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DNA Polymerase Activity Assay Using Near-infrared Fluorescent Labeled DNA Visualized by Acrylamide Gel Electrophoresis
Published on: October 6, 2017
Fluorescent xDNA nucleotides as efficient substrates for a template-independent polymerase
Sarah K Jarchow-Choy1, Andrew T Krueger, Haibo Liu
1Department of Chemistry, Stanford University, Stanford, CA 94305-5080, USA.
Nucleic Acids Research
|October 16, 2010
Summary
Terminal deoxynucleotidyl transferase (TdT) can incorporate novel, fluorescently-labeled expanded deoxynucleoside analogs (xDNA) into DNA. This enzymatic labeling method offers new possibilities for creating advanced DNA tags with unique fluorescence properties.
Area of Science:
- Biochemistry
- Molecular Biology
- Synthetic Biology
Background:
- Terminal deoxynucleotidyl transferase (TdT) is used for enzymatic DNA 3'-end labeling, often with fluorescent nucleotides.
- Existing fluorescent nucleotides can suffer from self-quenching and other undesirable fluorescence characteristics.
- Expanded deoxynucleoside analogs (xDNA) possess inherent fluorescence but are poorly accepted by template-dependent polymerases due to their size.
Purpose of the Study:
- To synthesize xDNA nucleoside triphosphates and evaluate their substrate capabilities with the template-independent enzyme TdT.
- To explore the potential of TdT-mediated incorporation of xDNA for novel DNA labeling applications.
- To characterize the fluorescence properties of incorporated xDNA monomers.
Main Methods:
- Synthesis of four benzo-expanded deoxynucleoside analogs (xDNA) as nucleoside triphosphates.
- Steady-state kinetic analysis of TdT's substrate efficiency for xDNA monomers.
- Enzymatic incorporation of xDNA monomers into DNA polymers using TdT.
- Fluorescence spectroscopy of xDNA polymers in solution and on a solid support.
Main Results:
- TdT efficiently incorporates all four xDNA monomers with kinetics comparable to natural nucleotides.
- Up to 30 consecutive xDNA monomers can be incorporated by TdT.
- Enzymatic incorporation of expanded adenine (dxATP) and cytosine (dxCTP) analogs resulted in observable fluorescence changes.
- Polymerized dxA showed monomer emission quenching and excimer formation, while polymerized dxC exhibited fluorescence enhancement.
Conclusions:
- TdT is a versatile enzyme capable of incorporating bulky xDNA monomers, overcoming limitations of template-dependent polymerases.
- TdT-mediated synthesis provides a novel route for creating fluorescent DNA labels and tags using xDNA.
- The distinct fluorescence behaviors of polymerized xDNA (dxA and dxC) offer unique properties for biotechnological applications.
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