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Schematic illustrating the a static and b dispersive one-step lysis and DNA extraction method using MILs
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![DNA extracted by placing 6 µL of (blue) [P6,6,6,14⁺][Ni(hfacac)3⁻],...](publication/359445276/figure/fig2/AS:1137352466530305@1648177367281/DNA-extracted-by-placing-6-L-of-blue-P6-6-6-14Nihfacac3-green_Q320.jpg)
![Amount of DNA recovered from the (green) [P6,6,6,14⁺][Ni(hfacac)3⁻] and...](publication/359445276/figure/fig3/AS:1137352466542598@1648177367304/Amount-of-DNA-recovered-from-the-green-P6-6-6-14Nihfacac3-and-blue_Q320.jpg)
Background
Plant DNA isolation and purification is a time-consuming and laborious process relative to epithelial and viral DNA sample preparation due to the cell wall. The lysis of plant cells to free intracellular DNA normally requires high temperatures, chemical surfactants, and mechanical separation of plant tissue prior to a DNA purification st...
Citations
... In 2022, Emaus et al. integrated hydrophobic ILs and MILs into a single step plant cell lysis and DNA extraction method resulting in significantly reduced extraction times. This study demonstrated that plant cells can be simultaneously lysed and DNA extracted by ILs and MILs alone without the need of elevated temperatures or chemical surfactants which can be inhibitory for enzymatic amplification assays [22]. In 2023, De Silva et al. developed a miniaturized vortex-assisted matrix solid phase dispersion approach by integrating an IL and a MIL to extract genomic DNA from plant tissue fragments of the model plant, Arabidopsis thaliana (L.) Heynh [23]. ...
... MIL as extraction solvents is based on previous studies where they have been used to successfully extract DNA from plant tissues and proven to be compatible with qPCR[15,22,23]. ...
Background
There is a growing demand for fast and reliable plant biomolecular analyses. DNA extraction is the major bottleneck in plant nucleic acid-based applications especially due to the complexity of tissues in different plant species. Conventional methods for plant cell lysis and DNA extraction typically require extensive sample preparation processes and large quantities of sample and chemicals, elevated temperatures, and multiple sample transfer steps which pose challenges for high throughput applications.
Results
In a prior investigation, an ionic liquid (IL)-based modified vortex-assisted matrix solid phase dispersion approach was developed using the model plant, Arabidopsis thaliana (L.) Heynh. Building upon this foundational study, the present study established a simple, rapid and efficient protocol for DNA extraction from milligram fragments of plant tissue representing a diverse range of taxa from the plant Tree of Life including 13 dicots and 4 monocots. Notably, the approach was successful in extracting DNA from a century old herbarium sample. The isolated DNA was of sufficient quality and quantity for sensitive molecular analyses such as qPCR. Two plant DNA barcoding markers, the plastid rbcL and nuclear ribosomal internal transcribed spacer (nrITS) regions were selected for DNA amplification and Sanger sequencing was conducted on PCR products of a representative dicot and monocot species. Successful qPCR amplification of the extracted DNA up to 3 weeks demonstrated that the DNA extracted using this approach remains stable at room temperature for an extended time period prior to downstream analysis.
Conclusions
The method presented here is a rapid and simple approach enabling cell lysis and DNA extraction from 1.5 mg of plant tissue across a broad range of plant taxa. Additional purification prior to DNA amplification is not required due to the compatibility of the extraction solvents with qPCR. The method has tremendous potential for applications in plant biology that require DNA, including barcoding methods for agriculture, conservation, ecology, evolution, and forensics.
... Plant biology poses an additional challenge in the isolation of high quality molecular weight DNA, a prerequisite for genomic library preparation. Presence of rigid cell walls in plant cells further makes the isolation of high quality and high molecular-weight DNA troublesome (Emaus et al., 2022). Plant cells also contain polysaccharides that often co-purify with DNA. ...
... Several methods have been proposed to overcome the difficulties when isolating DNA from tree species. Broadly, there are five main types of DNA isolation systems including organic extraction methods which use organic solvents like phenol and chloroform [26,27], solid-phase extraction methods that use solid matrices, such as silica to bind and purify the DNA [28], precipitation methods which use salts and ethanol to precipitate the DNA [29], enzymatic digestion methods that use individual or a combination of enzymes to digest the samples and release the DNA [30] and use of magnetic bead-based coated with an agent which binds with DNA and isolates it from the cellular suspension [31], or magnetic ionic liquids [32]. These methods are usually combined and modified depending on the experimenter's needs [17,33]. ...
