Molecular Biology

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    Protocols in Current Issue
    ATAC-Seq-based Identification of Extrachromosomal Circular DNA in Mammalian Cells and Its Validation Using Inverse PCR and FISH
    Authors:  Zhangli Su, Shekhar Saha, Teressa Paulsen, Pankaj Kumar and Anindya Dutta, date: 05/05/2021, view: 337, Q&A: 0

    Recent studies from multiple labs including ours have demonstrated the importance of extrachromosomal circular DNA (eccDNA) from yeast to humans (Shibata et al., 2012; Dillon et al., 2015; Møller et al., 2016; Kumar et al., 2017; Turner et

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    Identification of R-loop-forming Sequences in Drosophila melanogaster Embryos and Tissue Culture Cells Using DRIP-seq
    Authors:  Célia Alecki and Nicole J. Francis, date: 05/05/2021, view: 273, Q&A: 0

    R-loops are non-canonical nucleic structures composed of an RNA–DNA hybrid and a displaced ssDNA. Originally identified as a source of genomic instability, R-loops have been shown over the last decade to be involved in the targeting of

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    Identification of R-loop-forming Sequences in Drosophila melanogaster Embryos and Tissue Culture Cells Using DRIP-seq
    Authors:  Célia Alecki and Nicole J. Francis, date: 05/05/2021, view: 273, Q&A: 0
    [Abstract]

    R-loops are non-canonical nucleic structures composed of an RNA–DNA hybrid and a displaced ssDNA. Originally identified as a source of genomic instability, R-loops have been shown over the last decade to be involved in the targeting of proteins and to be associated with different histone modifications, suggesting a regulatory function. In

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    ATAC-Seq-based Identification of Extrachromosomal Circular DNA in Mammalian Cells and Its Validation Using Inverse PCR and FISH
    Authors:  Zhangli Su, Shekhar Saha, Teressa Paulsen, Pankaj Kumar and Anindya Dutta, date: 05/05/2021, view: 337, Q&A: 0
    [Abstract]

    Recent studies from multiple labs including ours have demonstrated the importance of extrachromosomal circular DNA (eccDNA) from yeast to humans (Shibata et al., 2012; Dillon et al., 2015; Møller et al., 2016; Kumar et al., 2017; Turner et al., 2017; Kim et al., 2020). More recently, it has been found that cancer cells obtain a selective

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    Optimized Recombinant Production of Secreted Proteins Using Human Embryonic Kidney (HEK293) Cells Grown in Suspension
    [Abstract]

    Recombinant proteins are an essential milestone for a plethora of different applications ranging from pharmaceutical to clinical, and mammalian cell lines are among the currently preferred systems to obtain large amounts of proteins of interest due to their high level of post-translational modification and manageable large-scale production. In

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    A Sensitive and Specific PCR-based Assay to Quantify Hepatitis B Virus Covalently Closed Circular (ccc) DNA while Preserving Cellular DNA
    Authors:  Benno Zehnder, Stephan Urban and Thomas Tu, date: 04/20/2021, view: 583, Q&A: 0
    [Abstract]

    Hepatitis B virus (HBV) is the major cause of liver diseases and liver cancer worldwide. After infecting hepatocytes, the virus establishes a stable episome (covalently closed circular DNA, or cccDNA) that serves as the template for all viral transcripts. Specific and accurate quantification of cccDNA is difficult because infected cells contain

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    Reconstitution of Chromatin by Stepwise Salt Dialysis
    Authors:  Grisel Cruz-Becerra and James T. Kadonaga, date: 04/05/2021, view: 338, Q&A: 0
    [Abstract]

    Chromatin, rather than plain DNA, is the natural substrate of the molecular machines that mediate DNA-directed processes in the nucleus. Chromatin can be reconstituted in vitro by using different methodologies. The salt dialysis method yields chromatin that consists of purified histones and DNA. This biochemically pure chromatin is well-suited for

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    A Novel Method to Construct Binary CRISPR Vectors for Plant Transformation by Single Round of PCR Amplification
    Authors:  Kang Li, Yuhui Wang and Chuanying Fang, date: 04/05/2021, view: 567, Q&A: 0
    [Abstract]

    CRISPR/Cas9 is an established and flexible tool for genome editing. However, most methods used to generate expression clones for the CRISPR/Cas9 are time-consuming. Hence, we have developed a one-step protocol to introduce sgRNA expression cassette(s) directly into binary vectors (Liu et al., 2020). In this approach, we have optimized the

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    In vitro Reconstitution Assays of Arabidopsis 20S Proteasome
    Authors:  Yanjun Li, Di Sun, Xingxing Yan, Zhiye Wang and Xiuren Zhang, date: 04/05/2021, view: 348, Q&A: 0
    [Abstract]

    The majority of cellular proteins are degraded by the 26S proteasome in eukaryotes. However, intrinsically disordered proteins (IDPs), which contain large portions of unstructured regions and are inherently unstable, are degraded via the ubiquitin-independent 20S proteasome. Emerging evidence indicates that plant IDP homeostasis may also be

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    Colorimetric RT-LAMP and LAMP-sequencing for Detecting SARS-CoV-2 RNA in Clinical Samples
    [Abstract]

    During pandemics, such as the one caused by SARS-CoV-2 coronavirus, simple methods to rapidly test large numbers of people are needed. As a faster and less resource-demanding alternative to detect viral RNA by conventional qPCR, we used reverse transcription loop-mediated isothermal amplification (RT-LAMP). We previously established colorimetric

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    Real-time Base Excision Repair Assay to Measure the Activity of the 8-oxoguanine DNA Glycosylase 1 in Isolated Mitochondria of Human Skin Fibroblasts
    Authors:  Daniel Schniertshauer, Daniel Gebhard and Jörg Bergemann, date: 03/20/2021, view: 642, Q&A: 0
    [Abstract]

    7,8-dihydro-8-oxoguanine (8-oxoG) is one of the most common and mutagenic oxidative DNA damages induced by reactive oxygen species (ROS). Since ROS is mainly produced in the inner membranes of the mitochondria, these organelles and especially the mitochondrial DNA (mtDNA) contained therein are particularly affected by this damage. Insufficient

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    Ligand and Carbohydrate Engagement (LACE) Assay and Fluorescence Quantification on Murine Neural Tissue
    Authors:  James M. Clegg and Thomas Pratt, date: 03/20/2021, view: 504, Q&A: 0
    [Abstract]

    The interaction between cell surface heparan sulphate and diffusible ligands such as FGFs is of vital importance for downstream signaling, however, there are few techniques that can be used to investigate this binding event. The ligand and carbohydrate engagement (LACE) assay is a powerful tool which can be used to probe the molecular interaction

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