Molecular Biology


Protocols in Current Issue
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0 Q&A 1024 Views Jun 5, 2022

Understanding the generation of mutations is fundamental to understanding evolution and genetic disease; however, the rarity of such events makes experimentally identifying them difficult. Mutation accumulation (MA) methods have been widely used. MA lines require serial bottlenecks to fix de novo mutations, followed by whole-genome sequencing. While powerful, this method is not suitable for exploring mutation variation among different genotypes due to its poor scalability with cost and labor. Alternatively, fluctuation assays estimate mutation rate in microorganisms by utilizing a reporter gene, in which Loss-of-function (LOF) mutations can be selected for using drugs toxic to cells containing the WT allele. Traditional fluctuation assays can estimate mutation rates but not their base change compositions. Here, we describe a new protocol that adapts traditional fluctuation assay using CAN1 reporter gene in Saccharomyces cerevisiae, followed by pooled sequencing methods, to identify both the rate and spectra of mutations in different strain backgrounds.

0 Q&A 2709 Views Jan 20, 2021

Antibacterial coatings have currently gained great importance in biomedical technology investigations. Because of the spatial arrangement of the film coatings, evaluation of antibacterial activity presents a new challenge regarding traditional bacterial counting methods. In this protocol, four clinically relevant pathogens, Salmonella typhimurium, Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus were incubated on titania mesostructured thin film coatings for 24 h. Then, cell viability was studied considering three methods: counting of the number of colony forming units (CFU), live/dead staining, and quantification of extracellular DNA in suspension. Firstly, bacterial count was determined by the standard plate-count technique. Secondly, bacteria membrane integrity was evaluated by utilization of two fluorescent dyes, which allow distinction between live (membrane intact) and dead (membrane disrupted) bacteria. Lastly, extracellular DNA was quantified by spectrophotometry. In this manner, the three aforementioned techniques enabled the study of bacterial viability by qualitative and quantitative analyses.

