Extraction Of High-quality DNA Research Articles

Extraction and analysis of high-quality chloroplast DNA with reduced nuclear DNA for medicinal plants

BackgroundObtaining high-quality chloroplast genome sequences requires chloroplast DNA (cpDNA) samples that meet the sequencing requirements. The quality of extracted cpDNA directly impacts the efficiency and accuracy of sequencing analysis. Currently, there are no reported methods for extracting cpDNA from Erigeron breviscapus. Therefore, we developed a suitable method for extracting cpDNA from E. breviscapus and further verified its applicability to other medicinal plants.ResultsWe conducted a comparative analysis of chloroplast isolation and cpDNA extraction using modified high-salt low-pH method, the high-salt method, and the NaOH low-salt method, respectively. Subsequently, the number of cpDNA copies relative to the nuclear DNA (nDNA ) was quantified via qPCR. As anticipated, chloroplasts isolated from E. breviscapus using the modified high-salt low-pH method exhibited intact structures with minimal cell debris. Moreover, the concentration, purity, and quality of E. breviscapus cpDNA extracted through this method surpassed those obtained from the other two methods. Furthermore, qPCR analysis confirmed that the modified high-salt low-pH method effectively minimized nDNA contamination in the extracted cpDNA. We then applied the developed modified high-salt low-pH method to other medicinal plant species, including Mentha haplocalyx, Taraxacum mongolicum, and Portulaca oleracea. The resultant effect on chloroplast isolation and cpDNA extraction further validated the generalizability and efficacy of this method across different plant species.ConclusionsThe modified high-salt low-pH method represents a reliable approach for obtaining high-quality cpDNA from E. breviscapus. Its universal applicability establishes a solid foundation for chloroplast genome sequencing and analysis of this species. Moreover, it serves as a benchmark for developing similar methods to extract chloroplast genomes from other medicinal plants.

Abstract 7248: Simultaneous isolation and parallel analysis of genomic DNA and RNA for gene therapy

Abstract Introduction Viral vectors, such as adeno-associated vector (AAV), play a crucial role in delivering transgenes for gene therapy research. The success of vector transduction is vital for efficacy, which is measured through vector copy number (VCN) and transgene expression. In preclinical studies, both VCN and transgene expression are commonly tested from the same sample to ensure accuracy. We have reported a method for simultaneous high-quality genomic DNA and RNA extraction without splitting the sample, utilizing SPRI bead-based technology that has consistent performance across diverse tissue types and brain regions. Methods AAV was administered to mouse and non-human primate (NHP) models. At the conclusion of each experiment, tissue samples from the adrenal gland, brain, gonads, heart, kidney, liver, lung, skeletal muscle, spinal cord, and spleen were harvested. All tissue samples that demonstrated around 15 mm3 were snap-frozen in liquid nitrogen before nucleic acid extraction. The extraction process was carried out manually or on an automated liquid handler. The quality and quantity of DNA and RNA were then assessed from all tested tissues before downstream assay. Results Our study found that all mouse tissue types (n=72/tissue type) and NHP (n=11/tissue type) had an average gDNA and RNA yield that exceeded the minimum yield cutoff of 25 ng/μL for DNA and 6.25 ng/μL for RNA. We also analyzed DNA from tissues treated with two different AAV serotype-treated tissues in the mouse model, which showed that transduction across all the tissues was highly efficient for both AAV serotypes. Furthermore, we observed variations in transgene expression levels (copies/1 μg RNA) among different tissue types. Similar patterns of VCN and transgene expression were also observed in NHP models. Conclusions Efficient gene therapy depends on a safe and effective viral vector. The simultaneous extraction of gDNA and RNA simplifies the process, saves time, ensures quality, and facilitates integration into existing lab automation. This leads to consistent and error-resistant performance in analyzing vector copy and transgene levels. Citation Format: Han Wei. Simultaneous isolation and parallel analysis of genomic DNA and RNA for gene therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7248.

