Analytical Methods
·
2024
·Vol. 16
·期 7
·1003-1011
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IF 2.7
Yongwei Fang,Chuang Wang,Yuli Wang,Jiandong Hu,Miao Lai,Pengfei Liu,Hong Zhang
Plant viral diseases can seriously affect the yield and quality of crops. In this work, a convenient and highly sensitive biosensor for the visual detection of plant viral disease is proposed by the PCR-induced generation of DNAzyme. In the absence of nucleic acid for a target plant virus, the primers prohibited the production of G-quadruplex by forming a hairpin structure. However, PCR amplification occurred and generated a number of specific PCR products with free G-quadruplex sequences at both ends in the presence of the target cDNA. A catalytically active G-quadruplex DNAzyme was formed with the help of K+ and hemin, resulting in the formation of colored products visible to the naked eye and a strong absorbance by the addition of ABTS2− and H2O2. The absorbance and the logarithm of target cDNA concentrations showed a good linear relationship in the range of 10 fM–1.0 nM, with a linear regression equation of A = 0.1402 lg c + 0.3761 (c: fM) and a detection limit of 0.19 fM. This method was successfully applied to the analysis of emerging tobacco mosaic virus (TMV) infections in tobacco leaf samples collected in the field due to its flexibility and convenience, indicating a potential application for the early detection of plant viral disease.
研究领域:
植物病理学
·
分子生物学
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DOI
PubMed
高影响因子
Biomaterials Science
·
2017
·Vol. 5
·期 7
·1279-1292
·
IF 4.21
Yawen Liu,Zhaozhu Zheng,He Gong,Meng Liu,Shaozhe Guo,Gang Li,Xiaoqin Wang,David L. Kaplan
The structure of DNA is susceptible to alterations at high temperature and on changing pH, irradiation and exposure to DNase. Options to protect and preserve DNA during storage are important for applications in genetic diagnosis, identity authentication, drug development and bioresearch. In the present study, the stability of total DNA purified from human dermal fibroblast cells, as well as that of plasmid DNA, was studied in silk protein materials. The DNA/silk mixtures were stabilized on filter paper (silk/DNA + filter) or filter paper pre-coated with silk and treated with methanol (silk/DNA + PT-filter) as a route to practical utility. After air-drying and water extraction, 50–70% of the DNA and silk could be retrieved and showed a single band on electrophoretic gels. 6% silk/DNA + PT-filter samples provided improved stability in comparison with 3% silk/DNA + filter samples and DNA + filter samples for DNA preservation, with ∼40% of the band intensity remaining at 37 °C after 40 days and ∼10% after exposure to UV light for 10 hours. Quantitative analysis using the PicoGreen assay confirmed the results. The use of Tris/borate/EDTA (TBE) buffer enhanced the preservation and/or extraction of the DNA. The DNA extracted after storage maintained integrity and function based on serving as a functional template for PCR amplification of the gene for zinc finger protein 750 (ZNF750) and for transgene expression of red fluorescence protein (dsRed) in HEK293 cells. The high molecular weight and high content of a crystalline beta-sheet structure formed on the coated surfaces likely accounted for the preservation effects observed for the silk/DNA + PT-filter samples. Although similar preservation effects were also obtained for lyophilized silk/DNA samples, the rapid and simple processing available with the silk–DNA–filter membrane system makes it appealing for future applications.
研究领域:
遗传学
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生物技术
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材料科学
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DOI
PubMed
高影响因子
TALANTA
·
2023
·
IF 6.1
Huizhen Li,Yunhui Li,Cenlin Gui,Daqi Chen,Lanfang Chen,Le Luo,Guobao Huang,Yang Yuan,Rong He,Fan Xia,Jiahai Wang
In this study, it is confirmed that without addition of organic solvent and embedding polymer hydrogel into glass nanopore, bare glass nanopore can faithfully measure various lengths of DNA duplexes from 200 to 3000 base pairs with 200 base pairs resolution, showing well-separated peak amplitudes of blockage currents. Furthermore, motivated by this readout capability of duplex DNA, amplicons from Polymerase Chain Reaction (PCR) amplification are straightforwardly discriminated by bare glassy nanopore without fluorescent labeling . Except simultaneous discrimination of up to 7 different segments of the same lambda genome, various pathogenic bacteria and viruses including SARS-CoV-2 and its mutants in clinical samples can be discriminated at high resolution. Moreover, quantitative measurement of PCR amplicons is obtained with detection range spanning from 0.75 aM to 7.5 pM and detection limit of 7.5 aM, which reveals that bare glass nanopore can faithfully disclose PCR results without any extra labeling.
