Molecular Biology


    Protocols in Current Issue
    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: 754, Q&A: 0

    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

    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: 735, Q&A: 0

    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

    SMART (Single Molecule Analysis of Resection Tracks) Technique for Assessing DNA end-Resection in Response to DNA Damage
    [Abstract] DNA double strand breaks (DSBs) are among the most toxic lesions affecting genome integrity. DSBs are mainly repaired through non-homologous end joining (NHEJ) and homologous recombination (HR). A crucial step of the HR process is the generation, through DNA end-resection, of a long 3′ single-strand DNA stretch, necessary to prime DNA synthesis ...
    Evaluation of the Efficiency of Genome Editing Tools by a Frameshift Fluorescence Protein Reporter
    Authors:  Balaji T. Moorthy, Akhilesh Kumar, Lauren X. Lotenfoe and Fangliang Zhang, date: 05/20/2020, view: 2147, Q&A: 0
    [Abstract] In the last decade, genome editing has been the center of attention as a novel tool for mechanistic investigations and for potential clinical applications. Various genome editing tools like meganucleases, zinc finger nucleases (ZFNs), transcription activator-like effector-based nucleases (TALEN), and the clustered regularly interspaced short ...
    Ex vivo Analysis of DNA Repair Capacity of Human Peripheral Blood Mononuclear Cells by a Modified Host Cell Reactivation Assay
    Authors:  Katja Matt and Jörg Bergemann, date: 08/05/2019, view: 2321, Q&A: 0
    [Abstract] The ability of humans to repair DNA damages decreases with increasing age. In order to be able to repair daily occurring DNA damages, it becomes more and more important to preserve repair capability of cells with aging. The preservation of DNA repair processes contributes to preventing DNA mutations and subsequently the onset of age-related ...
    Viral Chromosome Conformation Capture (V3C) Assays for Identifying Trans-interaction Sites between Lytic Viruses and the Cellular Genome
    Authors:  Kinjal Majumder, Maria Boftsi and David J Pintel, date: 03/20/2019, view: 2902, Q&A: 0
    [Abstract] The folding mechanisms of the mammalian genome package our genetic material into the nucleus, and in doing so, dictate its appropriate replication and expression. Chromosome conformation capture technology has enabled the dissection of the folding principles of the cellular genome. This has led to a better understanding of the role played by ...
    Microbial Mutagenicity Assay: Ames Test
    Authors:  Urvashi Vijay, Sonal Gupta, Priyanka Mathur, Prashanth Suravajhala and Pradeep Bhatnagar, date: 03/20/2018, view: 25901, Q&A: 2
    [Abstract] The Microbial mutagenicity Ames test is a bacterial bioassay accomplished in vitro to evaluate the mutagenicity of various environmental carcinogens and toxins. While Ames test is used to identify the revert mutations which are present in strains, it can also be used to detect the mutagenicity of environmental samples such as drugs, dyes, ...
    Determination of DNA Damage in the Retina Photoreceptors of Drosophila
    Authors:  Damulewicz Milena and Pyza Elzbieta, date: 02/05/2018, view: 4659, Q&A: 0
    [Abstract] The retina is sensitive for light damages, because of direct light exposure, especially intense blue and UV light, which increase level of ROS and other toxic phototransduction products in photoreceptor cells. In our previous work (Damulewicz et al., 2017a and 2017b), we used 8-oxo-deoxyguanosine (8-OHdG) as a marker for oxidative stress ...
    In vitro Assay to Measure DNA Polymerase β Nucleotide Insertion Coupled with the DNA Ligation Reaction during Base Excision Repair
    Authors:  Melike Çağlayan and Samuel H. Wilson, date: 06/20/2017, view: 5854, Q&A: 0
    [Abstract] We previously reported that oxidized nucleotide insertion by DNA polymerase β (pol β) can confound the DNA ligation step during base excision repair (BER) (Çağlayan et al., 2017). Here, we describe a method to investigate pol β nucleotide insertion coupled with DNA ligation, in the same reaction mixture including dGTP or 8-oxo-dGTP, pol β ...
    Targeted Nucleotide Substitution in Mammalian Cell by Target-AID
    Authors:  Takayuki Arazoe, Keiji Nishida and Akihiko Kondo, date: 06/05/2017, view: 12078, Q&A: 0
    [Abstract] Programmable RNA-guided nucleases based on CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated protein) systems have been applied to various type of cells as powerful genome editing tools. By using activation-induced cytidine deaminase (AID) in place of the nuclease activity of the CRISPR/Cas9 system, we have ...

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