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Some studies on the use of polynucleotide phosphorylase in the synthesis of polynucleotides
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Smith, John C. (1975) Some studies on the use of polynucleotide phosphorylase in the synthesis of polynucleotides. PhD thesis, University of Warwick.
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Official URL: http://webcat.warwick.ac.uk/record=b1747688~S15
Abstract
The purification of polynucleotide phosphorylase(E.C.2. 7. 7. 8) by affinity chromatography was investigated. Several affinity ligands were prepared and one of them, oligo(dT) Sepharose, was found to be an efficient adsorbent for the enzyme.
Polynucleotide phosphorylase purified from Micrococcusluteus and Escherichia coli, was immobilized by covalent attachment to a number of insoluble supports. The properties of the immobilized enzyme were compared with those of the enzyme in free solution. It was found that while there was no significant change in the kinetic parameters of the enzyme, there was a significant increase in the stability of the immobilized derivatives. Studies were made of the use of the immobilized enzyme in the synthesis of polynucleotides.
Polynucleotide phosphorylase was isolated from Bacillus Stearothermophilus and was used to incorporate CDP residues into long oligo nucleotide primers such as oligoi(1))and t-RNA. Primers were isolated by chromatography on dihydroxyboryl Sepharoso which ensured that the primers possessed an intact 3'-hydroxyl group. Although incorporation of CDP into the primer was achieved, the number of residues incorporated was low. However, the method could be used to label the 3-terminus of a polynucleotide, in an Appendix, file purification of an endonuclease from pig liver nuclei is described. The enzyme was used in the preparation of oligonucleotide primers.
| Item Type: | Thesis (PhD) | ||||
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| Subjects: | Q Science > QD Chemistry | ||||
| Library of Congress Subject Headings (LCSH): | Nucleosides -- Synthesis, Nucleic acids -- Synthesis, Nucleotides, RNA polymerases | ||||
| Official Date: | November 1975 | ||||
| Dates: |
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| Institution: | University of Warwick | ||||
| Theses Department: | Department of Chemistry | ||||
| Thesis Type: | PhD | ||||
| Publication Status: | Unpublished | ||||
| Supervisor(s)/Advisor: | Hutchinson, D. W. (David Wesley) | ||||
| Sponsors: | Science Research Council (Great Britain) ; Searle Research Laboratories | ||||
| Extent: | 78 leaves : illustrations | ||||
| Language: | eng |
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