Molecular Biology


    Protocols in Current Issue
    Estimation of the Minimum Number of Replication Origins Per Chromosome in any Organism
    Author:  Marcelo S. da Silva, date: 10/20/2020, view: 1119, Q&A: 0
    [Abstract] Eukaryote nuclear genomes predominantly replicate through multiple replication origins. The number of replication origins activated per chromosome during the S-phase duration may vary according to many factors, but the predominant one is replication stress. Several studies have applied different approaches to estimate the number and map the ...
    Nonenzymatic RNA-templated Synthesis of N3′→P5′ Phosphoramidate DNA
    Authors:  Derek K. O'Flaherty, Lijun Zhou and Jack W. Szostak, date: 09/05/2020, view: 1760, Q&A: 0
    [Abstract] The RNA world hypothesis describes a scenario where early life forms relied on RNA to govern both inheritance and catalyze useful chemical reactions. Prior to the emergence of enzymes capable of replicating the RNA genome, a nonenzymatic replication process would have been necessary to initiate Darwinian Evolution. However, the one-pot ...
    Unbiased and Tailored CRISPR/Cas gRNA Libraries by Synthesizing Covalently-closed-circular (3Cs) DNA
    Authors:  Martin Wegner, Koraljka Husnjak and Manuel Kaulich, date: 01/05/2020, view: 4092, Q&A: 0
    [Abstract] Simplicity, efficiency and versatility of the CRISPR/Cas system greatly contributed to its rapid use in a broad range of fields. Applications of unbiased CRISPR/Cas screenings are increasing and thus there is a growing need for unbiased and tailored CRISPR/Cas gRNA libraries. Conventional methods for gRNA library generation apply PCR and cloning ...
    Enhanced-ice-COLD-PCR for the Sensitive Detection of Rare DNA Methylation Patterns in Liquid Biopsies
    Authors:  Florence Mauger and Jörg Tost, date: 12/05/2019, view: 2583, Q&A: 0
    [Abstract] In the context of precision medicine, the identification of novel biomarkers for the diagnosis of disease, prognosis, predicting treatment outcome and monitoring of treatment success is of great importance. The analysis of methylated circulating-cell free DNA provides great promise to complement or replace genetic markers for these applications, ...
    In vitro Assays for Eukaryotic Leading/Lagging Strand DNA Replication
    Authors:  Grant Schauer, Jeff Finkelstein and Mike O’Donnell, date: 09/20/2017, view: 6660, Q&A: 0
    [Abstract] The eukaryotic replisome is a multiprotein complex that duplicates DNA. The replisome is sculpted to couple continuous leading strand synthesis with discontinuous lagging strand synthesis, primarily carried out by DNA polymerases ε and δ, respectively, along with helicases, polymerase α-primase, DNA sliding clamps, clamp loaders and many other ...
    EdU Based DNA Synthesis and Cell Proliferation Assay in Maize Infected by the Smut Fungus Ustilago maydis
    Authors:  Amey Redkar and Gunther Doehlemann, date: 03/20/2016, view: 7363, Q&A: 0
    [Abstract] The basidiomycetous smut fungus Ustilago maydis (U. maydis) infects all aerial parts of its host plant maize (Zea mays L.). Infection is seen in the form of prominent tumorous symptoms after the establishment of a biotrophic interaction with the host, usually around 5-6 days after infection. The fungus colonizes the ...
    In vitro DNA Polymerization Activity Assay Using Cell-free Extracts
    Authors:  Anurag K. Sinha and Malay K. Ray, date: 08/20/2014, view: 7129, Q&A: 0
    [Abstract] This protocol has been designed to measure the in-vitro DNA polymerization activity in crude cell extracts of the Antarctic bacterium Pseudomonas syrinagae Lz4W. This bacterium can grow at 4 °C with optimum growth rate at 22 °C. The slow growth rate of the bacterium observed at low temperature (4 °C) compared to higher ...
    EdU labeling of Trypanosome Cells and Their Kinetoplast DNA (kDNA)
    Author:  Jianyang Wang, date: 06/20/2013, view: 8459, Q&A: 0
    [Abstract] Trypanosome mitochondrial genome, known as Kinetoplast DNA (kDNA), is a massive network of interlocked DNA rings. The studies of kDNA replication and architecture are of major significance since kDNA is a valid drug target. However, DNA in procyclic trypanosomes can not be labeled with tracer concentrations of 3[H]-thymidine, possibly ...

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