Hybridization assay signal enhancement
First Claim
1. A method comprising steps ofadding a homopolymeric region to a target nucleic acid analyte, which target nucleic acid analyte has a target sequence;
- contacting the analyte under hybridizing conditions with a multiplicity of reporter probes, said probes comprising a signal region and an oligonucleotide sequence, said oligonucleotide sequence being complementary to said analyte homopolymeric region, so as to form a stable hybrid of multiple reporter probes with said analyte homopolymeric region; and
forming an analyte;
capture probe hybrid by contacting the analyte target sequence with a capture probe under hybridizing conditions.
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Abstract
A signal amplification method for detecting a target nucleic acid analyte having a homopolymeric region and a target sequence includes steps of: contacting an analyte under hybridizing conditions with a multiplicity of reporter probes, each reporter probe including a signal region and an oligonucleotide sequence which is complementary to and capable of forming a stable hybrid with the analyte homopolymeric region to form an analyte:reporter probe hybrid; and forming an analyte:capture probe hybrid by contacting the analyte target sequence with a capture probe under hybridizing conditions. The analyte:reporter probe hybrid may formed prior to contacting the analyte target sequence with the capture probe, so the result of contacting the analyte target sequence with the capture probe results in formation of an analyte:reporter probe:capture probe complex. The analyte:capture probe hybrid may be immobilized on a solid generally planar surface in an array format. Multiple reporter probes may form triple helix structures for further signal enhancement. Multiply-labeled hairpin reporter probes may be employed according to the invention. Also, a kit for carrying out the invention includes one or more capture probes immobilized on a surface, reporter probes each having a signal region and a sequence for binding analyte homopolymer regions, and, optionally, reagents for addition of homopolymer regions to nucleic acid analytes and for carrying out the hybridization reaction.
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Citations
24 Claims
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1. A method comprising steps of
adding a homopolymeric region to a target nucleic acid analyte, which target nucleic acid analyte has a target sequence; -
contacting the analyte under hybridizing conditions with a multiplicity of reporter probes, said probes comprising a signal region and an oligonucleotide sequence, said oligonucleotide sequence being complementary to said analyte homopolymeric region, so as to form a stable hybrid of multiple reporter probes with said analyte homopolymeric region; and forming an analyte;
capture probe hybrid by contacting the analyte target sequence with a capture probe under hybridizing conditions. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 19, 20, 21, 22, 23, 24)
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18. A method comprising steps of
adding a 3'"'"'-poly A region to a target mRNA analyte in a biological sample; -
contacting the sample under hybridization conditions with a capture probe immobilized on a solid surface and a multiplicity of reporter probes, said probes comprising a signal region linked to a homopolymeric sequence comprising (dT)n (U)n or (U'"'"')n, where n is an integer between 8 and 30, wherein said signal region is comprised of fluorescently-labeled linear or hairpin oligonucleotides, so as to form a stable hybrid of multiple reporter probes with said analyte poly A region; and detecting a signal from said signal region.
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Specification