Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-10-13
2004-10-12
Sisson, Bradley L. (Department: 1634)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091100, C435S091200, C435S183000, C435S287200, C436S501000, C436S507000, C436S532000, C436S094000, C436S172000, C436S800000, C436S809000, C536S023100, C536S024300, C530S387100, C530S391100, C530S810000
Reexamination Certificate
active
06803196
ABSTRACT:
This invention relates generally to methods and compounds for the detection of polymers and for amplifying detectable signals in specific binding assays, particularly nucleic acid hybridization assays. Nucleic acid hybridizations are commonly used in biochemical research and diagnostic assays. Generally a single stranded analyte nucleic acid is hybridized to labeled nucleic acid probe, and resulting nucleic acid duplexes are detected. Radioactive and nonradioactive labels have been used. Methods also have been developed to amplify the signal that is detected.
Methods for the detection of nucleic acid sequences have suffered from drawbacks including background noise, time and labor requirements, lack of specificity and lack of sensitivity. It is an object of the invention to provide materials for the detection of polymers, particularly nucleic acids. It is a particular object of the invention to provide methods for labeling nucleic acid sequences in specific binding assays. It is a further object of the invention to provide methods and compounds for amplifying signals used in the detection of nucleic acid sequences in specific binding assays. It is an even further object of the invention to provide methods and compounds that permit nucleic acid sequences to be detected specifically and rapidly with high sensitivity and high resolution.
SUMMARY OF THE INVENTION
Methods and compounds are provided for detecting target molecules using specific binding assays. Methods and compositions are provided that are useful in signal amplification in the detection of target molecules.
In one embodiment, methods are provided for detecting a target molecule, wherein the method comprises hybridizing a target to an immobilized probe, wherein the target comprises a binding ligand. The hybridized target is contacted with a receptor that is capable of binding the binding ligand. Attached to the receptor is a detectable microparticle. The presence of the complexed microparticle is then detected.
Optionally, the presently claimed invention provides methods for amplifying the detectable signal. The method comprises hybridizing a target molecule to a probe wherein the target comprises a binding ligand. The hybridized target is contacted with a first receptor comprising multiple sites capable of binding the binding ligand to complex the receptor to the binding ligand, the first receptor may or may not be attached to a detectable moiety. The first receptor is contacted with a microparticle that is conjugated to a plurality of anti-receptors that themselves comprise a binding ligand. The microparticle may or may not be detectable. The anti-receptors are exposed to a second receptor that is conjugated to a detectable moiety. The first and second receptor may or may not be the same.
In one preferred embodiment, the probe is immobilized on a surface. The surface may be, for example, Langmuir Blodgett film, glass, germanium, silicon, (poly)tetrafluorethylene, polystyrene, gallium arsenide, gallium phosphide, silicon oxide, silicon nitride, and combinations thereof.
In a further embodiment, a method for detecting a nucleic acid target is provided, wherein the method includes providing a surface comprising at least 100 nucleic acid probes each in an area of less than about 0.1 cm
2
, and each nucleic acid probe having a defined sequence and location on the surface, and contacting the surface with a nucleic acid target to permit the nucleic acid target to hybridize with at least one selected nucleic acid probe, wherein the target comprises a binding ligand. The method further includes contacting the hybridized target with a receptor that is conjugated to a detectable microparticle. Alternatively, the method comprises contacting the hybridized target with a receptor comprising a plurality of binding sites which may or may not be conjugated to a detectable moiety; contacting the receptor with an anti-receptor comprising multiple binding ligands wherein the anti-receptor is conjugated to a microparticle which may or may not be detectable; and exposing the above complex to a plurality of second receptors which are themselves complexed to detectable moieties.
Also provided are complexes including a nucleic acid comprising a binding ligand; a receptor; and microparticle.
In another embodiment, there is provided a substrate comprising a surface having immobilized thereon a nucleic acid probe, comprising a probe nucleic acid sequence, hybridized to a nucleic acid target, comprising a target nucleic acid sequence; wherein the target comprises a binding ligand, and wherein the binding ligand on the target is complexed with a receptor comprising multiple sites capable of binding the binding ligand, and wherein the receptor is complexed to a detectable microparticle.
In a further embodiment, there is provided a substrate comprising a surface having immobilized thereon a nucleic acid probe hybridized to a nucleic acid target; wherein the target comprises a binding ligand, and wherein the binding ligand on the target is complexed with a first receptor comprising a plurality of binding sites which may or may not be conjugated to a detectable moiety; the first receptor being complexed to an anti-receptor comprising multiple binding ligands wherein the anti-receptor is conjugated to a microparticle which may or may not be detectable; and the multiple binding ligands on the anti-receptor being complexed to a plurality of second receptors which are themselves complexed to detectable moieties.
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Lyon William A.
Tran Huu Minh
Affymetrix Inc.
McGarrigle Philip L.
Sisson Bradley L.
Wells Sandra E.
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