Molecular Biology

DNA extraction is a crucial step in molecular biology research, particularly for genetic and genomic analyses. These studies require a high concentration of high-quality DNA, which is often a challenge for underexplored species or when the available plant material consists of aged tissue. To address these challenges, the cetyltrimethylammonium bromide (CTAB)-based DNA extraction method has been optimized to improve efficiency and yield. The process begins with an overnight incubation of plant tissue macerated with liquid nitrogen in a solution containing a high concentration of CTAB (4%). Subsequently, the mixture undergoes two washes with chloroform: isoamyl alcohol. The nucleic acids are then precipitated using isopropanol, followed by a wash with 70% ethanol to ensure purity. Finally, the purified DNA is resuspended in ultrapure water. This optimized procedure produces high-quality DNA suitable for various downstream applications, including PCR and sequencing, even from older leaves of the three Theobroma species: T. cacao, T. bicolor, and T. grandiflorum. Additionally, this protocol significantly enhances throughput and allows for the parallel processing of a substantially larger number of samples compared to conventional techniques.

In molecular diagnosis, DNA extraction kits are sample-specific and proprietary, preventing lateral distribution among similar facilities from different sectors to alleviate supply shortages during a crisis. Previous fast extraction protocols such as detergent-based ones allow fast DNA extraction for nucleic acid amplification tests (NAAT), mainly polymerase chain reaction (PCR). The use of NaOH (dense alkali) to rupture cells and nuclei and destabilize the conformation of DNases might alleviate shortages and costs while retaining enough robustness to treat complicated samples with minimal environmental and logistical footprint. Biological samples are hand-crushed using a pestle in 1.5 mL tubes with 360 μL of 0.2 M NaOH for 3–5 min and incubated at 75 °C for 10 min. For immediate use, 115.2 μL of 1 M Tris (pH 8) and 364.8 μL nuclease-free water are added, and the sample is vortexed for 10 s and spun at 10,000× g for 3 min; then, 700 μL is transferred to a clean microtube. Two serial dilutions follow, and all concentrations are used as templates for PCR. A refined, storable extract can be produced by adding 70 μL of HCl 1 M (instead of Tris-HCl) and one volume of cold isopropanol to the extract for standard precipitation. This method can increase throughput in emergencies by field deployment in resource-limited settings (RLS) or allow benchtop backup in cases of acquisition disruption or sample surge in established facilities. The crude extract can be used for immediate PCR in both benchtop and portable thermocyclers, thus allowing NAAT in resource-limited settings with low costs and waste footprint or during prolonged crises, where supply chain failures may occur. The refined version produces alcohol-precipitated nucleic acids, suitable for both immediate use and for storage or dispatch for spatiotemporally separate analysis while offering much better amplification quality with a small increase in time and minimal increase in expendables/chemicals needed.

This manuscript details two modified protocols for the isolation of long-stranded or high molecular weight (HMW) DNA from Magnaporthaceae (Ascomycota) fungal mycelium intended for whole genome sequencing. The Cytiva Nucleon PhytoPure and the Macherey-Nagel NucleoBond HMW DNA kits were selected because the former requires lower amounts of starting material and the latter utilizes gentler methods to maximize DNA length, albeit at a higher requirement for input material. The Cytiva Nucleon PhytoPure kit successfully recovered HMW DNA for half of our fungal species by increasing the amount of RNase A treatment and adding in a proteinase K treatment. To reduce the impact of pigmentation development, which occurs toward later stages of culturing, extractions were run in quadruplicate to increase overall DNA concentration. We also adapted the Macherey-Nagel NucleoBond HMW DNA kit for high-quality HMW DNA by grinding the sample to a fine powder, overnight lysis, and splitting the sample before washing the precipitated DNA. For both kits, precipitated DNA was spooled out pre-washing, ensuring a higher percentage of high-integrity long strands. The Macherey-Nagel protocol offers advantages over the first through the utilization of gravity columns that provide gentler treatment, yielding >50% of high-purity DNA strands exceeding 40 kbp. The limitation of this method is the requirement for a large quantity of starting material (1 g). By triaging samples based on the rate of growth relative to the accumulation of secondary metabolites, our methodologies hold promise for yielding reliable and high-quality HMW DNA from a variety of fungal samples, improving sequencing outcomes.

