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US 2022/0177953 A1Published application
Detection of double stranded nucleic acids in situ and methods related thereto
AssigneeAdvanced Cell Diagnostics, Inc.
InventorsXiao-Jun MA, Farzaneh TONDNEVIS, Courtney TODOROV, Bingqing ZHANG
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Abstract
The invention provides methods for detecting target double stranded nucleic acids, including for in situ hybridization assays. The invention also provides samples of fixed and permeabilized cells with a detected target double stranded nucleic acids as well as slides containing such samples. The invention additionally provides kits for detecting target double stranded nucleic acids.
Claims70
A method of detecting a double stranded nucleic acid comprising:
- (A) contacting a sample comprising a cell comprising one or more double stranded nucleic acids with a target probe set, wherein the target probe set comprises a pair of target probes comprising a first probe that specifically hybridizes to a first strand of a target double stranded nucleic acid, and a second probe that specifically hybridizes to the second strand of the double stranded nucleic acid;
- (B) contacting the sample with a pre-amplifier comprising a binding site for the first target probe, a binding site for the second target probe, and a plurality of binding sites for an amplifier;
- (C) contacting the sample with a plurality of amplifiers, wherein the amplifiers comprise a binding site for the pre-amplifier and a plurality of binding sites for a label probe;
- (D) contacting the sample with a plurality of label probes, wherein the label probe comprises a label and a binding site for the amplifier;
- and
- (E) detecting the label probes bound to the target double stranded nucleic acid, thereby detecting the target double stranded nucleic acid.
3 more elements
A sample of fixed and permeabilized cells, comprising:
- (A) at least one fixed and permeabilized cell containing a target double stranded nucleic acid;
- (B) a target probe set, wherein the target probe set comprises a pair of target probes comprising a first probe specifically hybridized to a first strand of a target double stranded nucleic acid, and a second probe specifically hybridized to the second strand of the double stranded nucleic acid;
- (C) a pre-amplifier comprising a binding site for the first target probe, a binding site for the second target probe, and a plurality of binding sites for an amplifier, wherein the pre-amplifier is hybridized to the first and second target probes;
- (D) a plurality of amplifiers, wherein the amplifiers comprise a binding site for the pre-amplifier and a plurality of binding sites for a label probe, and wherein the amplifiers are hybridized to the pre-amplifier;
- and
- (E) a plurality of label probes, wherein the label probe comprises a label and a binding site for the amplifier, wherein the label probes are hybridized to the amplifiers;
- wherein the hybridizations provide a detectable label on the target double stranded nucleic acid.
4 more elements
A slide comprising:
- (A) a slide having immobilized thereon a plurality of fixed and permeabilized cells comprising at least one fixed and permeabilized cell containing a target double stranded nucleic acid;
- (B) a target probe set, wherein the target probe set comprises a pair of target probes comprising a first probe specifically hybridized to a first strand of a target double stranded nucleic acid, and a second probe specifically hybridized to the second strand of the double stranded nucleic acid;
- (C) a pre-amplifier comprising a binding site for the first target probe, a binding site for the second target probe, and a plurality of binding sites for an amplifier, wherein the pre-amplifier is hybridized to the first and second target probes;
- (D) a plurality of amplifiers, wherein the amplifiers comprise a binding site for the pre-amplifier and a plurality of binding sites for a label probe, and wherein the amplifiers are hybridized to the pre-amplifier;
- and
- (E) a plurality of label probes, wherein the label probe comprises a label and a binding site for the amplifier, wherein the label probes are hybridized to the amplifiers;
- wherein the hybridizations provide a detectable label on the target double stranded nucleic acid.
4 more elements
A kit for detection of a target double stranded nucleic acid, comprising:
- (A) a pre-amplifier comprising a binding site for the first target probe, a binding site for the second target probe, and a plurality of binding sites for an amplifier;
- (B) a plurality of amplifiers, wherein the amplifiers comprise a binding site for the pre-amplifier and a plurality of binding sites for a label probe;
- and
- (C) a plurality of label probes, wherein the label probe comprises a label and a binding site for the amplifiers.
