Patent ID: 6645721
Filing Date: 2003-11-11
Classification: B01J,B82B,B82Y,C07B,C07H,C12Q,C40B,Y10S

Abstract:
A method of detecting one or more nucleic acid targets, each having at least two portions, said method comprising:providing a substrate having one or more types of nanoparticles attached thereto, each type of nanoparticles having oligonucleotides attached thereto, the oligonucleotides of each type of nanoparticles having a sequence complementary to a first portion of the sequence of a specific nucleic acid target to be detected; providing one or more types of aggregate probes, each type of aggregrate probes comprising at least two types of nanoparticles having oligonucleotides attached thereto, the nanoparticles of each type of aggregate probe being bound to each other as a result of the hybridization of some of the oligonucleotides attached to them, at least one of the types of nanoparticles of each type of aggregate probes having oligonucleotides attached thereto which have a sequence complementary to a second portion of the sequence of said specific nucleic acid target to be detected; contacting one or more nucleic acid targets with one or more types of nanoparticles attached to the substrate under conditions effective to allow hybridization of the oligonucleotides on the nanoparticles with said nucleic acid targets; contacting said one or more nucleic acid targets bound to the substrate with one more types of aggregate probes under conditions effective to allow hybridization of the oligonucleotides on the aggregate probes with said nucleic acid targets, wherein in the presence of said nucleic acid targets and under hybridization conditions, the nanoparticles having oligonucleotides bound thereto form complexes with said nucleic acid targets, the resulting nanoparticle-nucleic acid target complexes having a sharp melting profile and an increased melting temperature, relative to a melting profile and a melting temperature of analogous complexes formed with said nucleic acid targets and an unlabeled or fluorophore-labeled oligonucleotides having a sequence identical to the oligonucleotides bound to the nanoparticles, to allow for selective discrimination of a single nucleotide insertion, deletion, or mismatch in said nucleic acid targets under stringent hybridization conditions, whereby the stringency of said hybridization conditions are higher than those possible for said analogous complexes; and observing a detectable change.