US 5,200,314 AGranted

Polynucleotide capture assay employing in vitro amplification

AssigneeChiron Corporation

InventorMichael Urdea

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Abstract

An analyte polynucleotide strand having an analyte sequence is detected within a sample containing polynucleotides by contacting the analyte polynucleotide with a capture probe under hybridization conditions, where the capture probe has a first binding partner specific for a solid-phase second binding partner. The resulting duplex is then immobilized by specific binding between the binding partners, and non-bound polynucleotides are separated from the bound species. The analyte polynucleotide is optionally displaced from the solid phase, then amplified by PCR. The PCR primers each have a polynucleotide region capable of hybridizing to a region of the analyte polynucleotide, and at least one of the primers further has an additional binding partner capable of binding a solid-phase binding partner. The amplified product is then separated from the reaction mixture by specific binding between the binding partners, and the amplified product is detected.

Claims24

Dependent claims

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.

8 more elements

An assay kit for amplifying and detecting an analyte polynucleotide strand having an analyte sequence within a sample containing polynucleotides, which kit comprises a package for:

a capture probe, said capture probe comprising an analyte-binding sequence complementary to a region of said analyte polynucleotide coupled to a displaceable first specific binding partner;
a first support, having bound thereto a second binding partner specific for said first partner;
a first primer complementary to a first primer-binding region of said analyte polynucleotide;
and
a second primer complementary to a second primer-binding region of said analyte-complementary strand, wherein said second primer-binding region does not substantially overlap said first primer-binding region and wherein said first binding partner comprises a first polynucleotide strand having a sequence which is not complementary to said analyte strand coupled to said primer by an arresting linker, and said second binding partner comprises a second polynucleotide strand having a sequence which is complementary to said first binding partner polynucleotide strand and is not complementary to said analyte strand.

2 more elements

Dependencies are inferred from the claim text.