Background
Genetic and genomic studies are seeing an increase in sample sizes together with a wider range of species investigated in response to environmental change concerns. In turn, these changes may come with challenges including the time and difficulty to isolate nucleic acids (DNA or RNA), the sequencing cost and environmental impacts of the growing amount of plastic waste generated in the process. Pseudotsuga menziesii var. menziesii (Mirbel) Franco (PM), Tsuga heterophylla (Raf.) Sarg. (TH) and Thuja plicata Donn ex D.Don (TP) are conifer species found in diverse woodlands both as natives and naturalized exotics. Our study was carried out whilst investigating their genetics to understand their population structure and potential for adaptation.
Results
In the present study, we compared two different DNA isolation methods, i.e., spin-column DNeasy plant mini kit (QIAGEN), and temperature-driven enzymatic cocktail Plant DNA Extraction (MicroGEM). The quantity of recovered DNA and the quality of DNA were assessed along with the plastic footprint and time needed for three tree species. Both methods were optimised and proven to provide enough DNA for each studied species. The yield of DNA for each method depended on the species: QIAGEN showed higher yield in P. menziesii and T. heterophylla, while T. plicata recovered similar amount of DNA for both methods. The DNA quality was investigated using DNA barcoding techniques by confirming species identity and species discrimination. No difference was detected in the PCR amplification of the two barcoding loci, (rbcL and trnH-psbA), and the recovered sequences between DNA isolation methods. Measurement of the plastic use and the processing time per sample indicated that MicroGEM had a 52.64% lower plastic footprint and was 51.8% faster than QIAGEN.
Conclusions
QIAGEN gave higher yields in two of the species although both methods showed similar quality results across all species. However, MicroGEM was clearly advantageous to decrease the plastic footprint and improve the time efficiency. Overall, MicroGEM recovers sufficient and reliable DNA to perform common downstream analyses such as PCR and sequencing. Our findings illustrate the benefits of research and efforts towards developing more sustainable methods and techniques to reduce the environmental footprint of molecular analyses.
... Recently, ionic liquids (ILs) and magnetic ionic liquids (MIL) have been explored as novel solvents for extracting DNA from complex biological matrices [54]. ILs and MILs promote DNA extraction through electrostatic interactions between cationic and negatively charged phosphate backbones, and hydrophobic interactions between the alkyl chains of the solvent and DNA bases [55,56]. Microscale electroporation is an emerging technology for the release of intracellular materials [57]. ...
Genetically modified (GM) maize is one of the earliest GM crops to have achieved large-scale commercial cultivation globally, and it is of great significance to excel in the development and implementation of safety policy regarding GM, and in its technical oversight. This article describes the general situation regarding genetically modified maize, including its varieties, applications, relevant laws and regulations, and so on. From a technical point of view, we summarize and critically analyze the existing methods for detecting nucleic acid levels in genetically modified maize. The nucleic acid extraction technology used for maize is explained, and the introduction of traditional detection techniques, which cover variable-temperature and isothermal amplification detection technology and gene chip technology, applications in maize are described. Moreover, new technologies are proposed, with special attention paid to nucleic acid detection methods using sensors. Finally, we review the current limitations and challenges of GM maize nucleic acid testing and share our vision for the future direction of this field.
... The panel of 143 inbred lines was genotyped by using the MaizeSNP50 chip, including 60,000 SNPs. At the five-leaf stage [60], leaves of each line were sampled in bulk to extract genomic DNA by the improved CTAB procedure. The genotyping work was completed by the Beijing Compass Biotechnology Company according to the Infinium1 HD assay ultra protocol guide. ...
