Probe density considerations and elongation of self-complementary looped probes where probes are attached to a solid phase

a probe and loop technology, applied in biochemical equipment and processes, specific use bioreactors/fermenters, biomass after-treatment, etc., can solve the problems of reducing specificity, reducing detection sensitivity, and reducing the likelihood of target capture, so as to enhance detection sensitivity, expand the range of stringencies compatible, and enhance detection sensitivity

a probe and loop technology, applied in biochemical equipment and processes, specific use bioreactors/fermenters, biomass after-treatment, etc., can solve the problems of reducing specificity, reducing detection sensitivity, and reducing the likelihood of target capture, so as to enhance detection sensitivity, expand the range of stringencies compatible, and enhance detection sensitivity

US20070243534A1Inactive Publication Date: 2007-10-18SEUL MICHAEL +4
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  • Probe density considerations and elongation of self-complementary looped probes where probes are attached to a solid phase
  • Probe density considerations and elongation of self-complementary looped probes where probes are attached to a solid phase
  • Probe density considerations and elongation of self-complementary looped probes where probes are attached to a solid phase

Examples

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Effect test

example i

Homogeneous Beadchip Assay Using Looped Probes

[0057] A homogenous BeadChip assay format, shown in FIG. 1, was implemented by providing a variable gap configuration set to a large value during target capture and a smaller value during recording of assay images from a random encoded array of beads displaying self-complementary probes as well as positive and negative controls. The reaction volume was sealed by encapsulation of the reaction with mineral oil (from Sigma-Aldrich).

[0058] BeadChips were prepared to contain a random array composed of 4,000 beads of four types of color-encoded microparticles (“beads”) on a 375-μm thick n-type Silicon substrate. Color-coding was achieved by staining the beads in accordance with a solvent tuning method described in U.S. application Ser. No. 10 / 348,165 (incorporated by reference). Stained beads were functionalized by covalent attachment of streptavidin to permit subsequent attachment of biotinylated self-complementary (“looped”) probes, illus...

example 2

Homogenous Assay in Suspension of Encoded Beads

[0062] The looped-probe design also can be used in a homogenous format with encoded beads in suspension, as described in U.S. Pat. No. 6,251,691; U.S. application Ser. No. 10 / 204,799 (incorporated by reference). As shown in FIG. 9, a reaction mixture in a sealed incubation chamber, or cartridge, may contain T7-tagged DNA template, components for in-vitro transcription reaction such as a T7 RNA polymerase, well known in the art, and looped-probe functionalized color-coded beads, each color corresponding to a unique capture probe sequence. Preferably, encoded magnetic beads are used (see U.S. application Ser. No. 11 / 218,838), and a random array of such beads is assembled in real time following completion of the assay, as described in U.S. Pat. No. 6,251,691; U.S. application Ser. No. 10 / 204,799.

[0063] Two sets of magnetic beads (Spherotech, 4.10 μm in diameter, ρ˜1.13 g / ml), one encoded with a green dye by solvent-tuning (REF—Solvent Tu...

example iii

Homogeneous Binding Assay in Suspension Using Looped Probes Immobilized on Magnetic Beads

[0067] Looped probes were immobilized on color-encoded magnetic microparticles (“beads”) for use in a homogeneous binding assay. Briefly, magnetic beads of ˜4 micron diameter were synthesized by standard methods and color-encoded as set forth in U.S. application Ser. No. 10 / 348,165, incorporated by reference. Next, encoded beads were modified by covalent attachment of Neutravidin to epoxy groups on the beads to permit: attachment of a “perfect-match (PM)” biotinylated looped probe, a “no-match (NM)” biotinylated looped probe, and a biotinylated positive control, in the form of a Cy3-labeled oligonucleotide.

[0068] As in the previous examples, looped-probes contain a donor dye and an acceptor dye at their respective 5′ and 3′ ends. Aliquots of probe-decorated, encoded magnetic beads were pooled in one test tube for determination of RNA target concentrations.

[0069] To determine the response of t...

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Abstract

In a multiplexed assay method carried out in solution, wherein the solution contains nucleic acid targets and, wherein several different types of oligonucleotide probes, each type having a different sequence in a region designated as a target binding domain, are used to detect the nucleic acid targets, said assay method including a method for increasing the effective concentration of the nucleic acid targets at the surface of a bead to which the oligonucleotide probes are bound, by one or more of the following steps: adjusting assay conditions so as to increase the effective concentration of the targets available for binding to the probes, by one or more of the following: (i) selecting a particular probe density on the surface of the bead; (ii) selecting a solution having an ionic strength greater than a threshold; (ii) selecting a target domain of a size less than a threshold; or (iii) selecting target domains within a specified proximity to a terminal end of the targets.

Description

BACKGROUND [0001] Molecular Stringency in Multiplexed Assays—A self-complementary oligonucleotide capture probe in a “looped” configuration may be used to adjust molecular stringency in an assay. Assay stringency relates to the positive results produced by an assay, such that high stringency conditions generate relatively fewer positive results than lower stringency conditions. Looped probes are described in WO 01 / 98765, entitled: “Multianalyte Molecular Analysis Using Application-Specific Random Particle Arrays” and U.S. Pat. No. 6,361,945 (assigned to Gen Probe, Inc.). Such a probe consists of a 5′-terminal subsequence and a complementary 3′-terminal subsequence, tethered by an unrelated subsequence, the two terminal subsequences capable of forming a duplex (“stem”), and the tether forming a loop, and either the 5′-terminal subsequence of the 3′-terminal subsequence capable of forming a duplex with a target nucleic acid. The probe may be attached to a solid phase such as an encode...

Claims

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Application Information

Patent Timeline
18 Oct 2007
Publication
US20070243534A1
IPC
C12Q1/68; C12M3/00
CPC
C12Q1/6818; C12Q1/6827; C12Q1/6837; C12Q2565/1015; C12Q2537/143; C12Q2533/101; C12Q2535/125; C12Q2525/301
Inventors
SEUL, MICHAEL; ZHANG, YI