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A kind of polyacrylamide microsphere for rapid extraction of nucleic acid and its preparation method and application

A technology of polyacrylamide microspheres and polyacrylamide, which is applied in the field of nucleic acid extraction and detection of biology, and can solve the problems of inability to improve machine automation, low DNA absorption and elution efficiency, etc.

Active Publication Date: 2021-07-27
SOUTHEAST UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although this is an equipment-free nucleic acid extraction dipstick method that is simple, rapid, and inexpensive, its low DNA uptake and elution efficiency may challenge its wide application
Also, unless multiplex PCR is used, one DNA adsorption strip can only be used to detect one target
Furthermore, the method cannot be improved for machine automation

Method used

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  • A kind of polyacrylamide microsphere for rapid extraction of nucleic acid and its preparation method and application
  • A kind of polyacrylamide microsphere for rapid extraction of nucleic acid and its preparation method and application
  • A kind of polyacrylamide microsphere for rapid extraction of nucleic acid and its preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Synthesis and characterization of embodiment 1 fPAMMP

[0053] method:

[0054] 1. Materials: acrylamide (AM), N-(3-aminopropyl)methacrylamide hydrochloride (APMA) were purchased from SigmaAldrich (USA). Glutaraldehyde (GTA), Span 80 and NaOH were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Mineral oil, ammonium persulfate (APS), N,N,N',N'-tetramethylethane-1,2-diamine (TEMED) and N,N'-methylenebisacrylamide (MBA) Purchased from Biosharp (Hefei, China). Polylysine (ε-PL) was purchased from Shanghai Shifeng Biotechnology Co., Ltd. (Shanghai, China). 2×Premix PrimerStar HS (product number: R040A) was purchased from Takara (Dalian). qPCR Master Mix (2x) was purchased from Promega. Oligonucleotides were produced by Sangon Biotech.

[0055] 2. Synthesis of fPAMMP by spot polymerization: The preparation scheme of PAMMP is as follows: 264mg AM, 80mg MBA and 12mg APMA were dissolved in 1mL deionized (DI) water under ultrasonic to obtain a unifo...

Embodiment 2

[0062] Example 2 The ability of fPAMMP to capture DNA

[0063] method:

[0064] Mix 2 μg of purified SiHa cell genomic DNA with 80 μL of fPAMMP aqueous solution and invert several times. fPAMMP was then washed to remove excess gDNA. The fPAMMP@SiHa DNA was detected by agarose gel electrophoresis.

[0065] result:

[0066] In order to develop a fast and simple DNA extraction method, the present invention first investigated the ability of fPAMMP to bind to DNA. Genomic DNA from SiHa cells was mixed with fPAMMP and inverted several times. fPAMMP was then washed to remove excess gDNA. Gel electrophoresis results show that fPAMMP can effectively bind or at least capture DNA within seconds due to its positive charge ( Figure 7 A, a). Negatively charged DNA binds to fPAMMP through electrostatic interactions. Due to the microscale size of fPAMMP, it cannot pass through the gel, so the positive results of DNA staining are shown in the gel wells, indicating that the DNA is firml...

Embodiment 3

[0067] Example 3 Carry out DNA extraction and direct PCR amplification with fPAMMP

[0068] method:

[0069] 1. DNA extraction with fPAMMP

[0070] Bacterial DNA extraction: Escherichia coli BL21 and DH5α in glycerol stocks were streaked onto Luria Bertani (LB) agar plates and incubated at 37°C for 16 hours, respectively. A single colony of Escherichia coli that was well separated was inoculated into a test tube containing 2 mL of LB liquid medium, and incubated at 37 °C for 8 hours with vigorous shaking at 220 rpm. The culture was then centrifuged at 12000 rpm (5415D, Eppendorf) for 5 minutes and the supernatant was discarded. Resuspend the obtained pellet in 200 µL of deionized water and mix with an equal volume of 0.4 M NaOH. Cells were lysed by gentle inversion several times. Then 100 μL fPAMMP aqueous solution was added to the mixture and incubated in a rotator at room temperature for 5-30 minutes. The fPAMMP (fPAMMP@DNA) that had captured bacterial genomic DNA was c...

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Abstract

The invention discloses a polyacrylamide microsphere for rapid nucleic acid extraction and its preparation method and application. The polyacrylamide microsphere is mainly composed of acrylamide, methylenebisacrylamide, N-(3-aminopropyl) Composed of methacrylamide, ammonium persulfate and polylysine, and further cross-linked by glutaraldehyde. The present invention prepares polyacrylamide microspheres of various sizes through spot polymerization and improved inverse microemulsion polymerization. It not only has visible fluorescence and near-infrared fluorescence, but also can withstand various harsh conditions such as strong acid, strong alkali and high temperature. It is morphologically stable and has a high positive charge, which can be used to efficiently capture nucleic acid molecules such as DNA. The present invention also provides a new method for rapidly extracting DNA (3 to 5 minutes) from various biological samples using polyacrylamide microspheres. The polyacrylamide microspheres adsorbed with DNA can be directly added to the PCR reaction for DNA molecular routine and quantitative PCR amplification detection.

Description

technical field [0001] The invention belongs to the field of nucleic acid extraction and detection biotechnology, and relates to a new technology for rapid DNA extraction and PCR detection based on polyacrylamide microspheres and its application, in particular to a polyacrylamide microsphere for rapid nucleic acid extraction and its application. Preparation methods and applications. Background technique [0002] Polymerase chain reaction (PCR) amplification is a powerful tool for DNA detection and has been widely used in various fields such as basic biological research, medical diagnosis, forensic science, and agricultural science. In these applications, DNA extraction is unavoidable for PCR detection. However, purification of DNA from samples is a complex and rate-limiting task, requiring highly trained technicians and involving many processing steps. In addition, some very specialized materials (such as filter tubes) and reagents (such as complex component lysis, binding...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C08F283/04C08F220/56C08F222/38C08F220/60C08F2/32C08J3/24C12N15/10C08L33/26C08L77/04
CPCC08F2/32C08F283/04C08J3/246C08J2333/26C08J2377/04C12N15/1006C08F220/56
Inventor 王进科王军
Owner SOUTHEAST UNIV
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