Foundation parents (FPs) play an irreplaceable role in maize breeding practices. Maize white spot (MWS) is an important disease in Southwest China that always seriously reduces production. However, knowledge about the genetic mechanism of MWS resistance is limited. In this paper, a panel of 143 elite lines were collected and genotyped by using the MaizeSNP50 chip with approximately 60,000 single nucleotide polymorphisms (SNPs) and evaluated for resistance to MWS among 3 environments, and a genome-wide association study (GWAS) and transcriptome analysis were integrated to reveal the function of the identity-by-descent (IBD) segments for MWS. The results showed that (1) 225 IBD segments were identified only in the FP QB512, 192 were found only in the FP QR273 and 197 were found only in the FP HCL645. (2) The GWAS results showed that 15 common quantitative trait nucleotides (QTNs) were associated with MWS. Interestingly, SYN10137 and PZA00131.14 were in the IBD segments of QB512, and the SYN10137-PZA00131.14 region existed in more than 58% of QR273′s descendants. (3) By integrating the GWAS and transcriptome analysis, Zm00001d031875 was found to located in the region of SYN10137-PZA00131.14. These results provide some new insights for the detection of MWS’s genetic variation mechanisms.
... [28], precipitation methods which use salts and ethanol to precipitate the DNA [29], enzymatic digestion methods that use individual or a combination of enzymes to digest the samples and release the DNA [30] and use of magnetic bead-based coated with an agent which binds with DNA and isolates it from the cellular suspension [31], or magnetic ionic liquids [32]. These methods are usually combined and modi ed depending on the experimenter's needs [17,33]. ...
Background
Genetic and genomic studies are seeing an increase in sample sizes together with a wider range of species investigated in response to environmental change concerns. In turn, these changes may come with challenges including the time and difficulty to isolate nucleic acids (DNA or RNA), the sequencing cost and environmental impacts of the growing amount of plastic waste generated in the process. Pseudotsuga menziesii var. menziesii (Mirbel) Franco (PM), Tsuga heterophylla (Raf.) Sarg. (TH) and Thuja plicata Donn ex D.Don (TP) are conifer species found in diverse woodlands both as natives and naturalized exotics. Our study was carried out whilst investigating their genetics to understand their population structure and potential for adaptation.
Results
In the present study, we compared two different DNA isolation methods, i.e., spin-column DNeasy plant mini kit (QIAGEN), and temperature-driven enzymatic cocktail Plant DNA Extraction (MicroGEM). The quantity of recovered DNA and the quality of DNA were assessed along with the plastic footprint and time needed for three tree species. Both methods were optimised and proven to provide enough DNA for each studied species. The yield of DNA for each method depended on the species: QIAGEN showed higher yield in PM and TH, while TP recovered similar amount of DNA for both methods. The DNA quality was investigated using DNA barcoding techniques by confirming species identity and species discrimination. No difference was detected on the PCR amplification of the two barcoding loci, (rbcL and trnH-psbA), and the recovered sequences between DNA isolation methods. Measurement of the plastic use and the processing time per sample indicated that MicroGEM had a 52.64% lower plastic footprint and was 51.8% faster than QIAGEN.
Conclusions
QIAGEN gave higher yields in two of the species although both methods showed similar quality results across all species. However, MicroGEM was clearly advantageous to decrease the plastic footprint and improve the time efficiency. Overall, MicroGEM recovers sufficient and reliable DNA to perform common downstream analyses such as PCR and sequencing. Our findings illustrate the benefits of research and efforts towards developing more sustainable methods and techniques to reduce the environmental footprint of molecular analyses.
... In this approach, a SDS based lysis step was performed at 100 • C to generate the plant lysate followed by extraction of DNA using MILs. Recently, Emaus et al. reported a one-step plant cell lysis and DNA extraction method incorporating hydrophobic ILs and MILs that circumvented the need for a lengthy temperature-controlled lysis step [27]. This study demonstrated that ILs and MILs alone are capable of lysing plant cells and extracting DNA from intact plant tissue and the amount of DNA extracted increases with longer times and higher temperatures. ...
... chosen. Selection of the solvents is based on previous studies where they were used to extract DNA from plants and shown to be compatible with qPCR[25,27,45]. Extraction of DNA directly from plant tissue was based on applying the IL/MIL to a 1.0 mg cut fragment of dried tissue followed by incubation and recovery of the DNA-enriched solvent for qPCR ...
