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Long Primer Extension by a Novel Inverse PCR Method
Bishop, Stephanie Cara
Bishop, Stephanie Cara
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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.
Description
Date
2009-05-21
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University of Kansas
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Keywords
Molecular biology, Atp synthase, Cloning, Gamma subunit, Insertion, Inverse pcr, Rotational kinetics