1 more element
A method of detecting a double stranded nucleic acid comprising:
- (A) contacting a sample comprising a cell comprising one or more double stranded nucleic acids with a target probe set, wherein the target probe set comprises a pair of target probes comprising a first probe that specifically hybridizes to a first strand of a target double stranded nucleic acid, and a second probe that specifically hybridizes to the second strand of the double stranded nucleic acid;
- (B) contacting the sample with a set of pre-pre-amplifiers comprising a first and second pre-pre-amplifier, wherein the first pre-pre-amplifier comprises a binding site for the first target probe, wherein the second pre-pre-amplifier comprises a binding site for the second target probe, and wherein the first and second pre-pre-amplifiers comprise a plurality of binding sites for a pre-amplifier;
- (C) contacting the sample with a plurality of pre-amplifiers, wherein the pre-amplifiers comprise a binding site for the first and second pre-pre-amplifiers and a plurality of binding sites for an amplifier;
- (D) contacting the sample with a plurality of amplifiers, wherein the amplifiers comprise a binding site for the pre-amplifiers and a plurality of binding sites for a label probe;
- (E) contacting the sample with a plurality of label probes, wherein the label probe comprises a label and a binding site for the amplifier;
- and
- (F) detecting the label probes bound to the target double stranded nucleic acid, thereby detecting the target double stranded nucleic acid.
4 more elements
A sample of fixed and permeabilized cells, comprising:
- (A) at least one fixed and permeabilized cell containing a target double stranded nucleic acid;
- (B) a target probe set, wherein the target probe set comprises a pair of target probes comprising a first probe specifically hybridized to a first strand of a target double stranded nucleic acid, and a second probe specifically hybridized to the second strand of the double stranded nucleic acid;
- (C) a set of pre-pre-amplifiers comprising a first and second pre-pre-amplifier, wherein the first pre-pre-amplifier comprises a binding site for the first target probe, wherein the second pre-pre-amplifier comprises a binding site for the second target probe, wherein the first and second pre-pre-amplifiers comprise a plurality of binding sites for a pre-amplifier, and wherein the pre-pre-amplifiers are hybridized to the first and second target probes;
- (D) a plurality of pre-amplifiers, wherein the pre-amplifiers comprise a binding site for the first and second pre-pre-amplifiers and a plurality of binding sites for an amplifier, wherein the pre-amplifiers are hybridized to the first and send pre-pre-amplifiers;
- (E) a plurality of amplifiers, wherein the amplifiers comprise a binding site for the pre-amplifiers and a plurality of binding sites for a label probe, wherein the amplifiers are hybridized to the pre-amplifiers;
- and
- (F) a plurality of label probes, wherein the label probe comprises a label and a binding site for the amplifiers, wherein the label probes are hybridized to the amplifiers;
- wherein the hybridizations provide a detectable label on the target double stranded nucleic acid.
5 more elements
A slide comprising:
- (A) a slide having immobilized thereon a plurality of fixed and permeabilized cells comprising at least one fixed and permeabilized cell containing a target double stranded nucleic acid;
- (B) a target probe set, wherein the target probe set comprises a pair of target probes comprising a first probe specifically hybridized to a first strand of a target double stranded nucleic acid, and a second probe specifically hybridized to the second strand of the double stranded nucleic acid;
- (C) a set of pre-pre-amplifiers comprising a first and second pre-pre-amplifier, wherein the first pre-pre-amplifier comprises a binding site for the first target probe, wherein the second pre-pre-amplifier comprises a binding site for the second target probe, and wherein the first and second pre-pre-amplifiers comprise a plurality of binding sites for a pre-amplifier, and wherein the pre-pre-amplifiers are hybridized to the first and second target probes;
- (D) a plurality of pre-amplifiers, wherein the pre-amplifiers comprise a binding site for the first and second pre-pre-amplifiers and a plurality of binding sites for an amplifier, wherein the pre-amplifiers are hybridized to the first and second pre-pre-amplifiers;
- (E) a plurality of amplifiers, wherein the amplifiers comprise a binding site for the pre-amplifiers and a plurality of binding sites for a label probe, wherein the amplifiers are hybridized to the first and second pre-amplifiers;
- and
- (F) a plurality of label probes, wherein the label probe comprises a label and a binding site for the amplifiers, wherein the label probes are hybridized to the amplifiers;
- wherein the hybridizations provide a detectable label on the target double stranded nucleic acid.
5 more elements
A kit for detection of a target double stranded nucleic acid, comprising:
- (A) a set of pre-pre-amplifiers comprising a first and second pre-pre-amplifier, wherein the first pre-pre-amplifier comprises a binding site for the first target probe, wherein the second pre-pre-amplifier comprises a binding site for the second target probe, and wherein the first and second pre-pre-amplifiers comprise a plurality of binding sites for a pre-amplifier;
- (B) a plurality of pre-amplifiers, wherein the pre-amplifiers comprise a binding site for the first and second pre-pre-amplifiers and a plurality of binding sites for an amplifier;
- (C) a plurality of amplifiers, wherein the amplifiers comprise a binding site for the pre-amplifiers and a plurality of binding sites for a label probe;
- and
- (D) a plurality of label probes, wherein the label probe comprises a label and a binding site for the amplifier.
