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US 5,683,875 AGranted
Method for detecting a target nucleic acid analyte in a sample
AssigneeHewlett-Packard Company
InventorKay Lichtenwalter
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Abstract
A method is provided for detecting a target nucleic acid analyte in a sample. The method involves preparing an immobilized capture oligonucleotide that is complementary to the target analyte using a magnetic cycling method, incubating the sample with the immobilized capture oligonucleotide to capture the target analyte thereby forming a capture oligonucleotide-analyte complex, and detecting the presence of the capture oligonucleotide-analyte complex.
Claims8
An assay method for detecting a nucleic acid analyte in a sample, comprising:
- (a) a capture oligonucleotide synthesis step of:
- (i) contacting a template oligonucleotide complementary to a target oligonucleotide and linked to a magnetically responsive particle with a primer oligonucleotide immobilized to a substrate, said primer oligonucleotide being complementary to a portion of the template oligonucleotide;
- (ii) incubating the template oligonucleotide and the primer oligonucleotide under hybridizing conditions to form a template-primer complex;
- (iii) adding a polynucleotide polymerase under polymerization conditions, whereby the primer oligonucleotide is extended to provide an immobilized capture oligonucleotide complementary to the template oligonucleotide;
- (iv) applying an electromagnetic field effective to de-hybridize the template oligonucleotide from the capture oligonucleotide;
- and (v) washing the immobilized capture oligonucleotide;
- (b) a capturing step of:
- (i) contacting the immobilized capture oligonucleotide synthesized in step (a) with a nucleic acid analyte containing or suspected of containing the target oligonucleotide;
- (ii) incubating the capture oligonucleotide with the nucleic acid analyte under hybridizing conditions to form a capture oligonucleotide-analyte complex;
- and (iii) washing to provide an immobilized capture oligonucleotide-analyte complex substantially free of unbound analytes;
- and
- (c) a detecting step of:
- (i) contacting the immobilized capture oligonucleotide-analyte complex obtained in step (b) with a detectably labeled analyte-binding molecule;
- and (ii) detecting the amount of bound or unbound label.
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Dependencies are inferred from the claim text.