Wastewater-based surveillance (WBS) can provide a wealth of information regarding the health status of communities from measurements of nucleic acids found in wastewater. Processing workflows for WBS typically include sample collection, a primary concentration step, and lysis of the microbes to release nucleic acids, followed by nucleic acid purification and molecular-based quantification. This manuscript provides workflows from beginning to end with an emphasis on filtration-based concentration approaches coupled with specific lysis and nucleic acid extraction processes. Here, two WBS processing approaches are presented, one focusing on RNA-specific pathogens and the other focused on DNA-specific pathogens found within wastewater: 1) The RNA-specific approach, employed for analyzing RNA viruses like severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) couples electronegative filtration of wastewater with the placement of the filter within a lysis buffer followed by direct RNA extraction. 2) The DNA-specific approach, employed for analyzing DNA pathogens like Candida auris, uses size selection membranes during filtration, subsequently followed by a lysis buffer, bead-beating, and DNA extraction. Separate workflows for RNA versus DNA isolations have the advantage of improving the detection of the target pathogen. A novel aspect of the RNA-specific workflow is the direct extraction of nucleic acids from filter lysates, which shows enhanced recoveries, whereas the DNA-specific approach requires bead beating prior to extraction. Novelty is also provided in a new qPCR approach called Volcano 2nd Generation (V2G), which uses a polymerase capable of using RNA as a template, bypassing the reverse transcriptase step normally required for qPCR.

In applications such as marker-assisted breeding and positional cloning, tissue sampling and plant tracking are vital steps in the genotyping pipeline. They enable the identification of desirable seedlings, saving time and reducing the cost, space, and handling required for growing adult plants, especially for greenhouses and winter nurseries. Small-scale marker-assisted selection laboratories rely heavily on leaf-based genotyping, which involves over-planting large, segregating populations followed by leaf sampling, genotyping, and backtracking to identify desired individuals, which is costly and laborious. Thus, there is a need to adopt seed-based genotyping to reduce costs and save time. Therefore, we developed a safe and cheap seed-chipping protocol using clipping pliers to chip seeds to genotype before planting. To identify a cost-effective and high-throughput DNA extraction method, we tested four extraction methods and assessed the quality of the seed DNA using PCR. For three of the methods, seed-based DNA was of comparable quality to DNA extracted from leaf punches. We also compared seed- and leaf-derived DNA from the same individuals in a segregating population to test for genotyping miscalls that could arise due to the presence of maternally derived pericarp in the seed samples. Out of 43 potential instances, we found zero miscalled samples and, therefore, no evidence supporting consequential pericarp inclusion. Germination rates of chipped and unchipped seeds were the same for the inbreds tested, B73 and Mo17. However, chipped seeds grew slower until ~14 days after sowing. Overall, seed sampling using clipping pliers provides a simple, reliable, and high-throughput method to identify specific genotypes before planting.

Fusarium oxysporum can cause many important plant diseases worldwide, such as crown rot, wilt, and root rot. During the development of strawberry crown rot, this pathogenic fungus spreads from the mother plant to the strawberry seedling through the stolon, with obvious characteristics of latent infection. Therefore, the rapid and timely detection of F. oxysporum can significantly help achieve effective disease management. Here, we present a protocol for the recombinase polymerase amplification– lateral flow dipstick (RPA–LFD) detection technique for the rapid detection of F. oxysporum on strawberry, which only takes half an hour. A significant advantage of our RPA–LFD technique is the elimination of the involvement of professional teams and laboratories, which qualifies it for field detection. We test this protocol directly on plant samples with suspected infection by F. oxysporum in the field and greenhouse. It is worth noting that this protocol can quickly, sensitively, and specifically detect F. oxysporum in soils and plants including strawberry.

Key features

• This protocol is used to detect whether plants such as strawberry are infected with F. oxysporum.

• This protocol has potential for application in portable nucleic acid detection.

• It can complete the detection of samples in the field within 30 min.

Graphical overview

In the field of molecular genetics, DNA extraction protocols and kits are sample-specific and proprietary, preventing lateral distribution among similar facilities from different sectors to alleviate supply shortages during a crisis. Expanding upon previous fast extraction protocols such as alkaline- and detergent-based ones, the use of boiling-hot water to rupture cells, virions, and nuclei, as proposed during the COVID-19 pandemic, might alleviate shortages and costs. Different soft, relatively abundant (highly enriched), and uncomplicated (genomically homogenous and with few inhibitors) biosamples are collected in 1.5 mL tubes, mixed with boiling-hot water, and stirred vigorously, so as to have membranes lysed and proteins deactivated; mechanical disruption may be used as well if necessary. Incubation in boiling water bath for 20–30 min follows. Depending on sample type and quantity, which affects the total extraction volume, 2–5 μL are pipetted off for direct PCR and the same volume for two decimal serial dilutions. The latter are intended to optimize the crude extract to a workable DNA/inhibitor concentration balance for direct PCR. Uncomplicated, highly enriched samples such as mycelial growth in fruits and human swab samples can be processed, contrary to complicated samples such as blood and physically unyielding samples such as plant tissue. The extract can be used for immediate PCR in both benchtop and portable thermocyclers, thus allowing nucleic acid amplification tests (NAAT) being performed in resource-limited settings with low cost and waste footprint or during prolonged crises, where supply chain failures may occur.