Testing of the Effect of Food Additives on the DNA Isolation from Mackerel and Fish Products

Authentication of fish products by DNA analysis requires the extraction of high quality DNA without the presence of inhibitors. Many nucleic acid isolation methods are currently available; silicate centrifugal columns have become very popular due to their speed and ease of extraction. However, their disadvantage may be the principle based on DNA charge, which may be affected by food composition, or clogging due to a poor sample pretreatment. The aim of this work was to compare three DNA isolation methods using different principles (silicate centrifugal columns, modified magnetic beads, Cetrimonium bromide and chloroform extraction) and to evaluate their suitability for DNA isolation from fish muscle. The criteria assessed were the recovery, purity and amplifiability of the isolated DNA. Mackerel tissue was analysed without and with the addition of additives commonly used in the manufacture of fish products, namely diphosphates (E 450) and colorants (E 110 and E 124), and the selected method was subsequently applied to commercial fish products. The modified method using the detergent CTAB proved to be the most suitable.

A multilevel core-shell structure Fe3O4@SiO2@InGaO@SiO2 magnetic particles for DNA extraction

DNA extraction is greatly significant in biological detection fields, including species identification, pathogen detection, environmental monitoring, biosafety, and food safety. Scientific research and practical application must conduct high-quality trace DNA separation, purification and extraction. Herein, a two-step strategy successfully prepares a multilevel core–shell structure Fe3O4@SiO2@InGaO@SiO2 magnetic particles for trace DNA extraction. The morphologies, structures, elemental compositions, components, and properties of the materials are systematically characterized. Fe3O4@SiO2@InGaO@SiO2 magnetic particles in the chaotropic salt solutions are used to directly target DNA binding due to the existence of Si-O–H bond. The maximum DNA adsorption rate can highly reach 83.1 % after 700 °C annealing in the air for 30 min, far exceeding the adsorption rate of commercial reagent kit, which is attributed to the increased specific surface area. The excellent result is conducive to rapid DNA extraction in practical applications, especially in daily criminal cases, further promoting social security and stability.

An optimal genomic DNA extraction method for shoots of four Dendrocalamus species based on membership function analysis.

High-quality genomic DNA extraction is fundamental for the study of gene cloning and expression in plants. Therefore, this study evaluated several methods for extracting genomic DNA from shoots of four Dendrocalamus species to determine the optimal technique. Genomic DNA was extracted using three different methods: a commercial DNA extraction kit method, a modified cetyltrimethylammonium bromide method and a sodium dodecyl sulfate method. A membership function analysis was employed to compare these methods. The results demonstrated that the commercial DNA extraction kit method was the most effective and comprehensive approach for extracting genomic DNA from shoots of four Dendrocalamus species. Furthermore, this study provided valuable insights into optimizing techniques for extracting genomic DNA in other bamboo species.

High-quality genomic DNA extraction methods of Yellow Spathoglottis Blume complex for next-generation sequencing

Abstract. Jasim JHM, Othman AS, Nordin FA, Talkah NSM. 2024. High-quality genomic DNA extraction methods of Yellow Spathoglottis Blume complex for next-generation sequencing. Biodiversitas 25: 654-663. Advancements in genomic research have spurred a growing interest in extracting high-quality genomic DNA, particularly from intricate plant species like the Yellow Spathoglottis Blume Complex. The preparation of genomic DNA high-quality from a plant sample is a crucial step for genomic and genetic analysis studies. A variety of genomic DNA extraction methods such as the traditional Hexadecyltrimethylammonium Bromide (CTAB) method and commercially available kits have been reported. Even so, they are either low purity and yield or costly. We develop a good-quality genomic DNA extraction method from fresh and dried leaves of the Yellow Spathoglottis Blume complex in Peninsular Malaysia, specifically tailored for Next-Generation Sequencing (NGS) applications. Three DNA extraction methods were compared traditional CTAB, modified CTAB, and the DNeasy Qiagen plant mini kit methods. The yield and quality of extracted DNA were assessed by PCR amplification using nuclear ETS and ITS genes performed to evaluate the success of DNA extraction. Additionally, the suitability of the modified CTAB method for NGS library preparation and sequencing was tested. It demonstrated better PCR amplification results, yielding high-concentration bands for both ETS and ITS primers. Furthermore, the modified CTAB method proved suitable for high molecular weight DNA extraction, without contamination. The extracted DNA from Spathoglottis aurea exhibited high molecular weight and passed the quality control assessment for NGS library preparation. Sequencing results demonstrated an average quality score of 36.0, with an accurate base call rate of approximately 99.9%. The developed method enables reliable genomic analysis and sequencing of the Yellow Spathoglottis Blume complex, contributing to further research and understanding of this species group.

