Application of Capillary Electrophoresis Laser Induced Fluorescence to the Detection of Nucleic Acid Probe Fragments

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Authors
  1. Boulet, C.A.
  2. Hung, G.
  3. Bader, D.E.
Corporate Authors
Defence Research Establishment Suffield, Ralston ALTA (CAN)
Abstract
The Canadian Forces require rapid, sensitive systems for identification of bacterial and viral biological warfare (BW) agents in environmental matrices such as air and water. The system must detect BW agents at extremely low concentrations with no false alarms and operate under battlefield conditions. Gene probes can be used to target signature sequences of BW agents for identification; Cycling Probe Technology (CPT (TRADEMARK)) is a gene probe identification technique based on the use of target nucleic acid as a catalyst for the conversion of chimeric probe moelcules to detectable products. Capillary zone electrophoresis with laser induced flurorescent detection (CE-LIF) is an attractive technology for the detection of nucleic acid fragments because of its ultra-low detection limits and relatively simple instrument design. Together, CE-LIF detection of gene probe fragments offers the potential for developing a sensitive BW agent identification capability. In this work, a chimeric 5' (DNA)8(RNA)4(DNA)16 3' probe for Bacillus globigii, an anthrax simulant, designated as DRES2A, was used. In a typical experiment, the 5' fluoresceinated DRES2A probe (10 fmoles/ mu l) and synthetic target DNA (E4 - E7 pmoles/mu l) were incubated at 65C for 30 min in the presence of RNase H. A 1:10 dilution of CPT reaction mixture was then analyzed by CE-LIF. TRUNCATED
Keywords
Genetic analysis;Capillary Electrophoresis (CE);Laser Induced Fluorescence (LIF);Cycling Probe Technology;CE-LIF;Nucelic acid probes;Oligonucleotide analysis;BWA identification;Biological Warfare Agents;Biological agent simulants;Bacillus globigii
Report Number
DRES-TM-1999-081 — Technical Memorandum
Date of publication
01 Sep 1999
Number of Pages
23
DSTKIM No
CA000078
CANDIS No
511989
Format(s):
Hardcopy;Document Image stored on Optical Disk

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