The isolation of high-quality plant genomic DNA is a major prerequisite in many plant biomolecular analyses involving nucleic acid amplification. Conventional plant cell lysis and DNA extraction methods involve lengthy sample preparation procedures that often require large amounts of sample and chemicals, high temperatures and multiple liquid transfer steps which can introduce challenges for high throughput applications. In this study, a simple, rapid, miniaturized ionic liquid (IL)-based extraction method was developed for the isolation of genomic DNA from milligram fragments of Arabidopsis thaliana plant tissue. This method is based on a modification of vortex-assisted matrix solid-phase dispersion (VA-MSPD) in which the trihexyl(tetradecyl)phosphonium bis(trifluoromethylsulfonyl)imide ([P6,6,6,14+][NTf2-]) IL or trihexyl(tetradecyl)phosphonium tris(hexafluoroacetylaceto)nickelate(II) ([P6,6,6,14+][Ni(hfacac)3-]) magnetic IL (MIL) was directly applied to treated plant tissue (∼1.5 mg) and dispersed in an agate mortar to facilitate plant cell lysis and DNA extraction, followed by recovery of the mixture with a qPCR compatible co-solvent. This study represents the first approach to use ILs and MILs in a MSPD procedure to facilitate plant cell lysis and DNA extraction. The DNA-enriched IL- and MIL-cosolvent mixtures were directly integrated into the qPCR buffer without inhibiting the reaction while also circumventing the need for additional purification steps prior to DNA amplification. Under optimum conditions, the IL and MIL yielded 2.87 ± 0.28 and 1.97 ± 0.59 ng of DNA/mg of plant tissue, respectively. Furthermore, the mild extraction conditions used in the method enabled plant DNA in IL- and MIL-cosolvent mixtures to be preserved from degradation at room temperature.
Monitoring nucleic acid contamination in laboratories is essential for ensuring the accuracy and reliability of polymerase chain reaction (PCR) assay results. Compared to traditional chemical reagent-based extraction, magnetic bead extraction is a targeted adsorption method for extracting DNA, offering greater efficiency and minor hazard. However, the effectiveness of this method in detecting nucleic acid contaminations in laboratory remains unclear. This study aimed to develop a PCR-based laboratory environmental monitoring method to investigate the effectiveness of magnetic bead extraction for detecting nucleic acid contamination. To mimic nucleic acid contamination, Staphylococcus aureus was aerosolized in a PCR laboratory. Following sampling, nucleic acid extraction was performed using the magnetic bead extraction method. Samples were analyzed using quantitative real-time fluorescence PCR (qPCR), and the concordance between the magnetic bead extraction results and theoretical results was assessed. This study presents a PCR laboratory nucleic acid contamination monitoring protocol. The results demonstrated that the nucleic acid detection outcomes obtained using the magnetic bead extraction method were consistent with the theoretical results across all regions tested using this protocol. The magnetic bead extraction method demonstrated comparable detection outcomes to the theoretical results in monitoring laboratory environments. This approach provides a more efficient and environmental friendly method of monitoring the laboratory environment.
Nucleic acid amplification testing facilitates the detection of disease through specific genomic sequences and is attractive for point-of-need testing (PONT); in particular, the early detection of microorganisms can alert early response systems to protect the public and ecosystems from widespread outbreaks of biological threats, including infectious diseases. Prior to nucleic acid amplification and detection, extensive sample preparation techniques are required to free nucleic acids and extract them from the sample matrix. Sample preparation is critical to maximize the sensitivity and reliability of testing. As the enzymatic amplification reactions can be sensitive to inhibitors from the sample, as well as from chemicals used for lysis and extraction, avoiding inhibition is a significant challenge, particularly when minimising liquid handling steps is also desirable for the translation of the assay to a portable format for PONT. The reagents used in sample preparation for nucleic acid testing, covering lysis and NA extraction (binding, washing, and elution), are reviewed with a focus on their suitability for use in PONT.
There is a trend in analytical chemistry towards development of eco-friendly methods of sample preparation without loss of efficiency. This book provides a general, critical, and updated vision of the different green sample preparation approaches that have been developed. These include miniaturisation of the extraction techniques that allow a reduction not only of the chemicals used during the process, but also of the sample amount; the use of greener solvents, such as certain ionic liquids (ILs) or deep eutectic solvents (DES), instead of conventional organic solvents; and the use of new selective sorbent materials that allow both extraction and clean-up in the same step. All of these strategies have been successfully applied to the determination of a wide variety of organic and inorganic compounds.
Advanced undergraduate and graduate students will find this book a good reference source and, because of the multidisciplinary nature of this topic, it will be of use to a broad audience including chemists, materials scientists, environmental analysts, forensic scientists, pharmacists, biologists and chemical engineers, who are involved and interested in the future frontiers of analytical chemistry.