A modified CTAB method for extracting high-quality genomic DNA from aquatic plants

This study introduces a streamlined approach for extractinghigh-quality DNA fromaquatic plants using CTAB, catering to molecular studies. Seven aquatic plant species(Hygrophilaauriculata, Limnophilarepens, Crinum malabaricum, Lagenandraovata, Ludwigiaperuviana, Eichhorniacrassipes, and Ipomoea aquatica) spanning six orderswere subjected toDNA extraction. The method combinesmechanical lysis and chemical treatments to effectively disrupt cells, coupled with RNase treatment and phenol extraction to mitigate RNA and protein contamination. The optimizedCTAB protocol facilitatesthe extraction of high-quality genomic DNA, suitable for amplifying plant barcode genes such as ITS and rbcL, as well asmarkerslike RAPD and ISSR, thereby enhancing the efficiency and reliability of genomic studies in aquatic plants.

An innovative optimized protocol for high-quality genomic DNA extraction from recalcitrant Shea tree (Vitellaria paradoxa, C.F. Gaertn) plant and its suitability for downstream applications.

It is not always easy to find a universal protocol for the extraction of genomic DNA (gDNA) from plants. Extraction of gDNA from plants such as shea with a lot of polysaccharides in their leaves is done in two steps: a first step to remove the polysaccharides and a second step for the extraction of the gDNA. In this work, we designed a protocol for extracting high-quality gDNA from shea tree and demonstrate its suitability for downstream molecular applications. Fifty milligrams of leaf and root tissues were used to test the efficiency of our protocol. The quantity of gDNA was measured with the NanoDrop spectrometer and the quality was checked on agarose gel. Its suitability for use in downstream applications was tested with restriction enzymes, SSRs and RAPD polymerase chain reactions and Sanger sequencing. The average yield of gDNA was 5.17; 3.96; 2.71 and 2.41µg for dry leaves, dry roots, fresh leaves and fresh roots respectively per 100mg of tissue. Variance analysis of the yield showed significant difference between all tissue types. Leaf gDNA quality was better compared to root gDNA at the absorbance ratio A260/280 and A260/230. The minimum amplifiable concentration of leaf gDNA was 1pg/µl while root gDNA remained amplifiable at 10pg/µl. Genomic DNA obtained was also suitable for sequencing. This protocol provides an efficient, convenient and cost effective DNA extraction method suitable for use in various vitellaria paradoxa genomic studies.

Low-cost, rapid and reliable DNA extraction from mammalian organoids using a C. elegans toolbox.

High-quality DNA extraction from organoids is an important step in molecular genetics research. Here, we show that a lysis buffer from the field of Caenorhabditis elegans research, called Single Worm Lysis Buffer (SWLB), is a low-cost, yet reliable method for DNA extraction from mammalian organoids. SWLB is superior in terms of price, storage, hands-on time and sustainability compared to current standardized DNA extraction protocols, while equally effective. This work indicates that it is useful to compare methods from different model systems, such as mammalian organoids and invertebrate nematodes, to find useful alternatives for research methodologies.

Protocol Development for Yielding High Quality DNA and RNA from Archived Formalin-Fixed Paraffin Embedded Tissues

