US 5,627,030 A · Method of amplification for increasing the sensitivity of detecting nucleic acid-probe target hybrids

Abstract

The present invention provides a method for simplifying and significantly enhancing the sensitivity of nucleic acid hybridization assays. A method is described whereby a single-stranded primary nucleic acid sequence that includes a region of sequences complementary to a single-stranded target nucleic acid sequence is hybridized to the target molecule. Stability of the double-stranded complex thereby formed can be enhanced by using RNA as the probe if DNA is the target or DNA as the probe if RNA is the target. The probe-target complex is subsequently immunocaptured for detection. After washing away extraneous material, a secondary nucleic acid sequence containing many repeating sequence units is hybridized to the probe component of the immobilized probe-target complex. Detection occurs following hybridization of many labelled nucleic acid sequence probes to each of the repeating sequence units of a nucleic acid amplification probe. Thus, attachment of multiple labelling probes to an amplification probe that is hybridized to an immobilized probe-target complex, provides a simplified method for amplifying the detection signal and therefore the sensitivity of nucleic acid hybridization assays.

Claims20

Dependent claims

A method for enhancing detectable labeling of probe-target complexes in nucleic acid hybridization assays comprising hybridizing a complex of a selected nucleic acid target and a selected primary probe with an amplification probe adapted to permit enhanced detectable labelling of said selected nucleic acid target, said probe comprising at least two regions of nucleic acid sequences:

a first region including a sequence complementary to a sequence on said selected primary probe which also contains a sequence complemental to a sequence of said selected nucleic acid target, and a second region including a plurality of discretely labelable mixed polynucleotide sequence units linearly arranged.

A method for detecting specific nucleic acid sequences comprising:

(a) hybridizing a first sequence of a primary polynucleotide probe to a selectable target nucleic acid sequence wherein the primary probe has a means for binding to an amplification probe comprising a nucleic acid sequence adapted to permit enhanced detectable labelling, the amplification probe being capable of hybridizing to at least one labelling probe comprising a nucleic acid sequence conjugated to a chemical label;
(b) immobilizing the target-probe complex;
(c) exposing the immobilized target-probe complex to said amplification probe, such probe comprising at least two regions of nucleic acid sequences:
a first region including a sequence complementary to a sequence on a selected primary probe which also contains a sequence complementary to a sequence of said selected nucleic acid target, and a second region including a plurality of discretely labelable mixed polynucleotide sequence units linearly arranged, under conditions that allow the amplification probe to hybridize to the target-probe complex;
(d) exposing the hybridized amplification probe to a labelling probe covalently attached to a detectable chemical label, such probe comprising sequences complementary to sequences on the amplification probe, under conditions that allow many labelling probes to hybridize to the amplification probe;
(e) observing the presence or absence of the detectable chemical label, covalently attached to said labelling probe, in association with the sample as indicating the presence or absence of the target sequence.

3 more elements

Dependencies are inferred from the claim text.

Method of amplification for increasing the sensitivity of detecting nucleic acid-probe target hybrids

Life of this patent

Method of amplification for increasing the sensitivity of detecting nucleic acid-probe target hybrids

Life of this patent

Abstract

Claims20