Nucleic acid hybridization assays employing dA-tailed capture probes. I. Multiple capture methods

Analytical Biochemistry
D V MorrisseyM L Collins

Abstract

A quantitative hybridization assay termed "reversible target capture" is described. The technique is designed to extensively purify the target nucleic acid from crude cell lysates in about 1 h without phenol extraction. Simple, rapid methods are described that explain how each process in the assay is optimized. The procedure involves hybridizing the target nucleic acid in solution with a dA-tailed capture probe and a labeled probe. The capture probe-target-labeled probe "ternary complex" is then captured on magnetic beads containing oligo(dT). After the excess unhybridized labeled probe, cell debris, and other sample impurities are washed away, the intact ternary complex is further purified by chemical elution from the beads and recapture on fresh beads. The ternary complex is then eluted thermally and recaptured on a third set of beads or on poly(dT) filters. This triple capture method results in a detection limit of approximately 0.2 amol (100 fg) of target with 32P-labeled riboprobes. This is approximately 1000 times more sensitive than sandwich assays employing only a single capture step. The method is illustrated by detecting Listeria cells in the presence of heterologous bacteria. With three rounds of target capture, as f...Continue Reading

References

Apr 1, 1985·Analytical Biochemistry·G BanfalviN Sarkar
Jun 1, 1989·Molecular and Cellular Probes·D V Morrissey, M L Collins
Jan 1, 1985·Gene·J A Langdale, A D Malcolm
Feb 1, 1986·Journal of Clinical Microbiology·A P KuritzaA A Salyers
Jul 1, 1965·Journal of Molecular Biology·D Gillespie, S Spiegelman
Oct 1, 1980·Gene·S G ArsenyanL L Kisselev
Jul 18, 1963·Biochemical and Biophysical Research Communications·A P NYGAARD, B D HALL

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Citations

Feb 1, 1992·Parasitology Today·S Dissanayake, W F Piessens
May 28, 1996·Proceedings of the National Academy of Sciences of the United States of America·S TyagiF R Kramer
Feb 1, 2005·Mycoses·E FaggiE Campisi
Feb 1, 1991·Molecular and Biochemical Parasitology·G X ChenJ W Zolg
Dec 29, 2017·European Journal of Clinical Microbiology & Infectious Diseases : Official Publication of the European Society of Clinical Microbiology·J S ShahR Ramasamy
Jan 1, 1990·Journal of Clinical Laboratory Analysis·C G Pritchard
Jun 1, 1997·Molecular and Cellular Probes·H J Blok, F R Kramer
Feb 1, 1991·Applied and Environmental Microbiology·H G Deneer, I Boychuk
Dec 17, 1990·Clinica Chimica Acta; International Journal of Clinical Chemistry·E P Diamandis
Oct 1, 1992·Clinical Microbiology Reviews·M J Wolcott
Oct 1, 1993·Journal of Clinical Microbiology·L J ChandlerB J Beaty
Jun 1, 1989·Molecular and Cellular Probes·D V Morrissey, M L Collins
Aug 1, 1990·Clinical Biochemistry·J D Thompson, D Gillespie

❮ Previous
Next ❯

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