Introduction: While genetic analysis of archived formalin-fixed paraffin embedded (FFPE) tissue specimens would be a significant research resource, extraction of high-quality DNA and RNA from these specimens is a significant challenge. Storage duration, tissue handling and tissue preservation processes as well as their interactions with the nucleic acids could influence the quality and integrity of the DNA or RNA required for genetic analysis.
 Objectives: The goal of this study was to establish a protocol to extract high quality DNA and RNA from the FFPE tissues for further use in quantitative PCR analysis.
 Methods: For the purposes of this study, human FFPE biopsy tissues from lung, liver, colon and kidney were obtained from a single center biorepository. GeneJET Genomic DNA Purification Kit (Catalog # K0722) obtained from Thermo Fisher Scientific and RecoverAll Total Nucleic Acid Isolation Kit obtained from Life Technologies were modified and used for extraction of DNA and RNA, respectively. Briefly, modifications involved extending reagent incubation times, increasing sample volumes and wash steps, and increased final nucleic acids recovery and concentration steps. Eight sections around 8-10 ?m thick were microtomed for each tissue sample and used for extraction. The purity of the nucleic acids obtained was verified using Nanodrop Spectrophotometer.
 Results: The average DNA yield from eight sections for each of the tissues was 270±184 ng/?l and for RNA was 296±188 ng/?l. Nucleic acid quality was assessed by measuring the 260nm/280nm absorbance ratio for protein contamination as well as the 260nm/230nm absorbance ratio for salt contamination. Both were found to be within acceptable ranges. RNA was reverse transcribed to cDNA and qPCR was successfully performed on both DNA and cDNA samples.
 Conclusions: These results indicate that protocols using the silica-based membrane technology can yield high quality DNA and RNA that can be successfully used for downstream genetic analysis.

Plant Sample Collection and Shipment for Multi-omic Analyses and Phytosanitary Evaluation.

Plant sample preparation for analyses is a fundamental step in high-throughput omics strategies. Especially for plant metabolomics, quenching of hydrolytic enzymes able to affect metabolite concentrations is crucial for the accuracy of results. Given that DNA is usually less labile than metabolites, most sampling and shipment procedures able to preserve the metabolome are also suitable for preventing the degradation of plant DNA or of DNA of pathogens in the plant tissue. In this article, we describe all the steps of sample collection, shipment (including the phytosanitary issues of moving plant samples), and processing for combined genomics and metabolomics from a single sample, as well as the protocols used in our laboratories for downstream approaches for crop plants, allowing collection of multi-omic datasets in large experimental setups. The protocols have been adjusted to apply to both freeze-dried and fresh-frozen material to allow the processing of crop plant samples that will require long-distance transport. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Preparation of freeze-dried leaf disks for multiplexed PCR or DArT-Seq genotyping Basic Protocol 2: Medium-throughput preparation of pathogen-free nucleic acids for most genotyping-resequencing applications or pathogen detection Alternate Protocol: Low-throughput extraction of high-quality DNA for resequencing using commercial kits Support Protocol: DNA quality control Basic Protocol 3: Preparation of freeze-dried plant material for metabolomics Basic Protocol 4: Preparation of fresh-frozen plant material for metabolomics Basic Protocol 5: Preparation and shipment of metabolite extracts for metabolomic analyses Basic Protocol 6: Sample shipping and long-term storage.

Automation protocol for high-efficiency and high-quality genomic DNA extraction from Saccharomyces cerevisiae.

Although many protocols have been previously developed for genomic DNA (gDNA) extraction from S. cerevisiae, to take advantage of recent advances in laboratory automation and DNA-barcode sequencing, there is a need for automated methods that can provide high-quality gDNA at high efficiency. Here, we describe and demonstrate a fully automated protocol that includes five basic steps: cell wall and RNA digestion, cell lysis, DNA binding to magnetic beads, washing with ethanol, and elution. Our protocol avoids the use of hazardous reagents (e.g., phenol, chloroform), glass beads for mechanical cell disruption, or incubation of samples at 100°C (i.e., boiling). We show that our protocol can extract gDNA with high efficiency both from cells grown in liquid culture and from colonies grown on agar plates. We also show results from gel electrophoresis that demonstrate that the resulting gDNA is of high quality.

Physical Pretreatments Applied in Three Commercial Kits for the Extraction of High-Quality DNA from Activated Sewage Sludge.

