Molecular Biology


Protocols in Current Issue
Protocols in Past Issues
0 Q&A 406 Views Jan 5, 2024

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

0 Q&A 630 Views Dec 20, 2023

Many organisms alternate the expression of genes from large gene sets or gene families to adapt to environmental cues or immune pressure. The single-celled protozoan pathogen Trypanosoma brucei spp. periodically changes its homogeneous surface coat of variant surface glycoproteins (VSGs) to evade host antibodies during infection. This pathogen expresses one out of ~2,500 VSG genes at a time from telomeric expression sites (ESs) and periodically changes their expression by transcriptional switching or recombination. Attempts to track VSG switching have previously relied on genetic modifications of ES sequences with drug-selectable markers or genes encoding fluorescent proteins. However, genetic modifications of the ESs can interfere with the binding of proteins that control VSG transcription and/or recombination, thus affecting VSG expression and switching. Other approaches include Illumina sequencing of the VSG repertoire, which shows VSGs expressed in the population rather than cell switching; the Illumina short reads often limit the distinction of the large set of VSG genes. Here, we describe a methodology to study antigenic switching without modifications of the ES sequences. Our protocol enables the detection of VSG switching at nucleotide resolution using multiplexed clonal cell barcoding to track cells and nanopore sequencing to identify cell-specific VSG expression. We also developed a computational pipeline that takes DNA sequences and outputs VSGs expressed by cell clones. This protocol can be adapted to study clonal cell expression of large gene families in prokaryotes or eukaryotes.

Key features

• This protocol enables the analysis of variant surface glycoproteins (VSG) switching in T. brucei without modifying the expression site sequences.

• It uses a streamlined computational pipeline that takes fastq DNA sequences and outputs expressed VSG genes by each parasite clone.

• The protocol leverages the long reads sequencing capacity of the Oxford nanopore sequencing technology, which enables accurate identification of the expressed VSGs.

• The protocol requires approximately eight to nine days to complete.

Graphical overview

0 Q&A 428 Views Nov 20, 2023

Rapid development in single-cell chromosome conformation capture technologies has provided valuable insights into the importance of spatial genome architecture for gene regulation. However, a long-standing technical gap remains in the simultaneous characterization of three-dimensional genomes and transcriptomes in the same cell. We have described an assay named Hi-C and RNA-seq employed simultaneously (HiRES), which integrates in situ reverse transcription and chromosome conformation capture (3C) for the parallel analysis of chromatin organization and gene expression. Here, we provide a detailed implementation of the assay, using mouse embryos and cerebral cortices as examples. The versatility of this method extends beyond these two samples, with the potential to be used in various other cell types.

Key features

• A multi-omics sequencing approach to profile 3D genome structure and gene expression simultaneously in single cells.

• Compatible with animal tissues.

• One-tube amplification of both DNA and RNA components.

• Requires three days to complete.

Graphical overview

Schematic illustration for the Hi-C and RNA-seq employed simultaneously (HiRES) workflow
0 Q&A 369 Views Nov 5, 2023

The precise and rapid detection of fungi is important in various fields, including clinics, industry, and agriculture. While sequencing universal DNA barcodes remains the standard method for species identification and phylogenetic analysis, a significant bottleneck has been the labor-intensive and time-consuming sample preparation for genomic DNA extraction. To address this, we developed a direct PCR method that bypasses the DNA extraction steps, facilitating efficient target DNA amplification. Instead of extracting genomic DNA from fungal mycelium, our method involves adding a small quantity of mycelium directly to the PCR mixture, followed by a heat shock and vortexing. We found these simple adjustments to be sufficient to lyse many filamentous fungal cells, enabling target DNA amplification. This paper presents a comprehensive protocol for executing direct PCR in filamentous fungi. Beyond species identification, this direct PCR approach holds promise for diverse applications, such as diagnostic PCR for genotype screening without fungal DNA extraction. We anticipate that direct PCR will expedite research on filamentous fungi and diagnosis of fungal diseases.

Key features

• Eliminates the time-consuming genomic DNA extraction step for PCR, enhancing the speed of molecular identification.

• Adds a small quantity of mycelium directly into the PCR mix.

• Emphasizes the crucial role of heat shock and vortexing in achieving efficient target DNA amplification.

• Accelerates the molecular identification of filamentous fungi and rapid diagnosis of fungal diseases.

