Molecular Biology

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    Protocols in Current Issue
    Minigene Assay to Evaluate CRISPR/Cas9-based Excision of Intronic Mutations that Cause Aberrant Splicing in Human Cells
    Authors:  David J. Sanz and Patrick T. Harrison, date: 06/05/2019, view: 871, Q&A: 0
    [Abstract] The construction of Hybrid minigenes provides a robust and simple strategy to study the effects of disease-causing mutations on mRNA splicing when biological material from patient cells is not available. Hybrid minigenes can be used as splicing reporter plasmids allow RNA expression and heterologous splicing reactions between synthetic splicing ...
    Construction and Cloning of Minigenes for in vivo Analysis of Potential Splice Mutations
    Authors:  Lisa Maria Riedmayr, Sybille Böhm, Stylianos Michalakis and Elvir Becirovic, date: 03/05/2018, view: 4867, Q&A: 0
    [Abstract] Disease-associated mutations influencing mRNA splicing are referred to as splice mutations. The majority of splice mutations are found on exon-intron boundaries defining canonical donor and acceptor splice sites. However, mutations in the coding region (exonic mutations) can also affect mRNA splicing. Exact knowledge of the disease mechanism of ...
    Production of Guide RNAs in vitro and in vivo for CRISPR Using Ribozymes and RNA Polymerase II Promoters
    Authors:  Tao Zhang, Yangbin Gao, Rongchen Wang and Yunde Zhao, date: 02/20/2017, view: 9589, Q&A: 0
    [Abstract] CRISPR/Cas9-mediated genome editing relies on a guide RNA (gRNA) molecule to generate sequence-specific DNA cleavage, which is a prerequisite for gene editing. Here we establish a method that enables production of gRNAs from any promoters, in any organisms, and in vitro (Gao and Zhao, 2014). This method also makes it feasible to conduct ...
    Detection and Cloning of Spliced Transcripts by RT-PCR
    Authors:  Bianca Hoffmann and Bastian Grewe, date: 04/20/2013, view: 9283, Q&A: 0
    [Abstract] Using a Reverse Transcriptase-PCR approach spliced transcripts can be converted to cDNA, amplified and cloned into an expression plasmid. Sequencing of the obtained cDNA allows identification of the splicing events that generated the detected RNA (Grewe et al., 2012).



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