Non-isotopic hybridization assays for bacterial DNA samples / by Sotirios A. Daskalakis.
| Author/creator | Daskalakis, Sotirios A. author. |
| Other author | Kennedy, Kathleen E., 1946- degree supervisor. |
| Other author | East Carolina University. Department of Biology. |
| Format | Theses and dissertations |
| Production | 1990. |
| Description | 39 pages, 28 unnumbered leaves : illustrations ; 28 cm |
| Supplemental Content | Access via ScholarShip |
| Subjects |
| Summary | Hybridization is a powerful technique that has a wide area of application in the diagnosis of infectious and genetic diseases. With the increasing concern over problems associated with radioactive probes, many scientists have turned to non-radioactive labels as an attractive alternative. The most common non-radioactive label has been biotin which is used in conjunction with streptavidin or avidin in order to visualize the formed hybrids. However, the high background associated with this system when crude DNA extracts are assayed, has discouraged many scientists from using it. Efforts to further purify the DNA sample make a diagnostic assay laborious, expensive and long. The purpose of this research was to develop a rapid non-isotopic hybridization assay for bacterial genes. Five labelling methods were evaluated: nick translation and random priming with biotinylated nucleotides, sulfonation, photobiotinylation and end labelling with biotin. The first three were chosen for the development of the assays, based on high sensitivity of the resulting probes. Three assays are presented. One utilizes sulfone-modified probe and anti-sulfone antibodies. With this system, the background is completely eliminated. The other is an improvement of the biotin-streptavidin system, where the background is controlled reliably by using a standardized development time. A third assay is also presented, sandwich hybridization. The background is eliminated with this method, but it is laborious and long. Possible improvements are discussed. |
| General note | Submitted to the faculty of the Dept. of Biology. |
| General note | Advisor: Kathleen E. Kennedy |
| Dissertation note | M.S. East Carolina University 1990 |
| Bibliography note | Includes bibliographical references (leaves 35-39). |
| Genre/form | Academic theses. |
| Genre/form | Academic theses. |
| Genre/form | Thèses et écrits académiques. |
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