研究领域:
生物物理学
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分子生物学
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医学诊断
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DOI
PubMed
高影响因子
ANALYST
·
2023
·Vol. 148
·期 7
·1492-1499
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IF 4.2
Jiahai Wang,Lanfang Chen,Cenlin Gui,Jianji Zhu,Baian Zhu,Zhuobin Zhu,Yunhui Li,Daqi Chen
DNA methylation has been considered an essential epigenetic biomarker for diagnosing various diseases, such as cancer. A simple and sensitive way for DNA methylation level detection is necessary. Inspired by the label-free and ultra-high sensitivity of solid-state nanopores to double-stranded DNA (dsDNA), we proposed a nanopore counter for evaluating DNA methylation by integrating a dual-restriction endonuclease digestion strategy coupled with polymerase chain reaction (PCR) amplification. Simultaneous application of BstUI/HhaI endonucleases can ensure the full digestion of the unmethylated target DNA but shows no effect on the methylated ones. Therefore, only the methylated DNA remains intact and can trigger the subsequent PCR reaction, producing a large quantity of fixed-length PCR amplicons, which can be directly detected through glassy nanopores. By simply counting the event rate of the translocation signals, the concentration of methylated DNA can be determined to range from 1 aM to 0.1 nM, with the detection limit as low as 0.61 aM. Moreover, a 0.01% DNA methylation level was successfully distinguished. The strategy of using the nanopore counter for highly sensitive DNA methylation evaluation would be a low-cost but reliable alternative in the analysis of DNA methylation.
研究领域:
生物化学
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分子生物学
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医学诊断
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DOI
PubMed
高影响因子
ACS Omega
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2021
·
IF 3.512
Zili Song,Ruilin Zhao,Hongjiao Zhang,Penglin Wei,Landa Qi,Guocan Chen,Wen-Bing Yin,Wei Li
Edible mushrooms are important nutraceutical sources of foods and drugs, which can produce various nutritional ingredients including all essential amino acids. The method of rapid screening for the strains producing specific functional components is very indispensable. Homocitrate synthase is one of the key enzymes in the α-aminoadipate pathway for lysine biosynthesis and has preferable sequence conservation in Agaricales. Based on the blast of homocitrate synthase homologous genes of strains of Agaricales, we achieved combinations of degenerate primers as molecular markers to rapidly screen the lysine-producing edible mushrooms. The experimental results revealed that the consistency between PCR amplification and HPLC analysis attained 82 and 75% in strains of Agaricales and Polyporales, respectively. The finding showed that the molecular marker has higher universality for screening edible mushroom resources of Agaricales. This PCR-based approach shows excellent potential in evaluating and discriminating edible wild-grown mushrooms with high lysine content in Agaricales.
研究领域:
真菌学
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分子生物学
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食品科学
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DOI
PubMed
高影响因子
RSC Advances
·
2021
·Vol. 11
·期 6
·3324-3332
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IF 3.361
Jia Xu,Dan Chen,Yuan Yang,Hongjian Gong,Wenqi Gao,Han Xiao
A simple and efficient approach for the rapid extraction of genomic DNA from blood using various amino-modified magnetic nanoparticles (AMNPs) has been described. The salmon sperm DNA was isolated from aqueous solution based on electrostatic interaction between the positively charged amino-groups of AMNPs and the negatively charged phosphate groups of the DNA. The results of ultraviolet-visible (UV-Vis) spectrometry showed that increasing number of amino groups on the AMNPs surface resulted in an improvement in DNA adsorption efficiency. Several variables including the extraction pH, adsorption time, ionic strength and quantity of AMNPs were optimized to achieve the best extraction efficiency with the proposed method. Acceptable adsorption efficiency of 92% and recovery of 91% were achieved using multi-amino modified MNPs (mAMNPs) with an extraction time of 10 min and an overall processing time of 30 min. The mAMNPs enabled genomic DNA capture from human whole blood, and the resulting mAMNP/DNA complexes could be directly used as templates for PCR amplification without the need for complex and time-consuming DNA elution and purification steps. Our results imply that this method can be used as an effective strategy for genomic DNA extraction and may be extended to other types of biological samples.
