Use this URL to cite or link to this record in EThOS: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.473163
Title: Some studies on the use of polynucleotide phosphorylase in the synthesis of polynucleotides
Author: Smith, John C.
ISNI:       0000 0001 2443 7539
Awarding Body: University of Warwick
Current Institution: University of Warwick
Date of Award: 1975
Availability of Full Text:
Access from EThOS:
Access from Institution:
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.
Supervisor: Not available Sponsor: Science Research Council ; Searle Research Laboratories
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.473163  DOI: Not available
Keywords: QD Chemistry
Share: