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Monomolecular gene typing method based on DNA paper folding probe specificity marking and application thereof

A genotyping method and specific technology, applied in the field of single-molecule genotyping based on specific markers of DNA origami probes, to achieve precise positioning, high-resolution haplotype maps, and improved accuracy

Inactive Publication Date: 2017-06-27
SHANGHAI INST OF APPLIED PHYSICS - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] The purpose of the present invention is to provide a single-molecule genotyping method and its application based on the specific labeling of DNA origami probes, so as to solve the problem that the super-resolution imaging of haplotype typing cannot be realized in the prior art

Method used

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  • Monomolecular gene typing method based on DNA paper folding probe specificity marking and application thereof
  • Monomolecular gene typing method based on DNA paper folding probe specificity marking and application thereof
  • Monomolecular gene typing method based on DNA paper folding probe specificity marking and application thereof

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Embodiment 1

[0033] According to this preferred embodiment, the design and preparation of 6 kinds of DNA origami probes are provided, and its specific operations are:

[0034] (1) Mix M13mp18 phage circular single-stranded DNA, more than 100 unmodified staple strands, and more than 100 staple strands with end-modified capture strands at a molar ratio of 1:10:10, that is, the added The volumes were 2.5 μL, 5 μL, 5 μL, and then 10 μL of 10×TAE-Mg was added 2+ Buffer (Mg 2+ Concentration 12.5mol / L), supplemented with ultrapure water to a final volume of 100 μL, shake well.

[0035] (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, Purified by agarose electrophoresis, concentrated and recovered using Freeze'N Squeeze DNA Gel Extraction Spin Columns, and stored at 4°C for use. When streptavidin (streptavidin, STA) is required ...

Embodiment 2

[0038] According to this preferred embodiment, there is provided a PhiX 174-based model constructed using "diblock" primers, the specific operations of which are as follows:

[0039] (1) figure 2 ① in ① is a single-stranded closed circular DNA with a length of 5386 bases (purchased from New England Biolabs Inc.), named PhiX 174. ② is the "double block" primer constructed, the M1 part binds to the specific site of the target DNA and effectively extends, the M3 part hybridizes with the DNA origami probe, and the M1 part and the M3 part are connected by a small spacer chain of the M2 part in the middle stand up. 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.

[0040] (2) Place the mixed solution in step (1) in a PCR instrument and start high-temperature denaturation at 92°C, anneal at 65°C to 80°C for 30 cycles of...

Embodiment 3

[0044] According to this preferred embodiment, the PhiX174 model provided in Example 2 is used for specific labeling of different sites using the DNA origami probe designed according to Example 1, and the specific operations are as follows:

[0045] (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.

[0046] (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 freshly peeled mica sheet, let it stand for about 3 minutes for adsorption, then dry it with nitrogen gas and observe it under the gas phase conditions of multi-mode 8 AFM (NanoScope 5 controller, Bruker Company), the schematic diagram is as follows figure 2 Shown in ④.

[0047] (3) After statistical analysis of the obtained AFM images, the highest label...

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Abstract

The invention provides a monomolecular gene typing method based on DNA paper folding probe specificity marking and application thereof. The method comprises the following steps: 1) preparing a DNA paper folding probe; 2) carrying out specificity marking on a target DNA through the DNA paper folding probe; 3) carrying out imaging observation on the marked target DNA under an atomic force microscope. Moreover, the monomolecular gene typing method can also be applied to haplotype typing of patient samples and haplotype typing of practical samples with unknown haplotype information. All in all, according to the method provided by the invention, single nucleotide polymorphism can be effectively detected, and the position of a single basic group is observed at high resolution under the atomic force microscope, so that the problem that fluorescent molecular marking is limited to optical diffraction limit under an electron microscope is successfully avoided, and the method can be widely applied to the research fields such as in situ test of chromogene variation and clinic gene diagnosis.

Description

technical field [0001] The invention relates to the field of detection analysis and biological application, and more specifically relates to a single-molecule genotyping method based on DNA origami probe specific labeling and its application. Background technique [0002] Single nucleotide polymorphism (single nucleotide polymorphism, SNP) refers to the increase, decrease and change of a single base on the life genome, and the frequency of occurrence in the population exceeds 1%. Except for a very small number of SNPs, most SNPs have only two alleles, so SNP typing is relatively easy. SNP is the most common genetic variation in the human genome, and approximately one polymorphic site occurs every 1000 base pairs in the human genome. Due to their wide distribution in the genome and easy typing, they have become important genetic markers in genome research. SNP genotyping (genotyping) is the process of determining the genotype (genotype) of a specific single nucleotide polym...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12Q1/6827C12Q2525/30C12Q2563/179C12Q2565/601
Inventor 樊春海晁洁张宏陆胡钧
Owner SHANGHAI INST OF APPLIED PHYSICS - CHINESE ACAD OF SCI
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