US 2011/0236997 A1Published application

Methods and Reagents for Shortening Incubation Times in Hybridization Assays

AssigneeSiemens Healthcare Diagnostics Inc.

InventorsThomas Battersby, Mark Baumeister, Jesse Brooks, Felix Kleshik, Stacey Tam

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Abstract

The methods and reagents described herein can be used to shorten incubation times in hybridization assays. As demonstrated in the examples, we have identified specific sulfonic acid polymers and hybridization conditions that lead to significantly shorter incubation times (e.g., signals after three hours that are comparable to signals that could traditionally only be obtained after overnight incubation). In some embodiments, shorter incubation times are achieved by adding the sulfonic acid polymer(s) during the hybridization process. Alternatively or additionally, in some embodiments, shorter incubation times are achieved via changes to the hybridizing conditions, e.g., by reducing the hybridization volume, increasing the salt concentration, and/or increasing the probe concentration (capture extender probe and/or label extender probe).

Claims39

Dependent claims

A solution nucleic acid hybridization assay for detecting a target nucleic acid that comprises first and second nucleic acid sequences comprising:

(a) contacting the target nucleic acid under hybridizing conditions with a sulfonic acid polymer or a salt thereof and an excess of (i) a label extender probe comprising first and second segments, wherein the first segment is complementary to the first nucleic acid sequence of the target nucleic acid and (ii) a capture extender probe comprising first and second segments, wherein the first segment is complementary to the second nucleic acid sequence of the target nucleic acid;
(b) contacting under hybridizing conditions the product of step (a) with a capture probe bound to a solid phase that is complementary to the second segment of the capture extender probe;
(c) removing materials not bound to the solid phase;
(d) contacting under hybridizing conditions the solid phase complex product of step (c) with a nucleic acid multimer that comprises (i) a preamplifier probe that is complementary to the second segment of the label extender probe and (ii) a multiplicity of amplifier probes;
(e) removing unbound nucleic acid multimer;
(f) contacting under hybridizing conditions the solid phase complex product of step (e) with a labeled oligonucleotide that is complementary to the amplifier probes of the nucleic acid multimer;
(g) removing unbound labeled oligonucleotide;
and
(h) detecting the presence of labeled oligonucleotide in the solid phase complex product of step (g),
wherein the sulfonic acid polymer or salt thereof in step (a) is of general formula (I) or (II):
or a salt thereof, wherein:
R1 is hydrogen or C1-6 aliphatic;
L is a covalent bond or C1-6 alkylene;
R3 is hydrogen or C1-6 alkyl;
and
n is an integer greater than 10.

13 more elements

A solution nucleic acid hybridization assay for detecting a target nucleic acid that comprises first and second nucleic acid sequences comprising:

(a) contacting the target nucleic acid under hybridizing conditions with a sulfonic acid polymer or a salt thereof and an excess of (i) a label extender probe comprising first and second segments, wherein the first segment is complementary to the first nucleic acid sequence of the target nucleic acid and (ii) a capture extender probe comprising first and second segments, wherein the first segment is complementary to the second nucleic acid sequence of the target nucleic acid;
(b) contacting under hybridizing conditions the product of step (a) with a capture probe bound to a solid phase that is complementary to the second segment of the capture extender probe;
(c) removing materials not bound to the solid phase;
(d) contacting under hybridizing conditions the solid phase complex product of step (c) with a nucleic acid multimer that comprises (i) a preamplifier probe that is complementary to the second segment of the label extender probe and (ii) a multiplicity of amplifier probes;
(e) removing unbound nucleic acid multimer;
(f) contacting under hybridizing conditions the solid phase complex product of step (e) with a labeled oligonucleotide that is complementary to the amplifier probes of the nucleic acid multimer;
(g) removing unbound labeled oligonucleotide;
and
(h) detecting the presence of labeled oligonucleotide in the solid phase complex product of step (g),
wherein the sulfonic acid polymer or salt thereof in step (a) is of general formula (III):
wherein:
R1 is hydrogen or C1-6 aliphatic;
R2 is a covalent bond, C1-6 aliphatic, phenyl, or a 6-membered heteroaryl ring having 1-3 nitrogens, wherein:
R2 is substituted with 1 or 2-SO3H groups or a salt thereof, provided that when R2 is a covalent bond, only 1 —SO3H group or a salt thereof is present, and
R2 is optionally substituted with 1-2 groups independently selected from —R3, halogen, —CN, —NO2, —OR3, —N(R3)2, and —SR3, wherein each R3 is independently hydrogen or a C1-6 alkyl group;
and
n is such that the sulfonic acid polymer or salt thereof has an average molecular weight of less than 10,000 Da.

14 more elements

Dependencies are inferred from the claim text.