Nanoparticles having oligonucleotides attached thereto and uses thereof

A method of detecting one or more nucleic acid targets, each having at least two portions comprising:

• providing a substrate having one or more types of first nanoparticles attached thereto, the first nanoparticles having oligonucleotides attached thereto, the oligonucleotides of each type of first nanoparticles having a sequence complementary to a first portion of the sequence of a specific nucleic acid target to be detected;
• contacting one or more nucleic acid targets with the first nanoparticles attached to the substrate under conditions effective to allow hybridization of the oligonucleotides on the first nanoparticles with said nucleic acid targets;
• providing one or more types of second nanoparticles having oligonucleotides attached thereto, the oligonucleotides of each type of second nanoparticles having a sequence complementary to one or more other portions of the sequence of said specific nucleic acid target to be detected;
• contacting said nucleic acid targets bound to the substrate with the second type of nanoparticles under conditions effective to allow hybridization of the oligonucleotides on the second type of nanoparticles with said nucleic acid targets;
• providing a binding oligonucleotide having a selected sequence having at least two portions, the first portion being complementary to at least a portion of the sequence of the oligonucleotides on the second nanoparticles;
• contacting the binding oligonucleotide with the second nanoparticles bound to the substrate under conditions effective to allow hybridization of the binding oligonucleotides to the oligonucleotides on the second nanoparticles;
• providing a third nanoparticles having oligonucleotides attached thereto, the oligonucleotides of said third nanoparticles having a sequence complementary to the sequence of a second portion of the binding oligonucleotide;
• contacting the third nanoparticles with the binding oligonucleotide bound to the substrate under conditions effective to allow hybridization of the binding oligonucleotide to the oligonucleotides on the nanoparticles;
• and
• observing a detectable change, wherein in the presence of said one or more nucleic acid targets and under hybridization conditions, said nanoparticles having oligonucleotides bound thereto form complexes with said nucleic acid targets and said binding oligonucleotides, 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, an unlabeled or fluorophore-labeled oligonucleotides having a sequence identical to the oligonucleotides bound to the nanoparticles, and said binding oligonucleotide, 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.

A method of detecting one or more nucleic acid targets, each having at least two portions comprising:

• contacting one or more nucleic acid targets to be detected with a substrate having one or more types of oligonucleotides attached thereto, each type of oligonucleotides having a sequence complementary to a first portion of the sequence of a specific nucleic acid target to be detected, the contacting taking place under conditions effective to allow hybridization of the oligonucleotides on the substrate with said nucleic acid targets;
• contacting one or more nucleic acid targets bound to the substrate with one or more types of first nanoparticles having one or more types of oligonucleotides attached thereto, at least one of the types of oligonucleotides bound to each type of nanoparticles having a sequence complementary to a second portion of the sequence of said nucleic acid target to be detected, the contacting taking place under conditions effective to allow hybridization of the oligonucleotides on the nanoparticles with said nucleic acid targets;
• contacting said first nanoparticles bound to the substrate with one or more types of second nanoparticles having oligonucleotides attached thereto, the oligonucleotides on each type of second nanoparticles having a sequence complementary to at least a portion of the sequence of one of the types of oligonucleotides on each type of said first nanoparticles, the contacting taking place under conditions effective to allow hybridization of the oligonucleotides on the first and second nanoparticles;
• and
• observing a detectable change, wherein in the presence of said one or more nucleic acid targets and under hybridization conditions, said first and second 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, 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.

A method of detecting one or more nucleic acid targets, each having at least two portions comprising:

• contacting one or more specific nucleic acid targets to be detected with a substrate having one or more types of oligonucleotides attached thereto, each type of oligonucleotides having a sequence complementary to a first portion of the sequence of a specific nucleic acid target to be detected, the contacting taking place under conditions effective to allow hybridization of the oligonucleotides on the substrate with said nucleic acid targets;
• contacting said nucleic acid targets bound to the substrate with one or more types of nanoparticles having oligonucleotides attached thereto, the oligonucleotides of each type of nanoparticles having a sequence complementary to a second portion of the sequence of said specific nucleic acid target to be detected, the contacting taking place under conditions effective to allow hybridization of the oligonucleotides on the nanoparticles with said nucleic acid targets;
• contacting the substrate with silver stain to produce a detectable change;
• and
• observing the detectable change, wherein in the presence of said one or more nucleic acid targets and under hybridization conditions, said nanoparticles having oligonucleotides bound thereto form complexes with said nucleic acid targets and said oligonucleotides bound to the substrate, 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 an unlabeled or fluorophore-labeled oligonucleotides having a sequence identical to the oligonucleotides bound to the nanoparticles, and oligonucleotides bound to the substrate, 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.