Obtaining sufficient and high-quality genomic DNA from sludge samples is a fundamental issue of feasibility and comparability in genomic studies of microbial diversity. Commercial kits for soil are often used for the extraction of gDNA from sludge samples due to the lack of specific kits. However, the evaluation of the performance of commercial kits for sludge DNA extraction is scarce and optimization of these methods to obtain a high quantity and quality of DNA is necessary, especially for downstream genomic sequencing. Sequential batch reactors (SBRs) loaded with lignocellulosic biomass are used for the synthesis of renewable resources such as levulinic acid (LA), adipic acid (AA), and polyhydroxyalkanoates (PHAs), and the biochemical synthesis of these compounds is conducted through the inoculation of microbes present in the residual activated sludge (AS) obtained from a municipal wastewater treatment plant. To characterize these microbes, the extraction of DNA from residual sewage sludge was conducted with three different commercial kits: Nucleospin® Soil from Macherey-Nagel, DNEasy® PowerSoil® from Qiagen, and E.Z.N.A.® Plant DNA Kit from Omega BIO-TEK. Nevertheless, to obtain the highest load and quality of DNA for next-generation sequencing (NGS) analysis, different pretreatments and different combinations of these pretreatments were used. The pretreatments considered were an ultrasonic bath and a temperature of 80 °C, together and separately with different incubation time periods of 30, 60, and 90 min. The results obtained suggest a significant improvement in the efficiency and quality of DNA extraction with the three commercial extraction kits when used together with the ultrasonic bath and 80 °C for 60 min. Here, we were able to prove that physical pretreatments are a viable alternative to chemical lysis for DNA extraction from complex samples such as sludge.

16S rRNA gene-based microbiota profiles from diverse avian faeces are largely independent of DNA preservation and extraction method.

The avian gut microbiota has been the subject of considerable recent attention, with potential implications for diverse fields such as the poultry industry, microbial ecology, and conservation. Faecal microbiotas are frequently used as a non-invasive proxy for the gut microbiota, however the extraction of high-quality microbial DNA from avian faeces has often proven challenging. Here we aimed to evaluate the performance of two DNA preservation methods (95% ethanol and RNAlater) and five extraction approaches (IndiSpin Pathogen Kit, QIAamp PowerFecal Pro DNA Kit, MicroGEM PrepGEM Bacteria Kit, ZymoBIOMICS DNA Miniprep Kit, and an in-house phase separation-based method) for studying the avian gut microbiota. Systematic testing of the efficacy of these approaches on faecal samples from an initial three avian species (chicken, ostrich, and the flightless parrot kākāpō) revealed substantial differences in the quality, quantity and integrity of extracted DNA, but negligible influence of applied method on 16S rRNA gene-based microbiota profiles. Subsequent testing with a selected combination of preservation and extraction method on 10 further phylogenetically and ecologically diverse avian species reiterated the efficacy of the chosen approach, with bacterial community structure clustering strongly by technical replicates for a given avian species. Our finding that marked differences in extraction efficacy do not appear to influence 16S rRNA gene-based bacterial community profiles provides an important foundation for ongoing research on the avian gut microbiota.

A modified CTAB method for high-molecular-weight DNA preparation from deep-sea holothurians

ABSTRACT With the features as global distribution and all depths dwelling, holothurians function an ideal model for the study of deep-sea adaptation. Acquisition of high-molecular-weight DNA is the basic requirement for molecular biology analyses. However, extraction of high-molecular-weight DNA from deep-sea holothurians is difficult by degradation and contamination. Here, we presented a modified CTAB (lysis buffer with cetyl trimethyl ammonium bromide) method and compared its extraction efficiency for deep-sea holothurians with other five common methods, i.e. the Tiangen Biotech kit, Qiagen kit, Omega Bio-tek kit, Sangon Biotech kit, and SDS (lysis buffer with sodium dodecyl sulfate). Based on the results of yield, integrity, and purity, we found that the modified CTAB method is more suitable to extract high-quality DNA for deep-sea holothurians, with a large amount of yield, minimized degradation, and higher purity. This study provides a high-quality DNA of deep-sea holothurians for genome sequencing and further studies.

