MARC 主機 00000nam 2200000 4500
001 AAI3253535
005 20071024130557.5
008 071024s2007 s eng d
035 (UMI)AAI3253535
040 UMI|cUMI
100 1 Silverman, Adam Phillip
245 10 Part I: Detection of RNA in cells with quenched
autoligation probes. Part II: Probing active site
tightness in nucleic acid replication enzymes|h[electronic
resource]
300 229 p
500 Source: Dissertation Abstracts International, Volume: 68-
02, Section: B, page: 0941
500 Adviser: Eric T. Kool
502 Thesis (Ph.D.)--Stanford University, 2007
520 This thesis concerns two research projects. In the first
project, the application of Quenched Autoligation (QUAL)
probes toward detection of RNA in bacteria is described.
QUAL probes are a class of self-reacting nucleic acid
probes that generate strong fluorescence signal only in
the presence of fully complementary RNAs and are highly
selective against single nucleotide mismatches. Probes
were introduced into live cells using small amounts of
detergent, thus eliminating the need for fixation, and
fluorescence signal was monitored by microscopy and flow
cytometry without any washing steps. Discrimination of the
closely related bacteria species E. coli, Salmonella
enterica, and Pseudomonas putida was achieved, based on
single nucleotide differences in their 16S rRNA. A FRET-
based QUAL system was developed to allow multiple colors
to be visualized under a single fluorescence filter set,
thereby simplifying detection protocols. The utility of
this approach was demonstrated in an application with
clinically important pathogens of the Shigella genus. The
results suggest that QUAL probes may be useful for rapid
identification of microorganisms in laboratory and
clinical settings
520 In the second project, a series of nonpolar thymidine and
uracil base mimics, varying in size over a 1.0-A range,
were used to probe the active site sterics of nucleic acid
replication enzymes. The nonpolar base analogs for
thymidine and uracil were dihalotoluene and dihalobenzene
C-nucleosides, respectively. The synthesis of these
compounds and their incorporation into DNA and RNA is
reported. Single nucleotide incorporation and extension
kinetic studies on human immunodeficiency virus type-1
reverse transcriptase (HIV-RT) were performed. Results
showed a large size dependence for the DNA polymerase
activity of HIV-RT, but a much smaller size dependence for
the reverse transcriptase activity. Kinetics were also
performed on E. coli Polymerases II and IV, two SOS-
induced replication enzymes. Results for these enzymes
showed generally poor incorporation and extension past
unnatural base pairs, but in the case of Pol II, very good
incorporation of the nonpolar dNTP analogs in a size-
dependent manner
590 School code: 0212
650 4 Chemistry, Analytical
690 0486
710 20 Stanford University
773 0 |tDissertation Abstracts International|g68-02B
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