Nucleic acid hybridization assays employing dA-tailed capture probes

Morrissey, David; Lombardo, Massimo; Eldredge, John; Kearney, Kevin R.; Groody, E. Patrick; Collins, Mark L.

doi:10.1016/0003-2697(89)90255-8

Cited by 54 publications

(22 citation statements)

References 19 publications

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“…For “sandwich” hybridization of sRNA-Capture Oligo and Oligo MagBeads, 50 μL of the pre-equilibriated (37 °C) Oligo MagBeads was added to the RNA/Capture Oligo mixture and incubated 37 °C for 15 min. Wash Solution prepared as described previously 45 was pre-equilibriated at 37 °C for 15 min before washing the beads. After magnetic capture with the Oligo MagBeads, they were washed three times by pipetting 300 μL of Wash Solution and discarding the supernatant from the collected Oligo MagBeads.…”

Section: Methodsmentioning

confidence: 99%

GRIL-seq provides a method for identifying direct targets of bacterial small regulatory RNA by in vivo proximity ligation

2016

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The first step in the post-transcriptional regulatory function of most bacterial small non-coding RNAs (sRNAs) is base-pairing with partially complementary sequences of targeted transcripts. We present a simple method for identifying sRNA targets in vivo and defining processing sites of the regulated transcripts. The technique (referred to as GRIL-Seq) is based on preferential ligation of sRNAs to ends of base-paired targets in bacteria co-expressing T4 RNA ligase, followed by sequencing to identify the chimeras. In addition to the RNA chaperone Hfq, the GRIL-Seq method depends on the activity of the pyrophosphorylase RppH. Using PrrF1, an iron-regulated sRNA in Pseudomonas aeruginosa, we demonstrate that direct regulatory targets of this sRNA can be readily identified. Therefore, GRIL-Seq represents a powerful tool not only for identifying direct targets of sRNAs in a variety of environments, but can also result in uncovering novel roles for sRNAs and their targets in complex regulatory networks.

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Section: Methodsmentioning

confidence: 99%

GRIL-seq provides a method for identifying direct targets of bacterial small regulatory RNA by in vivo proximity ligation

2016

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“…Reversible target capture is a solid-phase format that offers a distinct advantage over other formats in that it can be used with crude preparations without the generation of background noise that can lead to false-positive reactions (67,68,77). Magnetic microparticles are used to separate the reactants from the cell lysates.…”

Section: Filter Hybridizationsmentioning

confidence: 99%

“…The magnetic particle is removed from solution and washed free of contaminating materials. Chemical or thermal elution of the dA-dT pair and its recapture on fresh microparticles a few times leads to an exceedingly clean solid phase and increased sensitivity (67,68,77).…”

Section: Filter Hybridizationsmentioning

confidence: 99%

“…Another innovation in the use of Q3 replicase amplification came with the development of reversible target capture (14,68). A poly(dA)-tailed capture probe and magnetic microparticle beads containing poly(dT) are used to wash the reaction mixture clean of any residual contaminating materials, thereby lowering background noise (14,59,61).…”

Section: Cycling Probe Reactionmentioning

confidence: 99%

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Advances in nucleic acid-based detection methods

Wolcott

1992

Clin Microbiol Rev

151

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Laboratory techniques based on nucleic acid methods have increased in popularity over the last decade with clinical microbiologists and other laboratory scientists who are concerned with the diagnosis of infectious agents. This increase in popularity is a result primarily of advances made in nucleic acid amplification and detection techniques. Polymerase chain reaction, the original nucleic acid amplification technique, changed the way many people viewed and used nucleic acid techniques in clinical settings. After the potential of polymerase chain reaction became apparent, other methods of nucleic acid amplification and detection were developed. These alternative nucleic acid amplification methods may become serious contenders for application to routine laboratory analyses. This review presents some background information on nucleic acid analyses that might be used in clinical and anatomical laboratories and describes some recent advances in the amplification and detection of nucleic acids.

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“…In practice, however, their sensitivity has been limited by how well the probe-target hybrids can be separated from the large number of nonhybridized probes that are present to ensure that hybridization occurs rapidly. Despite the use of reversible target capture (16), which is an extremely efficient hybrid isolation procedure, we found that 10,000 nonhybridized probes could not be removed, generating a background signal that obscured the presence of rare targets (8).…”

mentioning

confidence: 99%

Extremely sensitive, background-free gene detection using binary probes and beta replicase.

Tyagi¹,

Landegren²,

Tazi³

et al. 1996

Proc. Natl. Acad. Sci. U.S.A.

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We have developed a specific and sensitive nucleic acid amplification assay that is suitable for routine gene detection. The assay is based on a novel molecular genetic strategy in which two different RNA probes are hybridized to adjacent positions on a target nucleic acid and then ligated to form an amplifiable reporter RNA. The reporter RNA is then replicated up to a hundred billion-fold in a 30-min isothermal reaction that signals the presence of the target. The assay can detect fewer than 100 nucleic acid molecules; it provides quantitative results over a wide range oftarget concentrations and it employs a universal format that can detect any infectious agent.

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Nucleic acid hybridization assays employing dA-tailed capture probes

Cited by 54 publications

References 19 publications

GRIL-seq provides a method for identifying direct targets of bacterial small regulatory RNA by in vivo proximity ligation

GRIL-seq provides a method for identifying direct targets of bacterial small regulatory RNA by in vivo proximity ligation

Advances in nucleic acid-based detection methods

Extremely sensitive, background-free gene detection using binary probes and beta replicase.

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