2 more elements
A method of detecting a double stranded nucleic acid comprising:
- (A) contacting a sample comprising a cell comprising one or more double stranded nucleic acids with a target probe set, wherein the target probe set comprises a pair of target probes comprising a first probe that specifically hybridizes to a first strand of a target double stranded nucleic acid, and a second probe that specifically hybridizes to the second strand of the double stranded nucleic acid;
- (B) contacting the sample with a pre-pre-amplifier comprising a binding site for the first target probe, a binding site for the second target probe, and a plurality of binding sites for a pre-amplifier;
- (C) contacting the sample with a plurality of pre-amplifiers comprising a binding site for the pre-pre-amplifier, and a plurality of binding sites for an amplifier;
- (D) contacting the sample with a plurality of amplifiers, wherein the amplifiers comprise a binding site for the pre-amplifier and a plurality of binding sites for a label probe;
- (E) contacting the sample with a plurality of label probes, wherein the label probe comprises a label and a binding site for the amplifier;
- and
- (F) detecting the label probes bound to the target double stranded nucleic acid, thereby detecting the target double stranded nucleic acid.
4 more elements
A sample of fixed and permeabilized cells, comprising:
- (A) at least one fixed and permeabilized cell containing a target double stranded nucleic acid;
- (B) a target probe set, wherein the target probe set comprises a pair of target probes comprising a first probe specifically hybridized to a first strand of a target double stranded nucleic acid, and a second probe specifically hybridized to the second strand of the double stranded nucleic acid;
- (C) a pre-pre-amplifier comprising a binding site for the first target probe, a binding site for the second target probe, and a plurality of binding sites for a pre-amplifier, wherein the pre-pre-amplifier is hybridized to the first and second target probes;
- (D) a plurality of pre-amplifiers comprising a binding site for the pre-pre-amplifiers and a plurality of binding sites for an amplifier, wherein the pre-amplifiers are hybridized to the pre-pre-amplifier;
- (E) a plurality of amplifiers, wherein the amplifiers comprise a binding site for the pre-amplifiers and a plurality of binding sites for a label probe, and wherein the amplifiers are hybridized to the pre-amplifiers;
- and
- (F) a plurality of label probes, wherein the label probe comprises a label and a binding site for the amplifier, wherein the label probes are hybridized to the amplifiers;
- wherein the hybridizations provide a detectable label on the target double stranded nucleic acid.
5 more elements
A slide comprising:
- (A) a slide having immobilized thereon a plurality of fixed and permeabilized cells comprising at least one fixed and permeabilized cell containing a target double stranded nucleic acid;
- (B) a target probe set, wherein the target probe set comprises a pair of target probes comprising a first probe specifically hybridized to a first strand of a target double stranded nucleic acid, and a second probe specifically hybridized to the second strand of the double stranded nucleic acid;
- (C) a pre-pre-amplifier comprising a binding site for the first target probe, a binding site for the second target probe, and a plurality of binding sites for a pre-amplifier, wherein the pre-pre-amplifier is hybridized to the first and second target probes;
- (D) a plurality of pre-amplifiers comprising a binding site for the pre-pre-amplifier and a plurality of binding sites for an amplifier, wherein the pre-amplifier is hybridized to the pre-pre-amplifier;
- (E) a plurality of amplifiers, wherein the amplifiers comprise a binding site for the pre-amplifiers and a plurality of binding sites for a label probe, and wherein the amplifiers are hybridized to the pre-amplifiers;
- and
- (F) a plurality of label probes, wherein the label probe comprises a label and a binding site for the amplifier, wherein the label probes are hybridized to the amplifiers;
- wherein the hybridizations provide a detectable label on the target double stranded nucleic acid.
5 more elements
A kit for detection of a target double stranded nucleic acid, comprising:
- (A) a pre-pre-amplifier comprising a binding site for the first target probe, a binding site for the second target probe, and a plurality of binding sites for a pre-amplifier;
- (B) a pre-amplifier comprising a binding site for the pre-pre-amplifier and a plurality of binding sites for an amplifier;
- (C) a plurality of amplifiers, wherein the amplifiers comprise a binding site for the pre-amplifier and a plurality of binding sites for a label probe;
- and
- (D) a plurality of label probes, wherein the label probe comprises a label and a binding site for the amplifiers.
2 more elements
Dependencies are inferred from the claim text.