Graphical overview

Direct PCR using filamentous fungal biomass

0 Q&A 764 Views Oct 20, 2023

An efficient and precise genome-editing approach is in high demand in any molecular biology or cell biology laboratory worldwide. However, despite a recent rapid progress in the toolbox tailored for precise genome-editing, including the base editors and prime editors, there is still a need for a cost-effective knock-in (KI) approach amenable for long donor DNA cargos with high efficiency. By harnessing the high-efficient double-strand break (DSB) repair pathway of microhomology-mediated end joining, we previously showed that a specially designed 3′-overhang double-strand DNA (odsDNA) donor harboring 50-nt homology arm (HA) allows high-efficient exogenous DNA KI when combined with CRISPR-Cas9 technology. The lengths of the 3′-overhangs of odsDNA donors could be manipulated by the five consecutive phosphorothioate (PT) modifications. In this protocol, we detail the stepwise procedures to conduct the LOCK (Long dsDNA with 3′-Overhangs mediated CRISPR Knock-in) method for gene-sized (~1–3 kb) KI in mammalian cells.

Graphical overview

Improvement of large DNA fragment knock-in rates by attaching odsDNA donors to Cas9-PCV2 fusion protein

0 Q&A 363 Views Sep 5, 2023

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.

0 Q&A 323 Views Sep 5, 2023

Magnaporthe oryzae is a filamentous fungus responsible for the detrimental rice blast disease afflicting rice crops worldwide. For years, M. oryzae has served as an excellent model organism to study plant pathogen interactions due to its sequenced genome, its amenability to functional genetics, and its capacity to be tracked in laboratory settings. As such, techniques to genetically manipulate M. oryzae for gene deletion range from genome editing via CRISPR-Cas9 to gene replacement through homologous recombination. This protocol focuses on detailing how to perform gene replacement in the model organism, M. oryzae, through a split marker method. This technique relies on replacing the open reading frame of a gene of interest with a gene conferring resistance to a specific selectable chemical, disrupting the transcription of the gene of interest and generating a knockout mutant M. oryzae strain.

Key features

• Comprehensive overview of primer design, PEG-mediated protoplast transformation, and fungal DNA extraction for screening.

Graphical overview

0 Q&A 808 Views Aug 20, 2023

Maintenance of genome integrity requires efficient and faithful resolution of DNA breaks and DNA replication obstacles. Dysfunctions in any of the processes orchestrating such resolution can lead to chromosomal instability, which appears as numerical and structural chromosome aberrations. Conventional cytogenetics remains as the golden standard method to detect naturally occurring chromosomal aberrations or those resulting from the treatment with genotoxic drugs. However, the success of cytogenetic studies depends on having high-quality chromosome spreads, which has been proven to be particularly challenging. Moreover, a lack of scoring guidelines and standardized methods for treating cells with genotoxic agents contribute to significant variability amongst different studies. Here, we report a simple and effective method for obtaining well-spread chromosomes from mammalian cells for the analysis of chromosomal aberrations. In this method, cells are (1) arrested in metaphase (when chromosome morphology is clearest), (2) swollen in hypotonic solution, (3) fixed before being dropped onto microscope slides, and (4) stained with DNA dyes to visualize the chromosomes. Metaphase chromosomes are then analyzed using high-resolution microscopy. We also provide examples, representative images, and useful guidelines to facilitate the scoring of the different chromosomal aberrations. This method can be used for the diagnosis of genetic diseases, as well as for cancer studies, by identifying chromosomal defects and providing insight into the cellular processes that influence chromosome integrity.

Graphical overview

0 Q&A 511 Views Aug 5, 2023

Study of gene function in eukaryotes frequently requires data on the impact of the gene when it is expressed as a transgene, such as in ectopic or overexpression studies. Currently, the use of transgenic constructs designed to achieve these aims is often hampered by the difficulty in distinguishing between the expression levels of the endogenous gene and its transgene equivalent, which may involve either laborious microdissection to isolate specific cell types or harvesting tissue at narrow timepoints. To address this challenge, we have exploited a feature of the Golden Gate cloning method to develop a simple, restriction digest–based protocol to differentiate between expression levels of transgenic and endogenous gene copies. This method is straightforward to implement when the endogenous gene contains a Bpi1 restriction site but, importantly, can be adapted for most genes and most other cloning strategies.

Key features

• This protocol was developed to determine the expression level of an ectopically expressed transcription factor with broad native expression in all surrounding tissues.

• The method described is most directly compatible with Golden Gate cloning but is, in principle, compatible with any cloning method.

• The protocol has been developed and validated in the model plant Arabidopsis thaliana but is applicable to most eukaryotes.

Graphical overview

0 Q&A 401 Views Aug 5, 2023

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

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