研究领域:
分子生物学
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纳米技术
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生物技术
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DOI
PubMed
高影响因子
CHEMICAL ENGINEERING JOURNAL
·
2022
·
IF 16.744
Xinglu Jiang,Kangjing Li,Beibei Xie,Jun Zhou
Taq DNA polymerase is the most widely used enzyme for PCR amplification and it contains 832 amino acids but no cysteines. Bovine serum albumin (BSA) is an 832-amino acid protein containing 35 cysteines, and at high temperature BSA can produce protein hydrogels through recombined disulfides and noncovalent bonds. When hydrogels are prepared using a mixture of Taq DNA polymerase and BSA, no disulfide bonds occur between them, and noncovalent bonds are the main forces holding together the Taq DNA polymerase with the hydrogels. During PCR amplification, temperature cycling can destroy the noncovalent bonds, thereby releasing Taq DNA polymerase from the hydrogels to synthesize DNA. The release rate, regulated through the variation of BSA concentration, allows for velocity-controlled amplification of DNA. As the amount of DNA products exceeds that of the Taq DNA polymerase released from hydrogels, a linear amplification replaces the exponential PCR amplification. When the amplification velocity begins to decrease, the time needed for reaching a plateau increase. This provides a broader detection window for the analysis of the end products, which correspond to the difference compared with the initial DNA concentration. The number of primers is continuously reduced with increasing cycles, which further causes a decline in the DNA amplification efficiency. The difference in the initial DNA template may cause a different consumption of primers, which can cause errors in the Ct values in the case of qPCR. Based on the slopes of the linear amplification curve, the Ct values can be calibrated to reduce this error, and a more accurate detection of the initial content of the target DNA can be implemented.
研究领域:
分子生物学
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生物技术
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生物化学
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DOI
高影响因子
ANALYTICA CHIMICA ACTA
·
2021
·
IF 6.558
Feixia Peng,Mei Liu,Xuelian Wang,Xueqin Ding
Fast extraction of high-purity nucleic acid from complex biological sample is the key to downstream nucleic acid analysis. In this work, two low-viscosity hydrophobic magnetic deep eutectic solvents (HMDESs) were synthesized for the selective extraction of DNA . The conformation of the HMDES was simulated by density functional theory (DFT) calculation. Characterization of HMDESs’ physical (magnetism, density, viscosity, and hydrophobicity) properties and thermal (melting point and decomposition temperature) properties were conducted. Based on the HMDESs, a vortex-assisted liquid-liquid micro-extraction (VALLME) DNA method was developed. Single stranded DNA that was extracted by HMDESs could be quickly collected by an external magnet. Factors that could impact the DNA extraction process, such as HMDESs volume, temperature, extraction time, and pH were systematically investigated via single-factor experimental analysis. Under the optimized condition, the proposed extraction method has been demonstrated with the extraction of DNA from a series of complex sample matrices, including metal mixture, protein mixture and E. coli cell lysate. The DNA extracted by using HMDES-based VALLME method was well suitable for PCR amplifications. After extraction, the retained DNAs could be readily recovered by simply using Britton-Robison (BR) buffer. In addition, the interaction and corresponding binding sites between HMDESs and DNA were investigated by FT-IR and DFT calculation. This work provides a new green magnetic solvent and a rapid and environmental-friendly extraction method for the enrichment of DNA and other biological macromolecules .
研究领域:
分析化学
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分子生物学
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生物技术
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纳米技术
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DOI
PubMed
高影响因子
RSC Advances
·
2019
·Vol. 9
·期 40
·23119-23128
·
IF 3.049
Mei Liu,Xueqin Ding,Xuelian Wang,Jianzhong Li,Huansheng Yang,Yulong Yin
A series of guanidinium ionic liquid modified magnetic chitosan/graphene oxide (GIL-MCGO) nanocomposites have been prepared for DNA extraction via magnetic solid-phase extraction technology. These nanocomposites are of only 20 nanometers in diameter. Single stranded DNA or DNA sodium salts that were absorbed by GIL-MCGO could be quickly collected by an external magnet and extracted. The DNA extraction efficiency of 11 GIL-MCGO nanocomposites was evaluated using NanoDrop. Factors that could impact the DNA extraction process, such as pH, temperature, extraction time, and ionic strength were systematically investigated via single-factor experimental analysis. Under the optimum extraction conditions, a maximum DNA extraction capacity of 233.0 ± 0.4 mg g−1 of GIL-MCGO nanocomposite was achieved. The solid phase extraction method based on GIL-MCGO nanocomposites has been demonstrated with the extraction of DNA from a series of complex sample matrices, including single stranded DNA samples, salmon sperm DNA sodium salt, human whole blood and E. coli cell lysate. The DNA extracted by using the GIL-MCGO nanocomposites are well suitable for PCR amplifications. In addition, an initial study on the interaction between GIL-MCGO and DNA was conducted: the preference of GIL-MCGO on DNA absorption with varying base composition was tested. Only a slight loss in the DNA extraction efficiency of GIL-MCGO was observed after four extraction–desorption cycles, proving excellent regeneration performance and recyclability of the GIL-MCGO nanocomposites in the DNA extraction process.
研究领域:
生物技术
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纳米技术
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分子生物学
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分析化学
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DOI
PubMed
高影响因子
危险品化学品经营许可证（带存储）