Tracing genetic variation of Gelidium canariense (Rhodophyta) based on new and historical collections

ABSTRACT Genetic sequencing of herbarium specimens provides invaluable information on species genetic history. However, several factors hinder the extraction of high-quality DNA from long-term preserved specimens. Our goal was to study the genetic variability of the endemic and habitat-forming macroalga Gelidium canariense over the last 40 years using the mitochondrial intergenic marker cox2-3 spacer. We also studied the genetic diversity at the mesoscale (<100 km), i.e. between two localities on the north coast of Tenerife and at the macroscale (>100 km) using specimens collected on the island of La Palma. We found the presence of the same haplotype at the same location for the last 40 years. This haplotype also coincided within populations of Puerto de la Cruz and Garachico (Tenerife Island) and between populations of different islands (Tenerife and La Palma). This study provides a baseline (i.e. extraction method, PCR protocol for cox2-3 spacer molecular marker, level of DNA degradation of herbarium samples) of the genetic characterization of G. canariense that can be used in future molecular studies to better understand the distribution of genetic diversity in this vulnerable species.

Isolation of Genomic DNA from Mammalian Cells and Fixed Tissue.

Examination of DNA variation is central to understanding the function of mammalian cells, tissues, and whole bodies. Extraction of high-quality DNA from cells and tissues is necessary for innumerable different experiments. We present protocols for the extraction of DNA from both fresh samples and formalin-fixed tissue. Methods for extracting DNA have been standardized and streamlined over the past couple of decades and many extraction kits are available for a reasonable cost. In addition, many of the extraction procedures can also be automated for even higher throughput sample preparation. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: DNA isolation from whole blood, tissue, and cultured cells Alternate Protocol: DNA extraction using automated machines Basic Protocol 2: DNA isolation from saliva and buccal swabs Basic Protocol 3: DNA isolation from formalin-fixed, paraffin-embedded tissue.

A New and Profitable Protocol to DNA Extraction in Limnospira maxima.

Limnospira maxima is a remarkable organism showing great potential as a versatile and sustainable food source, offering a powerful solution to address the pressing issues of malnutrition and undernourishment worldwide. L. maxima contains high amounts of proteins, vitamins, minerals, and essential fatty acids. It can be grown in both bioreactors and open systems; however, before considering industrial production, optimization studies of the cultivation must be conducted to obtain knowledge about the ideal environmental conditions. Additionally, for the molecular typing of L. maxima strains and their industrial scaling, high-quality and large quantity DNA extraction is required. Notwithstanding, DNA extraction from L. maxima can be challenging due to the low amount of DNA in cells and the presence of difficult-to-remove substances such as polysaccharides and polyphenols. In this study, the quality and quantity of DNA extracted from two types of L. maxima samples (Limnospira maxima strain SISCA accession GenBank: OR195505.1) were evaluated using three commercially available DNA extraction kits and two types of input biological material. The results showed that Pbact-P kit had the highest quantity and quality of DNA, while CTAB-P allowed for a higher quantity and quality of RNA, making them optimal protocols for nucleic acid extraction to improve PCR, rt-PCR, and genome sequencing of L. maxima compared with other extraction methods.

An improved Shorea robusta genomic DNA extraction protocol with high PCR fidelity.

Shorea robusta (Dipterocarpaceae), commonly known as Sal, is an economically and culturally important timber species, known to contain a wide spectrum of polyphenols, polysaccharides, and other secondary metabolites in the tissues, which can interfere with the extraction of high-quality genomic DNA. In order to screen simple sequence repeat (SSR) markers and carry out other DNA-based analyses for this species in our laboratory, a high-throughput DNA extraction methodology was needed. Hence, we have optimized a simple, rapid, safe, and reliable high-throughput protocol for DNA extraction suitable for both fresh and dry leaves. The standardized protocol delivered good DNA yield of ∼1500 µg from 1 g of leaf tissue, with purity indicated by a 260 nm/280 nm absorbance ratio ranging from 1.70 to 1.91, which validated the suitability of extracted DNA and revealed reduced levels of contaminants. Additionally, the protocol that we developed was found to be suitable for polymerase chain reaction (PCR) amplification using microsatellite markers. Genome-wide characterization with SSR markers has been established in S. robusta, which further validates the protocol and its usefulness in DNA-based studies across the genus and/or family.

Extraction of high-quality genomic DNA from leaf tissues of marigold

Extraction of high-quality genomic DNA from leaf tissues of marigold