0 Q&A 3045 Views Aug 20, 2020
Extracellular DNA is studied as a diagnostic biomarker, but also as a factor involved in the pathophysiology of several diseases due to its pro-inflammatory properties. Extracellular DNA can be extracted from plasma, urine, saliva or other biofluids using standard DNA isolation procedures and specialized commercial kits. Sample preparation for isolation is important, freezing and thawing may affect the amount of extracellular DNA extracted. Subsequent centrifugations remove cells and cell debris from the samples to obtain true extracellular DNA. Small volume of samples especially from animal experiments is often an issue and it affects the DNA yield. Very short fragments (˂ 100 bp) can be lost during isolation and are difficult to quantify using PCR. Fluorometric methods asses all stained DNA fragments. Selecting the method for quantification of extracellular DNA is crucial and combination of at least two methods is ideal. Standardization of procedures or at least their reporting in research papers is of utmost importance for comparison of results.
0 Q&A 4260 Views Feb 5, 2020
Chlamydia trachomatis is an obligate human pathogen. It infects the genital tract of humans ascending into the fallopian tube, exacerbated by chronic pelvic pain, pelvic inflammatory disease, and fallopian tube scaring resulting in infertility and other malignancies. The major hurdle in controlling chlamydial spread is that the infection remains asymptomatic, thus leading to chronic, recurrent and persistent infections, with no vaccines developed so far. Being a human pathogen, we do not have an in vivo model of C. trachomatis infection. C. trachomatis do not cause ascending infections and fallopian tube pathology in the mouse urogenital tract when infected vaginally. To overcome this hurdle trans cervical method of infection must be adapted. In this protocol the method of establishing trans-cervical chlamydial infection with the procedure to determine the bacterial load is detailed. This method will facilitate to deliver the bacteria past the cervix establishing an ascending infection into the uterine horns reciprocating human fallopian tube infections.
0 Q&A 4071 Views Dec 5, 2019
Chromatin consists of compacted DNA in complex with proteins and contributes to the organization of DNA and its stability. Furthermore, chromatin plays key roles in regulating cellular processes such as DNA replication, transcription, DNA repair, and mitosis. Chromatin assumes more compact (inaccessible) or decondensed (accessible) conformations depending on the function that is being supported in the genome, either locally or globally. The activity of nucleases has been used previously to assess the accessibility of specific genomic regions in vitro, such as origins of replication at varying points in the cell cycle. Here, we provide an assay to determine the accessibility of specific human genomic regions (example used herein: Lamin B2 origin of DNA replication) by measuring the effect of DNase I nuclease on qPCR signal from the studied site. This assay provides a powerful method to interrogate the molecular mechanisms that regulate chromatin accessibility, and how these processes affect various cellular functions involving the human genome that require manipulation of chromatin conformation.
0 Q&A 4303 Views Oct 20, 2019
Time to AIDS infection is longer with HIV-2, compared to HIV-1, but without antiretroviral therapy both infections will cause AIDS-related mortality. In HIV-2 infection, monitoring of antiretroviral treatment (ART) efficacy is challenging since a large proportion of HIV-2-infected individuals displays low or undetectable plasma RNA levels. Hence, quantification of cellular DNA load may constitute an alternative method for monitoring ART efficacy. Moreover, sensitive HIV-2 DNA quantification protocols are also important for the characterization of the HIV-2 reservoirs, and ultimately for the development of HIV-2 cure strategies. We have developed a sensitive and robust HIV-2 DNA quantification protocol based on whole blood as DNA source, including normalization of leukocyte cell numbers using parallel quantification of the single copy porphobilinogen deaminase gene. The specificity and sensitivity of the assay was 100%. The limit of detection was 1 copy and limit of quantification was 5 copies. When applying this protocol to HIV-2 infected, it was found that HIV-2 viral DNA was detectable in individuals in whom viral RNA was undetectable or under quantification level. Thus, this method provides a sensitive approach to HIV-2 DNA viral quantification from whole blood of HIV-2 infected patients.
0 Q&A 4646 Views Feb 20, 2019
Upon entry into a host cell, the HIV-1 virus undergoes a series of critical early replication events including reverse transcription, nuclear import, and integration of its cDNA into the host genome. Molecular assays used to detect and analyze changes in HIV-1 early phase replication events are valuable tools in developing potential antiretroviral drugs, as well as studying the pathogenesis of HIV. Described here are the molecular assays utilized to detect and quantify HIV-1 early, intermediate, and late reverse transcription (RT) products. In addition to this, protocols for quantifying HIV-1 2-LTR circle DNA and proviral DNA after integration are also included. In these protocols, the optimized TaqMan Real-time PCR reagent is used to increase assay sensitivity and reproducibility. Furthermore, a nested PCR is applied to HIV-1 integration quantification with increased accuracy.
0 Q&A 11135 Views Jan 20, 2017
The binding and internalization of adeno-associated virus (AAV) is an important determinant of viral infectivity and tropism. The ability to dissect these two tightly connected cellular processes would allow better understanding and provide insight on virus entry and trafficking. In the following protocol, we describe a quantitative PCR (qPCR) based method to determine the amount of vector bound to the cell surface and the amount of subsequent virus internalization based on viral genome quantification. This protocol is optimized for studying AAV. Nevertheless, it can serve as a backbone for studying other viruses with careful modification.
0 Q&A 7506 Views Jul 5, 2016
Cyanobacteria are prokaryotic organisms that perform oxygenic photosynthesis. Freshwater cyanobacteria, such as Synechococcus elongatus PCC 7942 and Synechocystis sp. PCC 6803, are model organisms for the study of photosynthesis, gene regulation, and biotechnological applications because they are easy to manipulate genetically. However, while studying these cyanobacteria, care has to be taken with respect to genetic heterogeneity in the establishment of gene disruptants, because these cyanobacteria contain multiple chromosomal copies per cell. Here, we describe a method for the estimation of chromosomal copy number in Synechococcus 7942. Using this method, we have recently observed that the chromosomal copy number of Synechococcus 7942 significantly changes during its growth phases. This technique is available for studying polyploidy not only in cyanobacteria, but also in other polyploid organisms.
0 Q&A 7838 Views Jan 20, 2015
Drug-induced mitochondrial injury can be caused by many different mechanisms including inhibition of mitochondrial DNA replication, transcription, translation, and altered protein function. Determination of the level of mitochondrial DNA relative to the nuclear DNA levels provides important information on potential mitochondrial toxicity.



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