Nucleic acid hybridization assays employing dA-tailed capture probes. I. Multiple capture methods.
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 few as six Listeria cells have been detected in the presence of 1.25 x 10(7) control cells.
Morrissey
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Gene-Trak Systems, Framingham, Massachusetts 01701.
Lombardo
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M
Eldredge
J K
JK
Kearney
K R
KR
Groody
E P
EP
Collins
M L
ML
D002021:Buffers; D002167:Campylobacter / Q000235:genetics; D002851:Chromatography, High Pressure Liquid; D015342:DNA Probes; D003839:Deoxyadenosines / Q000032:analysis*; D005374:Filtration; D006146:Guanidines; D008089:Listeria monocytogenes / Q000235:genetics; D008280:Magnetics; D008863:Microspheres; D009693:Nucleic Acid Hybridization; D009838:Oligodeoxyribonucleotides / Q000032:analysis; D011071:Poly T / Q000138:chemical synthesis; D011067:Poly dA-dT / Q000032:analysis; D012335:RNA, Ribosomal / Q000032:analysis; D013861:Thiocyanates / Q000032:analysis
D002021:Buffers; D015342:DNA Probes; D003839:Deoxyadenosines; D006146:Guanidines; D009838:Oligodeoxyribonucleotides; D012335:RNA, Ribosomal; D013861:Thiocyanates; C016069:poly (dA).oligo((d)T); D011071:Poly T; D011067:Poly dA-dT; C054436:guanidine thiocyanate