Dataset Information

Fast DNA Extraction with Polyacrylamide Microspheres for Polymerase Chain Reaction Detection.

ABSTRACT: The fast and cost-effective DNA extraction is critical for all DNA-based detections. Here, we fabricated a new kind of polyacrylamide microsphere (PAMMP) in various sizes with two methods, spot polymerization (large size but low yield) and modified inverse microemulsion polymerization (small size but high yield). The fabricated PAMMPs have strong autofluorescence (fPAMMPs), including both visible fluorescence (VF) and near-infrared fluorescence (NIRF), which can remain very stable in various stringent conditions including strong acid and alkali and high temperature. The fabricated fPAMMPs were also highly positively charged, which could be used to effectively capture various biomolecules such as IRDye 800-labeled streptavidin and DNA. We thus developed a new method for rapid extraction (3-5 min) of DNA from various samples including bacteria, mammalian cells, plant and animal solid tissues, and human blood plasma using fPAMMPs. Moreover, the DNA captured on fPAMMPs (fPAMMP@DNA) could be effectively detected by both normal and quantitative PCR amplifications. Finally, we showed that NaBH₄ treatment removed autofluorescence in fPAMMPs (PAMMPs), which could also be applied to DNA extraction and PCR detection. In conclusion, we here fabricated new kinds of fPAMMPs and PAMMPs, developed a new rapid DNA extraction method, and demonstrated their useful applications in PCR detection.

SUBMITTER: Wang J

PROVIDER: S-EPMC7301550 | biostudies-literature | 2020 Jun

REPOSITORIES: biostudies-literature

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Fast DNA Extraction with Polyacrylamide Microspheres for Polymerase Chain Reaction Detection.

Wang Jun J Zhang Shuyan S Xu Xinhui X Xing Yujun Y Li Zongru Z Wang Jinke J

ACS omega 20200604 23

The fast and cost-effective DNA extraction is critical for all DNA-based detections. Here, we fabricated a new kind of polyacrylamide microsphere (PAMMP) in various sizes with two methods, spot polymerization (large size but low yield) and modified inverse microemulsion polymerization (small size but high yield). The fabricated PAMMPs have strong autofluorescence (fPAMMPs), including both visible fluorescence (VF) and near-infrared fluorescence (NIRF), which can remain very stable in various str ...[more]

PMID: 32566849

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Project description:Polymerase chain reaction (PCR) is an in vitro technology in molecular genetics that progressively amplifies minimal copies of short DNA sequences in a fast and inexpensive manner. However, PCR performance is sensitive to suboptimal processing conditions. Compromised PCR conditions lead to artifacts and bias that downgrade the discriminatory power and reproducibility of the results. Promising attempts to resolve the PCR performance optimization issue have been guided by quality improvement tactics adopted in the past for industrial trials. Thus, orthogonal arrays (OAs) have been employed to program quick-and-easy structured experiments. Profiling of influences facilitates the quantification of effects that may counteract the detectability of amplified DNA fragments. Nevertheless, the attractive feature of reducing greatly the amount of work and expenditures by planning trials with saturated-unreplicated OA schemes is known to be relinquished in the subsequent analysis phase. This is because of an inherent incompatibility of ordinary multi-factorial comparison techniques to convert small yet dense datasets. Treating unreplicated-saturated data with either the analysis of variance (ANOVA) or regression models destroys the information extraction process. Both of those mentioned approaches are rendered blind to error since the examined effects absorb all available degrees of freedom. Therefore, in lack of approximating an experimental uncertainty, any outcome interpretation is rendered subjective. We propose a profiling method that permits the non-linear maximization of amplicon resolution by eliminating the necessity for direct error estimation. Our approach is distribution-free, calibration-free, simulation-free and sparsity-free with well-known power properties. It is also user-friendly by promoting rudimentary analytics. Testing our method on published amplicon count data, we found that the preponderant effect is the concentration of MgCl2 (p<0.05) followed by the primer content (p<0.1) whilst the effects due to either the content of the deoxynucleotide (dNTP) or DNA remained dormant (p>0.1). Comparison of the proposed method with other stochastic approaches is also discussed. Our technique is expected to have extensive applications in genetics and biotechnology where there is a demand for cheap, expedient, and robust information.

Dataset Information

Fast DNA Extraction with Polyacrylamide Microspheres for Polymerase Chain Reaction Detection.

Publications

Fast DNA Extraction with Polyacrylamide Microspheres for Polymerase Chain Reaction Detection.

Similar Datasets

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