Evaluation of "through bond" energy transfer cassettes, 2',3' - dideoxyribonucleotide triphosphate analogs, and a plasmid vector as tools for DNA analysis /
Three fluorescent energy transfer cassettes (BET-1, 2, and 3) were conjugated to a DNA oligonucleotide primer and evaluated as tools for DNA analysis. These dyes were made of three BODIPYO dyes with distinct emission maxima. Both the fluorescence energy transfer and the intensity of the emission sig...
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| Format: | Thesis Book |
| Language: | English |
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[Place of publication not identified] :
[publisher not identified] ;
1999.
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| Subjects: | |
| Online Access: | http://proxy.library.tamu.edu/login?url=http://proquest.umi.com/pqdweb?did=730298221&sid=1&Fmt=2&clientId=2945&RQT=309&VName=PQD |
| Summary: | Three fluorescent energy transfer cassettes (BET-1, 2, and 3) were conjugated to a DNA oligonucleotide primer and evaluated as tools for DNA analysis. These dyes were made of three BODIPYO dyes with distinct emission maxima. Both the fluorescence energy transfer and the intensity of the emission signal were reduced drastically in aqueous media once bound to DNA. Micelle formation was proved to be the reason, at least in part, of the observed results. Amplification of a 1.7 kb DNA fragment was attempted using the Polymerase Chain Reaction (PCR) where the BET-labeled oligonucleotides were one of the primers. Non-specific products were obtained and the overall yield of the reaction was reduced. These specificity problems were somewhat solved by reducing the annealing temperature in the thermalcycling conditions. Furthermore, BET-1 primer was used for the efficient labeling of the restriction fragments produced by Bam H I on lambda DNA. The samples were resolved by both agarose and polyacrylamide gel electrophoresis and thus its potential in DNA fingerprinting demonstrated. In addition, five new kinds of chain terminators for DNA sequencing were studied. Peptide nucleic acids (PNA) based dideoxynucleoside triphosphate (ddNTP) analogs containing thymine or adenine as the heterophile were incorporated by AmpliTaq[] DNA polymerase FS (Taq FS), Taquenase, and HIV-1 RT with different efficiencies. 3-nitropyrrole and 5-nitroindole analogs were not incorporated by any of the polymerases tested. A fifth terminator with a more rigid backbone was studied that was a good substrate for almost all the enzymes tested with Taq FS giving the best results. A redesigned double-stranded vector for the preparation of subclone shotgun libraries for DNA sequencing was prepared. New primer sites were introduced closer to the insert starting positions and their utility measured based on the average read length obtained when using them as sequencing primers. Finally, the synthesis of a photocleavable amino acid for a mass laddering method is described. This amino acid was incorporated into the sequence of several reptiles synthesized on a solid support. Prove of concept was achieved by analyzing the fragments produced after photolysis using Matrix-Assisted Laser Disorption/Ionization mass spectrometry. |
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| Item Description: | Vita. "Major Subject: Chemistry". In title numerals are used. |
| Physical Description: | xiii, 175 leaves : illustrations ; 28 cm. Issued also on microfiche from University Microfilm Inc. |
| Bibliography: | Includes bibliographical references (leaves 107-112). |