Nucleic acid hybridization assays employing dA-tailed capture probes. II. Advanced multiple capture methods.

Analytical Biochemistry
W R HunsakerM L Collins

Abstract

A fourth capture is added to the reversible target capture procedure of the preceding paper. This results in an improved radioisotopic detection limit of 7.3 x 10(-21) mol of target. In addition, the standard triple capture method is converted into a nonradioactive format with a detection limit of under 1 amol of target. The principal advantage of nonradioactive detection is that the entire assay can be performed in about 1 h. Nucleic acids are released from cells in the presence of the ('capture probe') which contains a 3'-poly(dA) sequence and the ('labeled probe') which contains a detectable nonradioactive moiety such as biotin. After a brief hybridization in solution, the target is captured on oligo(dT) magnetic particles. The target is further purified from sample impurities and excess labeled probe by recapture either once or twice more on fresh magnetic particles. The highly purified target is then concentrated to 200 nl by recapture onto a poly(dT) nitrocellulose filter and rapidly detected with streptavidin-alkaline phosphatase using bromochloroindolyl phosphate and nitroblue tetrazolium. Using this procedure, as little as 0.25 amol of a target plasmid has been detected nonradioactively in crude samples in just 1 h wit...Continue Reading

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Citations

Jun 1, 1989·Molecular and Cellular Probes·D V Morrissey, M L Collins
Jun 1, 1997·Molecular and Cellular Probes·H J Blok, F R Kramer
Aug 1, 1990·Clinical Biochemistry·J D Thompson, D Gillespie
Dec 29, 2017·European Journal of Clinical Microbiology & Infectious Diseases : Official Publication of the European Society of Clinical Microbiology·J S ShahR Ramasamy
Jan 4, 2008·The Journal of Infectious Diseases·Utpal PalErol Fikrig
Jan 1, 1990·Journal of Clinical Laboratory Analysis·C G Pritchard
Jan 29, 2002·Analytical Biochemistry·León F García-MartínezRobert Kovelman
Dec 17, 1990·Clinica Chimica Acta; International Journal of Clinical Chemistry·E P Diamandis

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