complex composition of the actual sample and the extremely low content of target DNA, this requires the detection method to have extremely high sensitivity and specificity. Conventional fluorescently labeled nucleic acid amplification The sensitivity and selectivity of enhanced detection technology cannot meet its detection needs

Method used

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  • DNA molecular machine and nucleic acid detection method
  • DNA molecular machine and nucleic acid detection method
  • DNA molecular machine and nucleic acid detection method

Examples

Experimental program
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Effect test

Embodiment 1

[0080] Step 1: Synthesize the DNA molecular machine and configure it into a solution with a concentration of 20nM,

[0081] Step 2: Pulverize mouse liver cancer tissue at room temperature, filter and extract the juice, and prepare a sample solution with a concentration of 2 μM,

[0082] Step 3: Take 4 μL of sample solution and 2 μL of DNA molecular machine solution, mix evenly by micro-oscillation to obtain a mixed solution,

[0083] Step 4: Add 10 μL buffer, 6 μL polymerase, 6 μL nickase, 6 μL Thioflavin T, 6 μL dNTP, 6 μL Mg 2+ Add to the mixed solution obtained in step 3 to obtain a reaction solution.

[0084] Wherein, the above-mentioned dNTP concentration is 0.12mM; the polymerase concentration is 0.112U / uL; the nickase concentration is 0.01U / uL; the thioflavin T concentration is 0.1uM; HCL, tween20 at a concentration of 0.1%, KCl at a concentration of 130 mM, (NH4) at a concentration of 10 mM 2 SO 4 and NaCl at a concentration of 30 mM; Mg 2+ The concentration is 1m...

Embodiment 2

[0101] Step 1: Synthesize the DNA molecular machine and configure it into a solution with a concentration of 60nM,

[0102] Step 2: Grinding the mouse liver cancer tissue at room temperature, filtering and extracting the juice, and preparing a sample solution with a concentration of 8 μM,

[0103] Step 3: Take 8 μL of sample solution and 4 μL of DNA molecular machine solution, mix evenly by micro-oscillation to obtain a mixed solution,

[0104] Step 4: Add 40 μL buffer, 10 μL polymerase, 10 μL nickase, 10 μL Thioflavin T, 10 μL dNTP, 10 μL Mg 2+ Add to the mixed solution obtained in step 3 to obtain a reaction solution.

[0105] Wherein, the above-mentioned dNTP concentration is 0.50mM; the polymerase concentration is 0.236U / uL; the nickase concentration is 0.06U / uL; the buffer solution includes Tris HCL with a concentration of 70mM, pH9.0, and tween20 with a concentration of 0.6%. KCl at a concentration of 160mM, (NH4) at a concentration of 60mM 2 SO 4 , NaCl at a concent...

Embodiment 3

[0115] Step 1: Synthesize the DNA molecular machine and configure it into a solution with a concentration of 100nM,

[0116] Step 2: Pulverize mouse liver cancer tissue at room temperature, filter and extract the juice, and prepare a sample solution with a concentration of 2 μM,

[0117] Step 3: Take 4 μL of the sample solution and 4 μL of the DNA molecular machine solution, and mix evenly by micro-oscillation to obtain a mixed solution.

[0118] Step 4: Add 20 μL buffer, 6 μL polymerase, 7 μL nickase, 10 μL Thioflavin T, 3 μL dNTP, 36 μL Mg 2+ Add to the mixed solution obtained in step 3 to obtain a reaction solution.

[0119] Wherein, the above-mentioned dNTP concentration is 0.3mM; the polymerase concentration is 0.212U / uL; the nickase concentration is 0.03U / uL; the thioflavin T concentration is 0.1uM; HCL, tween20 at a concentration of 0.4%, KCl at a concentration of 145mM, (NH4) at a concentration of 45mM 2 SO 4 , a concentration of 46mM NaCl; Mg 2+ The concentration...

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Abstract

The invention belongs to the field of biotechnology, and particularly relates to a DNA molecular machine. The DNA molecular machine is a nucleotide sequence and comprises the following regions a target sequence recognition region, a stable region, a nickase recognition region and a G-quadruplex synthesis template. The target sequence recognition region, the stabile region, the nickase recognitionregion and the G-quadruplex synthesis template are arranged in order from the 3' end to the 5' end of the nucleotide sequence. The method is used for converting nucleic acid signals in a sample into G-quadruplex, and can achieve continuous amplification of the G-quadruplex at normal temperature to amplify the nucleic acid signal height and achieve rapid qualitative and semi-quantitative analyticaldetermination of nucleic acid with high sensitivity.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a DNA molecular machine and a nucleic acid detection method. Background technique [0002] Nucleic acid isothermal amplification technology is one of the new nucleic acid amplification technologies. It does not need to go through three stages of temperature changes, denaturation, annealing and extension like PCR, and it can quickly and stably realize nucleic acid amplification at a single constant temperature. increase. In addition, the conditions required for the isothermal amplification technique are very simple, without relying on sophisticated instruments and equipment, it can be performed under a water bath, even on the cell surface or in living cells. In view of these advantages of isothermal detection technology, a series of nucleic acid isothermal amplification technologies have been developed and researched, matured, completed the transition from laboratory to p...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6825C12Q1/6844
CPCC12Q1/6825C12Q1/6844
Inventor 张晓婷张芳张艳丽杜鹏强高飞谢源周琳李洪连
Owner HENAN AGRICULTURAL UNIVERSITY
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