A Comparison of Detection Systems for an Enzyme-Linked Oligonucleotide Assay and the Chemical Biotinylation of Nucleic Acid Complexes

The detection and quantification of nucleic acids over a range of concentrations presents an analytical challenge. Thus, there is an urgency to develop more sensitive technologies that can quantify specific nucleic acid sequences with lower detection and quantification limits than traditional methods. Amplification methods such as polymerase chain reaction (PCR) are often non-linear and frequently make a transition from undetectable to a saturated reaction over a narrow concentration range. Biotinylation of deoxynucleic acid (DNA) is a precondition for detection with the universal alkaline phosphatase-streptavidin (AP-SA) enzyme detection conjugate. Conditions for biotinylating DNA in in vitro were explored with the observation that pre-heating or detergent had little effect on the access of biotinylation reagents to any reactive amines as assayed by colorimetric detection on polyvinylidene fluoride (PVDF) membrane. Biotin on a polyethylene glycol (PEG) spacer arm with the amine reactive Nhydroxysuccinimide (NHS) was a cost-effective and rapid method to chemically biotinylate oligonucleotides as a cost-effective, rapid, method of biotinylation for the purpose of direct serological detection of plasmid. Biotinylated DNA in buffer versus fetal bovine serum (FBS) were compared on PVDF and polystyrene solid support surfaces using colorimetric UV-Vis or enhanced chemiluminescence (ECL). At present, colorimetric or enhanced chemiluminescent detection systems may lack sensitivity with high occurrences of false positive and negative results. The focus of this thesis is to compare the quantification of DNA using a novel and robust enzyme-linked oligonucleotide hybridization assay using colorimetric, ECL and mass spectrometric (LC-ESI-MS) detection systems will be compared.