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Nanoprobe used for specific recognition of DNA sequences, and application method thereof

A DNA sequence and application method technology, applied in the field of detection analysis and molecular biology, can solve the problem of limited resolution optical diffraction limit and so on

Inactive Publication Date: 2017-10-13
NANJING UNIV OF POSTS & TELECOMM
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

DNA molecular labeling methods based on fluorescent molecular labels, whose resolution is limited by the optical diffraction limit compared to specific labeling of DNA origami probes

Method used

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  • Nanoprobe used for specific recognition of DNA sequences, and application method thereof
  • Nanoprobe used for specific recognition of DNA sequences, and application method thereof
  • Nanoprobe used for specific recognition of DNA sequences, and application method thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0041] This embodiment is about the establishment of a closed circular single-stranded PhiX 174DNA model with diblock primers of different lengths, and its specific operations are as follows:

[0042] (1) figure 1 A in A is the constructed diblock primer, the Primer part binds to the specific site of the target DNA and effectively extends, the Capturer part hybridizes with the DNA origami probe, and is connected by a small single strand of the Spacer part in the middle. The sequence length of the fixed Capturer part is 20nt (Y=20nt), and the sequence length of the Primer part is set to 20nt, 17nt, 14nt, 12nt, 10nt, 8nt, 6nt (X=20nt, 17nt, 14nt, 12nt, 10nt, 8nt, 6nt), to explore the extension effect of primers of different lengths on PhiX 174DNA. In a 30 μL reaction system, 6 μL of PhiX 174 template strand, Vent(exo - ) enzyme 0.8 μL, 10×ThermoPol buffer 3 μL, diblock primer (10 μM) 2 μL, dNT mixture (2.5 mM) 2 μL, ultrapure water 16.2 μL. well mixed.

[0043] (2) Place the...

Embodiment 2

[0048] This embodiment is about the design and preparation of triangular and cross-shaped DNA origami probes, and its specific operations are:

[0049] (1) Mix M13mp18 bacteriophage circular single-stranded DNA, more than 100 unmodified staple strands, and staple strands with end-modified capture strands at a molar ratio of 1:10:10, that is, the added volumes are 2.5 μL, 5 μL, 5 μL, then 10 μL 10×TAE-Mg 2+ Buffer (Mg 2+ Concentration 12.5mol / L), supplemented with ultrapure water to a final volume of 100 μL, shake well.

[0050] (2) Place the mixed solution in step (1) in a PCR instrument and anneal at a rate of 0.1°C / 10s from 95°C to 20°C. After the reaction, use a 100kDa ultrafiltration tube to centrifuge to remove excess staple chains or pass through agar The sugar was purified by electrophoresis, concentrated and recovered using Freeze'N Squeeze DNA Gel Extraction Spin Columns, and stored at 4°C for use.

[0051] Among them, for the triA65, triB65, and triC65 end-modifie...

Embodiment 3

[0053] This example is about the specific labeling of the DNA origami probe to the same gene fragment of PhiX 174 and the high-resolution labeling at close range. The specific operations are as follows:

[0054] (1) Mix excess DNA origami probes with target DNA molecules labeled with specific diblock primers, place them in a closed foam box filled with 2L of water at 45°C, and react with natural cooling for more than 12 hours. From the extension of diblock primers to the labeling of origami probes, such as figure 2 Shown in B.

[0055] (2) The product obtained in step (1) is diluted to a certain number of times and observed under an atomic force microscope. Drop 3 μL of the sample on the newly peeled mica sheet, let it stand for adsorption for about 3 minutes, blow it dry with nitrogen gas, and observe it under the gas phase condition of multi-mode 8 AFM (NanoScope 5 controller, Bruker Company).

[0056] (3) For the close-range high-resolution markers of 3427 and 3460, 1900...

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Abstract

The invention belongs to the technical field of detection analysis and molecular biology, and discloses a nanoprobe used for specific recognition of DNA sequences, and an application method thereof. The application method comprises following steps: 1, a DNA origami probe is prepared, wherein the DNA origami probe is prepared based on Rothemund and Seeman method; 2, a PhiX 174 model based on closed circular single-chain DNA or a Lambda model of linear double chain DNA is constructed, wherein in model construction, a double segmented DNA primer composed of three parts is adopted; 3, specific marking of the DNA origami probe is carried out, wherein an excess amount of the DNA origami probe and a target DNA molecular are subjected to mixed reaction for 12h or longer; and 4, imaging observation is carried out using an atomic force microscope. The application method is capable of realizing accurate positioning of same gene segments on a single-molecular DNA, observation of corresponding sites at high resolution using an atomic force microscope can be realized, a problem that fluorescent molecular marking is limited by optical diffraction limit under electron microscopes is avoided; and the application method can be adopted in the fields such as in situ testing of chromosome genovariation and clinical genetic diagnosis.

Description

technical field [0001] The invention belongs to the technical field of detection analysis and molecular biology, and specifically relates to a triangular and cross-shaped DNA origami structure used as a probe to specifically mark a target gene of the same sequence, so as to realize high-resolution gene sequence positioning of circular and linear DNA. Background technique [0002] Genemapping is an important means in the fields of genome sequencing, medical diagnosis and identification of pathogenic bacteria, including the two aspects of locating gene fragments on chromosomes and determining the order and distance of linear arrangement of genes on chromosomes. Marking according to different morphological traits and using isozyme marking are the two main traditional gene mapping methods, which indirectly reflect the marking effect based on the results of gene expression. In recent decades, with the vigorous development of modern molecular biology technology, a new genetic mark...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6844C12Q2531/113C12Q2565/601
Inventor 汪联辉樊春海邢益康晁洁
Owner NANJING UNIV OF POSTS & TELECOMM
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