Asymmetric polymerase chain reaction technology based on nano particles

A nanoparticle and magnetic nanoparticle technology, applied in the field of asymmetric polymerase chain reaction technology, can solve the problems of lack of preparation and separation of single-stranded DNA, etc.

Inactive Publication Date: 2009-04-08
SHANGHAI NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0008] However, prior to the present invention, there were no reports of combining nanotechnology with asymmetric PCR, and the art lacked a simple and efficient method for preparing and isolating single-stranded DNA

Method used

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  • Asymmetric polymerase chain reaction technology based on nano particles
  • Asymmetric polymerase chain reaction technology based on nano particles
  • Asymmetric polymerase chain reaction technology based on nano particles

Examples

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preparation example Construction

[0053] In a preferred example, the preparation method of the PCR primer based on nanoparticles of the present invention comprises the following steps:

[0054] (1) Preparation of magnetic nanoparticles

[0055] Double distilled H2O 2 O separately prepared FeSO 4 ·7H 2 O and FeCl 3 ·6H 2 O mixed solution and NaOH solution. In a mixed solution of iron salts, Fe 2+ The ion concentration is 0.1~0.2mol / l, Fe 3+ The concentration of ions is 0.1-0.3 mol / l, and the concentration of NaOH solution is 2-3 mol / l. Under vigorous stirring, the NaOH solution whose volume is half of the volume of the mixed salt solution was slowly added dropwise into the mixed salt solution. Aging the obtained solid precipitate at 40°C to 60°C for 12 hours, washing the precipitate several times with twice distilled water, filtering and drying at 40°C to 80°C for 24 hours, grinding it in an agate mortar That is the product.

[0056] (2) Silica in γ-Fe 2 o 3 surface modification

[0057] Mix Triton...

Embodiment 1

[0077] Preparation method of inner core layer of magnetic nanoparticles

[0078] The core layer of magnetic particles was prepared by improved chemical co-precipitation, the specific method is as follows: prepare FeSO 4 ·7H 2 O and FeCl 3 ·6H 2 O mixed solution and NaOH solution. In a mixed solution of iron salts, Fe 2+ The ion concentration is 0.1~0.2mol / l, Fe 3+ The concentration of ions is 0.1-0.3 mol / l, and the concentration of NaOH solution is 2-3 mol / l. Under vigorous stirring, the NaOH solution whose volume is half of the volume of the mixed salt solution was slowly added dropwise into the mixed salt solution. Aging the obtained solid precipitate at 40°C to 60°C for 12 hours, washing the precipitate several times with double distilled water, filtering and drying at 40°C to 80°C for 24 hours, and grinding it in an agate mortar Get the product.

Embodiment 2

[0080] Preparation method of core-shell nucleic acid magnetic nanoparticles

[0081] Mix TritonX-100, n-hexanol and cyclohexane uniformly at a ratio of 1:2:5 to form a transparent and stable microemulsion system. Put the microemulsion system above into ultrasonic treatment for 30-60 minutes, then add 0.5g of γ-Fe 2 o 3After 6 minutes of ultrasonic treatment, the supernatant was taken out and poured into a three-necked flask, and stirred for 30 minutes to make it uniform. Take 1ml of a certain concentration of concentrated ammonia water and dilute it with 2ml of double distilled water, slowly add it into the microemulsion that is constantly stirring after 30 minutes, and keep stirring for 30 minutes to make the ammonia water evenly dispersed in the microemulsion. After 1 hour, 1-3 ml of tetraethyl orthosilicate was added dropwise to the microemulsion, while stirring continuously for 10 hours, and the temperature of the system was kept between 15-30°C. Add acetone to the sy...

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Abstract

The present invention relates to one kind of asymmetrical PCR technology based on nanometer particles. The nonrestriction primer adopted with PCR primer based on nanometer particle in the structure of Z-(X)n, where X expresses single stranded oligonucleotide primer of 10-100 bp length, Z expresses solid particles with average size of 1-100 nm, '-' expresses the covalent bond between X and Z, and n is integral number of 1-1000. The present invention also relates to PCR based on nanometer particles. The present invention makes it possible to prepare and separate single stranded DNA proliferation product simply, fast and effectively.

Description

technical field [0001] The invention relates to the field of biotechnology, more specifically to a nanoparticle-based asymmetric polymerase chain reaction (PCR) technology. The invention also relates to the preparation of primer sets for this asymmetric PCR. Background technique [0002] Polymerase chain reaction (PCR) is an extremely important technique in the field of molecular biology technology. It can pass a very small number of target sequences in a sample through multiple "annealing-extension-denaturation" cycles (usually 20-50 Cycle), so that the number of target sequences increases exponentially, and finally millions or even more target sequences are obtained, which is convenient for detection and operation. [0003] However, the separation of PCR amplification products is basically performed by electrophoresis-gel tapping at present, and there is a lack of simple and effective methods for separating PCR amplification products. [0004] As we all know, the field o...

Claims

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Application Information

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IPC IPC(8): C12N15/00C12P19/34
Inventor 沈鹤柏周海清朱龙章
Owner SHANGHAI NORMAL UNIVERSITY
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