Sequential injection analysis system for the sandwich hybridization-based detection of nucleic acids

Analytical Chemistry
Katie A Edwards, Antje J Baeumner

Abstract

A sequential injection analysis lab-on-valve (SIA-LOV) system was developed for the specific detection of single-stranded nucleic acid sequences via sandwich hybridization of specific DNA probes to the target sequence. One DNA probe was tagged with fluorescein; the other was biotinylated and immobilized to streptavidin-coated porous beads. The system was optimized with respect to buffer composition, length of hybridization and wash steps, and volumes and concentrations of components used. On-bead oligonucleotide hybridization was studied using UV detection at 260 nm, while a final dose response curve was quantified using fluorescence detection. A dynamic range of 1-1000 pmol was obtained for a synthetic DNA sequence that was homologous to a segment in the B. anthracis atxA mRNA. A within-day variation of 7.2% and a day-to-day variation of 9.9% was observed. Each analysis was completed within 20 min. Subsequently, the system was applied to the detection of atxA mRNA expressed in a surrogate organism and amplified using NASBA. The SIA-LOV will find its application in routine laboratory-based analysis of specific single-stranded DNA/RNA sequences. Future improvements will include the integration of dye-encapsulating liposomes for ...Continue Reading

References

Jul 1, 1992·Analytical Chemistry·C H PollemaA Lernmark
Mar 1, 1990·Journal of Clinical Microbiology·R BoomJ van der Noordaa
Jul 10, 1987·Nucleic Acids Research·T R GingerasG R Davis
Apr 16, 1999·Analytical Chemistry·J Ruzicka, L Scampavia
Sep 17, 1999·International Journal of Food Microbiology·E de Boer, R R Beumer
Feb 22, 2000·Annals of the New York Academy of Sciences·J A HigginsE A Henchal
Sep 20, 2000·Analytical Chemistry·C J Bruckner-LeaD P Chandler
Nov 18, 2000·Analytical Chemistry·E ThordarsonJ Emneus
Feb 24, 2001·Biosensors & Bioelectronics·S S IqbalJ P Chambers
Mar 6, 2002·Molecular Biotechnology·Birgit DeimanPeter Sillekens
Apr 4, 2003·International Journal of Food Microbiology·Burkhard MalornyJeffrey Hoorfar
May 7, 2003·Analytical and Bioanalytical Chemistry·Harriet A Hartley, Antje J Baeumner
May 25, 2004·Nucleic Acids Research·Fang YuWolfgang Knoll
Nov 13, 2004·Journal of Agricultural and Food Chemistry·M J Reis LimaAntónio O S S Rangel
Nov 30, 2004·The Lancet Infectious Diseases·Remco P H PetersChristina M J E Vandenbroucke-Grauls

❮ Previous
Next ❯

Citations

Mar 11, 2008·Analytical and Bioanalytical Chemistry·Katie A EdwardsAntje J Baeumner

❮ Previous
Next ❯

Related Concepts

Related Feeds

Anthrax Vaccines

Three different types of anthrax vaccines are available; a live-attenuated, an alum-precipitated cell-free filtrate and a protein recombinant vaccine. The effectiveness between the three is uncertain, but the live-attenuated have shown to reduce the risk of anthrax with low adverse events. Here is the latest research on anthrax vaccines.

Anthrax

Anthrax toxin, comprising protective antigen, lethal factor, and oedema factor, is the major virulence factor of Bacillus anthracis, an agent that causes high mortality in humans and animals. Here is the latest research on Anthrax.

Anthrax Vaccines (ASM)

Three different types of anthrax vaccines are available; a live-attenuated, an alum-precipitated cell-free filtrate and a protein recombinant vaccine. The effectiveness between the three is uncertain, but the live-attenuated have shown to reduce the risk of anthrax with low adverse events. Here is the latest research on anthrax vaccines.