Key features

• DNA extraction from different sample types using only boiling water and occasional mechanical assistance.

• Crude extract serially diluted twice, 10- and 100-fold, to bypass purification and quantification steps.

• Direct PCR for 2–10 μL of crude lysate and dilutions (conditional to sample type and quantity) to enhance probability of workable DNA-inhibitors’ concentrations.

• Lowers the cost and curtails the overall footprint of testing to increase sustainability in field operations and in standard lab environments under supply chain derailment.

High yield of good quality plasmid DNA from gram -ve bacteria (Agrobacterium tumefaciens, A. rhizogenes, and Rhizobium sp.) and gram +ve bacterium (Bacillus thuringiensis) is difficult. The widely used plasmid extraction kits for Escherichia coli yield a low quantity of poor-quality plasmid DNA from these species. We have optimized an in-house modification of the QIAprep Spin Miniprep kit protocol of Qiagen, consisting of two extraction steps. In the first, the centrifugation after adding neutralization buffer is followed by ethanol (absolute) precipitation of plasmid DNA. In the second extraction step, the precipitated DNA is dissolved in Tris-EDTA (TE) buffer, followed by an addition of 0.5 volumes of 5 M sodium chloride and 0.1 volumes of 20% (w/v) sodium dodecyl sulfate. After incubation at 65 °C for 15 min, the plasmid DNA is extracted with an equal volume of chloroform:isoamyl alcohol (CIA). RNase (20 mg/mL) is added to the upper phase retrieved after centrifugation and is incubated at 37 °C for 15 min. The extraction of the plasmid DNA with an equal volume of CIA is followed by centrifugation and is precipitated from the retrieved upper phase by adding an equal volume of absolute ethanol. The pellet obtained after centrifugation is washed twice with 70% (v/v) ethanol, air dried, dissolved in TE buffer, and quantified. This easy-to-perform protocol is free from phenol extraction, density gradient steps, and DNA binding columns, and yields high-quality plasmid DNA. The protocol opens an easy scale up to yield a large amount of high-quality plasmid DNA, useful for high-throughput downstream applications.

Key features

• The protocol is free from density gradient steps and use of phenol.

• The protocol is an extension of the QIAprep Spin Miniprep kit (Qiagen) and is applicable for plasmid DNA isolation from difficult-to-extract bacterial species.

• The protocol facilitates the direct transformation of the ligation product into Agrobacterium by skipping the step of E. coli transformation.

• The plasmids isolated are of sequencing grade and the method is useful for extracting plasmids for metagenomic studies.

Graphical overview

Overview of the plasmid isolation protocol (modified QIAprep Spin Miniprep kit) of the present study

We present a safe and low-cost method suitable for DNA extraction from mycelium and tree tissue samples. After sample preparation, the extraction takes about 60 min. Method performance was tested by extracting DNA from various tree tissue samples and from mycelium grown on solid and liquid media. DNA was extracted from juvenile and mature host material (Picea abies, Populus trichocarpa, Pseudotsuga menziesii) infected with different pathogens (Heterobasidion annosum, Heterobasidion parviporum, Leptographium wagenerii, Sphaerulina musiva). Additionally, DNA was extracted from pure cultures of the pathogens and several endophytic fungi. PCR success rate was 100% for young poplar material and fungal samples, and 48-72% for conifer and mature broadleaved plant samples. We recommend using 10-50 mg of fresh sample for the best results. The method offers a safe and low-cost DNA extraction alternative to study tree-fungus interactions, and is a potential resource for teaching purposes.

The purification of nucleic acids is one of the most common procedures employed in modern molecular biology laboratories. Typically, commercial column-based protocols are utilized to isolate DNA or RNA from various sources. However, these methods not only require specialized equipment, but are also extremely expensive for high-throughput applications. Although an elegant answer to this issue can be provided by paramagnetic beads, bead-based open-source protocols have been limited in the past. Here, we provide an easy to follow step-by-step manual for the synthesis of paramagnetic beads, as well as their functionalization with either a silica- or a carboxyl-surface that can be used to replace the commercial columns with self-made magnetic beads. Together with a variety of detailed protocols for their use in high-throughput nucleic acids extractions, this bead synthesis method forms the recently published open platform Bio-On-Magnetic-Beads (BOMB), which is available on PLOS Biology (Oberacker et al., 2019). Updated protocols can be found on the associated webpage (https://bomb.bio).