Polynucleotide capture assay employing in vitro amplification

Claim 1

A method for detecting an analyte polynucleotide strand having an analyte sequence within a sample containing polynucleotides, which method comprises: a) contacting said analyte polynucleotide with a capture probe under hybridization conditions to form an analyte-capture probe complex, said capture probe comprising an analyte-binding region and a first specific binding partner, said analyte-binding region hybridizable with a region of said analyte polynucleotide, and said first specific binding partner having specificity for a second binding partner; b) contacting said first specific binding partner with said second binding partner, wherein said second binding partner is immobilized on a first support, whereby said analyte-capture probe complex is immobilized at said first support to provide an immobilized analyte-capture probe complex; c) separating nonbound polynucleotides from said immobilized analyte-capture probe complex; d) contacting said analyte polynucleotide with a first primer complementary to a first primer-binding region of said analyte polynucleotide under hybridizing conditions, said first primer comprising an analyte-hybridizing region and a third specific binding partner separated from one another by means for halting transcription therebetween; e) initiating nucleotide polymerization with polymerization means at said first primer to form an analyte-complementary strand duplex said complementary strand complementary to said analyte but not to said third specific binding partner; f) denaturing said analyte-complementary strand duplex to yield said complementary strand separated from said analyte polynucleotide; g) contacting said complementary strand with a second primer capable of hybridizing to a second primer-binding region of said complementary strand, and contacting said analyte polynucleotide with said first primer; h) initiating nucleotide polymerization with polymerization means to form an analyte-copy duplex having a strand complementary to a region of said complementary strand containing said first primer-binding region, and an analyte-complementary strand duplex; i) repeating steps f-h using the product of step h in place of the analyte-complementary strand duplex of step e to provide an amplified product comprising said first and second primers, amplified amounts of said analyte polynucleotide and a polynucleotide sequence complementary to said analyte sequence; and j) detecting said amplified product.

Claim 2

A method for detecting an analyte polynucleotide strand having an analyte sequence within a sample containing polynucleotides, which method comprises: a) contacting said analyte polynucleotide with a capture probe under hybridization conditions to form an analyte-capture probe complex, said capture probe comprising an analyte-binding region and a first specific binding partner, said analyte-binding region hybridizable with a region of said analyte polynucleotide, and said first specific binding partner having specificity for a second binding partner; b) contacting said first specific binding partner with said second binding partner, wherein said second binding partner is immobilized on a first support, whereby said analyte-capture probe complex is immobilized at said first support to provide an immobilized analyte-capture probe complex; c) separating nonbound polynucleotides from said immobilized analyte-capture probe complex; d) contacting said analyte polynucleotide with a first primer complementary to a first primer-binding region of said analyte polynucleotide under hybridizing conditions, said first primer comprising an analyte-hybridizing region and a third specific binding partner separated from one another by means for halting transcription therebetween; e) initiating nucleotide polymerization with polymerization means at said first primer to form an analyte-complementary strand duplex said complementary strand complementary to said analyte but not to said third specific binding partner; f) denaturing said analyte-complementary strand duplex to yield said complementary strand separated from said analyte polynucleotide; g) contacting said complementary strand with a second primer capable of hybridizing to a second primer-binding region of said complementary strand, and contacting said analyte polynucleotide with said first primer; h) initiating nucleotide polymerization with polymerization means to form an analyte-copy duplex having a strand complementary to a region of said complementary strand containing said first primer-binding region, and an analyte-complementary strand duplex; i) repeating steps f-h using the product of step h in place of the analyte-complementary strand duplex of step e to provide an amplified product comprising said first and second primers, amplified amounts of said analyte polynucleotide and a polynucleotide sequence complementary to said analyte sequence; and j) detecting said amplified product.