Nanoparticle single chain DNA amplification method based on streptavidin coating

A streptavidin and nanoparticle technology, applied in DNA preparation, recombinant DNA technology, etc., can solve the problems of cumbersome operation and long separation operation time, and achieve the effects of improving economy, efficient preparation, and simple and efficient action.

Inactive Publication Date: 2018-06-08
FOURTH MILITARY MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] The purpose of the present invention is to provide a single-stranded DNA amplification method based on streptavidin-coated nanoparticles, which can complete a large amount of single-stranded DNA amplification within 60-120min, and significantly improve the separation operation time in the prior art Long and cumbersome to operate, improve the efficiency of single-stranded DNA preparation

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  • Nanoparticle single chain DNA amplification method based on streptavidin coating
  • Nanoparticle single chain DNA amplification method based on streptavidin coating
  • Nanoparticle single chain DNA amplification method based on streptavidin coating

Examples

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Effect test

Embodiment 1

[0050] 1. Preparation of solid-phase primers;

[0051] Take 5 μl of 100 μM biotin-labeled primer and 100 μl streptavidin-coated magnetic nanoparticles and incubate at room temperature for 30 minutes, remove the liquid phase under the action of a magnetic separator, rinse with deionized water, and then use magnetic separation again to remove the rinse solution. Then add 5 μl deionized water to suspend for later use.

[0052] 1. Single-stranded DNA amplification system:

[0053]

[0054] Single-stranded DNA amplification reaction conditions:

[0055] According to the Tm value of the primers, the single-stranded DNA amplification reaction conditions were set according to the conventional conditions of the PCR reaction.

[0056] 3. Separation of single-stranded DNA:

[0057] After the amplification reaction is completed, use a magnetic separator to separate the solid and liquid of the reaction system, and absorb the liquid phase for storage. After adding an appropriate amo...

Embodiment 2

[0059] 1. Preparation of solid-phase primers;

[0060] Take 5 μl of 100 μM biotin-labeled primers and 100 μl of streptavidin-coated non-magnetic nanoparticles and incubate at room temperature for 30 minutes, centrifuge to remove the liquid phase, rinse with deionized water, and centrifuge again to remove the rinse solution . Then add 5 μl deionized water to suspend for later use.

[0061] 2. Single-stranded DNA amplification system:

[0062]

[0063] Single-stranded DNA amplification reaction conditions:

[0064] According to the Tm value of the primers, the single-stranded DNA amplification reaction conditions were set according to the conventional conditions of the PCR reaction.

[0065] 3. Separation of single-stranded DNA:

[0066] After the amplification reaction is completed, use a centrifuge to separate the solid and liquid of the reaction system, and absorb the liquid phase for storage. After adding an appropriate amount of deionized water to rinse the nanopart...

Embodiment 3

[0068] 1. Preparation of solid-phase primers;

[0069] Take 5 μl of 100 μM biotin-labeled primer and 100 μl streptavidin-coated magnetic nanoparticles and incubate at room temperature for 30 minutes, remove the liquid phase under the action of a magnetic separator, rinse with deionized water, and then use magnetic separation again to remove the rinse solution. Then add 5 μl deionized water to suspend for later use.

[0070] 2. Single-stranded DNA amplification system:

[0071]

[0072] Single-stranded DNA amplification reaction conditions:

[0073] According to the Tm value of the primers, the single-stranded DNA amplification reaction conditions were set according to the conventional conditions of the PCR reaction.

[0074] 3. Separation of single-stranded DNA:

[0075] After the amplification reaction is completed, use a magnetic separator to separate the solid and liquid of the reaction system, and absorb the liquid phase for storage. After adding an appropriate amo...

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Abstract

The invention relates to a nanoparticle single chain DNA amplification method based on streptavidin coating. The conventional PCR technology is suitable for double chain DNA amplification and enrichment; in single chain DNA amplification, problems that amplification products are mixed with double chain DNA, and the steps are complex are induced. According to the nanoparticle single chain DNA amplification method, nanoparticles coated by streptavidin are used for fixing biotin labeled PCR amplimers onto the surfaces of metal nanoparticles, and then single chain DNA amplification reaction is carried out. According to the nanoparticle single chain DNA amplification method, single chain DNA amplification is realized via irreversible combination of biotin with streptavidin coated nanoparticles,selective obtaining of sample DNA positive chains or inverse chain DNA can be realized, the nanoparticle single chain DNA amplification method is simple and rapid, is high in efficiency, and is capable of improving the amplification efficiency of single chain DNA obviously.

Description

technical field [0001] The invention relates to a method for amplifying single-stranded DNA, in particular to a method for amplifying single-stranded DNA based on streptavidin-coated nanoparticles. Background technique [0002] The semi-conservative replication of DNA is an important way of biological inheritance and evolution. The double-stranded DNA can be denatured and unzipped into a single strand under the action of various enzymes, and with the participation of DNA polymerase and promoter, it can be copied into the same two molecular copies according to the principle of complementary base pairing. The polymerase chain reaction (PCR) technology is a biological technology that realizes the in vitro replication of specific genes through temperature control in vitro. This technology can amplify a specific DNA fragment millions of times after a few hours of reaction in a test tube. This technology of rapidly obtaining a large number of single nucleic acid fragments is of p...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10
CPCC12N15/1006C12N15/1013C12N15/1017
Inventor 宦怡任静黄旭方文娣娣仲津漫张振华赵娓娓马婉玲
Owner FOURTH MILITARY MEDICAL UNIVERSITY
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