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Fast DNA Extraction with Polyacrylamide Microspheres for Polymerase Chain Reaction Detection
Jun Wang1, Shuyan Zhang1, Xinhui Xu1
1State Key Laboratory of Bioelectronics, Southeast University, Nanjing 210096, China.
ACS Omega
|June 23, 2020
Summary
We developed fluorescent polyacrylamide microspheres (fPAMMPs) for rapid DNA extraction in 3-5 minutes. These microspheres enable effective DNA capture and subsequent PCR detection across diverse biological samples.
Area of Science:
- Biomaterials Science
- Molecular Biology
- Analytical Chemistry
Background:
- Fast and cost-effective DNA extraction is crucial for DNA-based detection methods.
- Existing DNA extraction techniques can be time-consuming and expensive.
- Development of novel materials for efficient biomolecule isolation is an ongoing research area.
Purpose of the Study:
- To fabricate novel polyacrylamide microspheres (PAMMPs) with autofluorescence properties.
- To develop a rapid and efficient DNA extraction method using these fluorescent PAMMPs (fPAMMPs).
- To demonstrate the utility of fPAMMPs for subsequent Polymerase Chain Reaction (PCR) detection.
Main Methods:
- Fabrication of polyacrylamide microspheres (PAMMPs) using spot polymerization and modified inverse microemulsion polymerization.
- Characterization of PAMMPs' autofluorescence (visible and near-infrared) and stability under harsh conditions (acid, alkali, high temperature).
- Assessment of fPAMMPs' positive charge for capturing biomolecules like DNA and streptavidin.
- Development and validation of a rapid DNA extraction protocol (3-5 min) from various sample types.
- Evaluation of DNA captured on fPAMMPs for PCR amplification (normal and quantitative).
- Investigation of NaBH4 treatment to remove autofluorescence for DNA extraction and PCR.
Main Results:
- Successfully fabricated PAMMPs in various sizes, with fluorescent variants (fPAMMPs) exhibiting stable visible and near-infrared autofluorescence.
- fPAMMPs demonstrated strong positive charge, enabling effective capture of DNA and labeled streptavidin.
- Developed a rapid DNA extraction method (3-5 min) applicable to bacteria, mammalian cells, plant/animal tissues, and human plasma.
- DNA extracted using fPAMMPs was successfully amplified by both normal and quantitative PCR.
- NaBH4 treatment effectively removed autofluorescence, yielding PAMMPs suitable for DNA extraction and PCR.
Conclusions:
- New fluorescent polyacrylamide microspheres (fPAMMPs) and non-fluorescent PAMMPs were fabricated.
- A novel, rapid DNA extraction method utilizing fPAMMPs was developed and validated.
- The fabricated microspheres and the developed method show significant potential for efficient DNA extraction and downstream PCR detection across diverse biological samples.
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