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    Long Primer Extension by a Novel Inverse PCR Method

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    Bishop_ku_0099M_10232_DATA_1.pdf (2.245Mb)
    Issue Date
    2009-05-21
    Author
    Bishop, Stephanie Cara
    Publisher
    University of Kansas
    Format
    44 pages
    Type
    Thesis
    Degree Level
    M.A.
    Discipline
    Biochemistry & Molecular Biology
    Rights
    This item is protected by copyright and unless otherwise specified the copyright of this thesis/dissertation is held by the author.
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    Abstract
    An inverse polymerase chain reaction (PCR) was employed to construct an engineered F1-ATPase by means of inserting the repressor of primer (Rop) DNA sequence into the region of the ATP synthase gamma (γ) subunit DNA sequence encoding a regulatory dithiol-containing domain. A two-step PCR approach was developed to insert two unusually long (>100 base pairs each) primers encoding 189 base pairs of exogenous DNA into a single site within a pACYC multiple cloning host vector. The construct was verified by means of DNA sequencing. This approach allowed direct insertion of large pieces of DNA into a host DNA molecule without introducing restriction enzyme sites, thus avoiding common shortcomings such as inclusion or omission of base pairs that were associated with traditional subcloning methods. The engineered gamma subunit was designed for assembly with the recombinant alpha (α) and beta (β) subunits into a core F1-ATPase. The rigid twisted helical structure of the Rop protein extended the regulatory domain of the gamma subunit by approximately 60 Ångstroms, thus creating a rigid, rotating armature within the enzyme. The armature is intended for use as a site for attachment of gold particles to monitor rotation of the gamma subunit during ATP hydrolysis.
    URI
    http://hdl.handle.net/1808/5448
    Collections
    • Theses [3710]
    • Molecular Biosciences Dissertations and Theses [182]

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    785-864-8983

    KU Libraries
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    Lawrence, KS 66045
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    Contact KU ScholarWorks
    785-864-8983
    KU Libraries
    1425 Jayhawk Blvd
    Lawrence, KS 66045
    785-864-8983

    KU Libraries
    1425 Jayhawk Blvd
    Lawrence, KS 66045
    Image Credits
     

     

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