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Gene mutation multi-detecting method based on signal amplification DNA logic gate

A signal and fluorescent signal technology, which is applied in the determination/inspection of microorganisms, biochemical equipment and methods, etc., can solve the problems that the background signal affects the detection sensitivity and specificity of nucleic acid targets, it is difficult to apply DNA logic gates, and the output of wrong results, etc. , to achieve the effects of improved detection sensitivity, high signal-to-noise ratio output, and simplified analysis process

Active Publication Date: 2017-12-29
NANJING GENERAL HOSPITAL NANJING MILLITARY COMMAND P L A
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Excessive background signal affects the detection sensitivity and detection specificity of the cascade nucleic acid invasion reaction for nucleic acid targets
At the same time, the background signal may lead to wrong result output, so it is difficult to apply to the construction of DNA logic gate

Method used

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  • Gene mutation multi-detecting method based on signal amplification DNA logic gate
  • Gene mutation multi-detecting method based on signal amplification DNA logic gate
  • Gene mutation multi-detecting method based on signal amplification DNA logic gate

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1: Highly sensitive detection of artificially synthesized oligonucleotide fragments using a controllable extension reaction bridging two-step nucleic acid invasion signal amplification reaction detection method

[0038] In this example, artificially synthesized single-stranded DNA targets of different concentrations were detected to verify the feasibility of the nucleic acid detection method stated in the present invention and examine the sensitivity of the method.

[0039] Reaction conditions:

[0040] The reaction system included 10mmol / L MOPS (pH 7.5), 0.05% (v / v) NP-40, 0.05% (v / v) Tween-20, 12μg / mL BSA, 7.5mmol / L MgCl 2 , 6.675ng / μL Afu endonuclease, 50nmol / L upstream probe (sequence is: 5'-ATG TCA CTT CCC CTT GGT TCT CTC C-3', namely SEQ ID NO.1), 600nmol / L downstream probe Needle (sequence: 5'-CAT AGT CCA GGA GGC TTG TTA AGT GTC TTA GCG TTT TCG CTA AGA TCTGGC CTG GTG C(Spacer C3)-3', namely SEQ ID NO.2), 600nmol / L fluorescent probe ( The sequence is: 5'...

Embodiment 2

[0043] Example 2: Using a controllable extension reaction bridging two-step nucleic acid invasion signal amplification reaction detection method to achieve high specificity detection of single-base differential targets

[0044] In this example, six artificially synthesized mutation targets that have a single base difference with the target nucleic acid sequence at different positions are detected, and the positions of each mutation point are shown in the attached Figure 5 As indicated by the middle letter, the base type of each mutation site is the same as the corresponding site in the probe sequence, which is used to examine the specificity of the method of the present invention for single-base differential target detection.

[0045] Reaction conditions for detection of single base mutation targets at different positions:

[0046] The reaction system included 10mmol / L MOPS (pH 7.5), 0.05% (v / v) NP-40, 0.05% (v / v) Tween-20, 12μg / mL BSA, 7.5mmol / L MgCl 2 , 6.675ng / μL Afu endo...

Embodiment 3

[0059] Example 3: Construction of basic DNA logic gates using controllable extension reaction bridging two-step nucleic acid invasion signal amplification reaction detection method

[0060] In this example, by adjusting the composition of the controllable extension reaction bridging two-step nucleic acid invasion signal amplification reaction system, a DNA logic gate is constructed to verify the logic operation of the method of the present invention.

[0061] (1) "AND" logic gate response condition

[0062] The reaction system included 10mmol / L MOPS (pH 7.5), 0.05% (v / v) NP-40, 0.05% (v / v) Tween-20, 12μg / mL BSA, 7.5mmol / L MgCl 2 , 6.675ng / μL Afu endonuclease, 600nmol / L downstream probe (ie SEQ ID NO.2), 600nmol / L fluorescent probe (ie SEQ ID NO.3), 0.005U DNA polymerase, 10 μmol / L of dATP, dCTP and dTTP, 10 pmol / L of the DNA target (ie SEQ ID NO.4) and 50nmol / L of the upstream probe (ie SEQ ID NO.1) were used as input (input 1 and input 2), add water to make up the volume t...

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Abstract

The invention discloses a gene mutation multi-detecting method based on a signal amplification DNA logic gate. The method comprises the following steps: performing high specificity recognition on a detecting target by invading into a signal amplification reaction by utilizing nucleic acid, and simultaneously, invading into the signal amplification reaction by utilizing a controllable DNA polymerase extension reaction bridging two-step nucleic acid to form signal amplification. The method disclosed by the invention has high detecting sensitivity and high detecting specificity to a nucleic acid target. A DNA logic gate is constructed by invading into the signal amplification reaction by adopting the controllable DNA polymerase extension reaction bridging two-step nucleic acid disclosed by the invention, a background signal is inhibited by introducing an intervening sequence, and meanwhile, a two-step signal amplification reaction exists, so that the defecting sensitivity is improved, and high signal-to-noise ratio output can be realized under the conditions of trace amount of DNA input and short reaction time. Moreover, through the method established by the invention, a plurality of gene mutation points can be detected simultaneously and results can be subjected to logical judgment, and an analyzing process of gene mutation detection in a clinically individualized medicating process is simplified.

Description

technical field [0001] The invention relates to a gene mutation detection method, in particular to a method for simultaneously detecting multiple gene mutations by using a signal amplification reaction to construct a DNA logic gate. Background technique [0002] Gene mutation detection has been used in a variety of clinical medicine fields such as disease diagnosis and individualized treatment. At present, many gene mutation detection methods have been developed, such as mutation arrest amplification system-polymerase chain reaction (Amplification refractory mutation system-polymerase chain reaction, ARMS-PCR), competitive allele-specific Probe polymerase chain reaction (Competitive allele-specific TaqManpolymerase chain reaction, castPCR), digital polymerase chain reaction (Digital polymerase chain reaction, dPCR), high resolution melting curve (Highresolutionmelting, HRM) and other methods. Although many of these methods can realize simultaneous detection of multiple gen...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/682C12Q2537/143C12Q2533/101C12Q2563/107
Inventor 周国华刘云龙邹秉杰武海萍
Owner NANJING GENERAL HOSPITAL NANJING MILLITARY COMMAND P L A
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