A Novel Approach to DNA Sequencing Employing Near -IR Fluorescence Detection Coupled With Microscale Reaction Vessels With Capillary Gel Separations.
Date of Award
Doctor of Philosophy (PhD)
Steven A. Soper
Electropherograms of oligonucleotides labeled with near-IR fluorescent dyes, separated by capillary gel electrophoresis (CGE) and detected using an ultrasensitive near-IR fluorescence detection system will be presented. A Universal M13 sequencing primer was labeled on the 5' end with a near-IR dye containing an isothiocyanate functional group. Comparison of the on-column detection limits in capillary gel electrophoresis for the near-IR-labeled sequencing primer to that of a visible fluorescein-labeled primer indicated improved sensitivity for the near-IR case. The detection limit was found to be 3.4 x 10-20 moles (SNR = 3) for the near-IR-labeled primer while the on-column detection limit for the fluorescein analog was 1.5 x 10-18 moles (SNR = 3). The sequence of nucleotide bases in an M13mp18 template was determined using a single lane, single dye technique. The molar concentrations of the ddNTP's used during chain extension were varied in order to achieve a ratio of 4:2:1:0 (A:C:G:T) which allowed the identification of each terminal base via fluorescence intensity measurements. Comparison of the known sequence of the M13mp18 plasmid to that obtained using this protocol yielded a base-calling accuracy of 84%. A catfish gene DNA template was attached to the interior wall of an aminoalkysilane derivatized (100 mum, 50 mum or 20 mum) fused-silica capillary tube via a biotin/streptavidin linkage. The DNA modified capillary reactor was then used to perform DNA sequencing reactions employing standard Sanger dideoxynucleotide termination protocols and the VentRTM DNA polymerase enzyme. Preliminary analysis of nano-reactor products was performed using a commercial fluorescence-based DNA sequencing instrument, the DuPont Genesis 2000. The nano-reactor was then directly interfaced to a capillary gel electrophoresis system to take advantage of the small sample requirements of CGE and create and integrated near-IR-LIF-based DNA analysis system. An investigation of the effect of varying capillary internal diameter (i.d.) on the separation efficiency is presented. Gel-filled columns with i.d.s of 20 mum, 50 mum, 75 mum and 100 mum were tested with an oligonucleotide standard, until failure, to determine the efficiency obtainable using these columns.
Williams, Daryl Clifford, "A Novel Approach to DNA Sequencing Employing Near -IR Fluorescence Detection Coupled With Microscale Reaction Vessels With Capillary Gel Separations." (1999). LSU Historical Dissertations and Theses. 7033.