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Fluorescent Based Assay for Group I Ribozymes using an ABI 310 Genetic Analyzer
Author(s) -
Stevens Timothy,
Rohlman Christopher
Publication year - 2010
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.24.1_supplement.882.5
Subject(s) - ribozyme , group ii intron , ligase ribozyme , rna , tetrahymena , rna splicing , intron , biology , capillary electrophoresis , fluorescence , computational biology , substrate (aquarium) , chemistry , biochemistry , microbiology and biotechnology , gene , ecology , physics , quantum mechanics
Group I introns are catalytic RNAs capable of performing a range of phosphotransesterification reactions including self splicing and RNA cleavage. To better understand their nature, variants of the Tetrahymena, Anabaena, and Twort Group I ribozymes and fluorescently tagged substrates were synthesized. We are currently developing an assay to utilize the capillary electrophoresis (CE) and spectroscopy capabilities found in an ABI 310 Genetic Analyzer. The goal of this assay is to acquire kinetic data for reactions catalyzed by wild‐type and variant Group I introns to better understand their catalytic nature. The ABI 310 was designed to function with fluoroscein based tags and a similar tag was selected for the substrate during these assays. CE will separate the bound ribozyme, unbound ribozyme, and free substrate. When combined with the fluorescent spectroscopy capabilities of the ABI produced quantifiable multiple turnover kinetics data with picomolar levels of ribozyme. This research was supported by the Albion College Foundation for Undergraduate Research, Scholarly and Creative Activity.