Date of Award

1999

Publication Type

Doctoral Thesis

Degree Name

Ph.D.

Department

Chemistry and Biochemistry

Keywords

Biology, Molecular.

Rights

info:eu-repo/semantics/openAccess

Abstract

There is a growing demand for assay systems which permit detection of substances present at very low concentrations or in very small sample volumes. To this end we have used in vitro expression as part of an effective signal amplification system. In vitro expression contributes two levels of amplification; transcription of a DNA label to generate multiple mRNAs and translation of the mRNAs to generate multiple protein molecules which are used to initiate signal generation. We used a DNA label encoding the alpha-peptide of beta-galactosidase in an immunoassay. This label was linked to an analyte-specific antibody and, following immune complex formation, the immobilised label was expressed in vitro and alpha-complementation was performed. This assay facilitated detection of 3 fmol of analyte immobilised on the microtitre well surface. Various strategies were employed in attempts to increase the expression efficiency of the DNA label, including: (i) switching from a prokaryotic to a eukaryotic expression system, (ii) incorporation of a T7 promoter, (iii) addition of 5'-untranslated leader sequences and (iv) addition of downstream [dA/dT]30 sequences. In no case was an improvement in expression observed. A DNA label, containing the coding sequence for apoacquorin under the control of the T7 promoter and a downstream [dA/dT]30 sequence, was engineered and used in sensitive nucleic acid hybridisation assays. Cell-free expression of this template in a eukaryotic system, followed by aequorin regeneration, permitted production of, on average, 156 aequorin molecules per DNA. The DNA label, linked to a DNA probe and used in captured target and sandwich hybridisation assays, allowed detection of 0.5 and 0.25 amol of target DNA, respectively. We expanded the concept of expression as part of an amplification system in a technique for production of protein from traces of DNA. An isothermal, transcription-dependent amplification system was developed and linked to in vitro translation. The devised system allowed production of approximately 21 million molecules of aequorin from each DNA template in two hours. This work demonstrates that in vitro expression of an appropriate DNA can form part of an extremely efficient amplification system which may be successfully incorporated in a variety of applications.Dept. of Philosophy. Paper copy at Leddy Library: Theses & Major Papers - Basement, West Bldg. / Call Number: Thesis1999 .W55. Source: Dissertation Abstracts International, Volume: 61-09, Section: B, page: 4581. Thesis (Ph.D.)--University of Windsor (Canada), 1999.

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