Solid phase based nucleic acid assays combining high affinity and high specificity
First Claim
1. A solid phase based nucleic acid assay that can distinguish alleles that comprise a single nucleotide polymorphism in a sample containing a target nucleic acid molecule comprising a) immobilizing the target nucleic acid molecule to a solid support using i) one or more capture oligonucleotides that are immobilized to the solid support, that hybridize to the target molecule and that have terminal nucleotides blocked and/or unphosphorylated terminal nucleotides and ii) a discrimination oligonucleotide that are immobilized to the solid support, that hybridize to the target molecule wherein a nucleotide at a terminus of the discrimination oligonucleotide is complementary to the single nucleotide polymorphism position and is unblocked and/or phosphorylated;
- b) performing a reaction on any unblocked and/or phosphorylated terminal nucleotide hybridized to a complementary nucleotide, the reaction being specific for unblocked and/or phosphorylated terminal nucleotide perfectly hybridized to a complementary nucleotide; and
c) determining if any reaction occurred, wherein detecting a reaction of unblocked and/or phosphorylated terminal nucleotide perfectly hybridized to a complementary nucleotide indicates the discrimination oligonucleotide hybridized perfectly to the target nucleic acid molecule at the single nucleotide polymorphism position.
5 Assignments
0 Petitions
Accused Products
Abstract
The invention relates to methods for detection of nucleic acids on a solid phase, combining high affinity and high specificity. More particularly, the invention relates to methods combining high-affinity hybridization with highly specific enzymatic discrimination in solid phase based nucleic acid assays. This invention further relates to kits containing the reagents necessary for carrying out the disclosed assays.
-
Citations
24 Claims
-
1. A solid phase based nucleic acid assay that can distinguish alleles that comprise a single nucleotide polymorphism in a sample containing a target nucleic acid molecule comprising
a) immobilizing the target nucleic acid molecule to a solid support using i) one or more capture oligonucleotides that are immobilized to the solid support, that hybridize to the target molecule and that have terminal nucleotides blocked and/or unphosphorylated terminal nucleotides and ii) a discrimination oligonucleotide that are immobilized to the solid support, that hybridize to the target molecule wherein a nucleotide at a terminus of the discrimination oligonucleotide is complementary to the single nucleotide polymorphism position and is unblocked and/or phosphorylated; -
b) performing a reaction on any unblocked and/or phosphorylated terminal nucleotide hybridized to a complementary nucleotide, the reaction being specific for unblocked and/or phosphorylated terminal nucleotide perfectly hybridized to a complementary nucleotide; and
c) determining if any reaction occurred, wherein detecting a reaction of unblocked and/or phosphorylated terminal nucleotide perfectly hybridized to a complementary nucleotide indicates the discrimination oligonucleotide hybridized perfectly to the target nucleic acid molecule at the single nucleotide polymorphism position. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20)
-
-
21. A kit comprising a solid support selected from the group consisting of a solid support that comprises:
-
a) a capture probe and one or more target capture probes linked to the solid support at the 3′
terminus directly or with spacers, one or more target capture extenders with blocked 3′
termini and sequences complementary to a sequence on a target capture probe and a target nucleic acid molecule, and a discrimination extender with an unblocked 3′
terminus that and a sequence complementary to a sequence on a capture probe and a target nucleic acid molecule wherein the nucleotide at the 3′
terminus of the discrimination extender is complementary to a single nucleotide polymorphism position of an allele;
b) a capture probe and one or more target capture probes linked to the solid support at the 5′
terminus directly or with spacers, one or more target capture extenders with unphosphorylated 5′
termini and sequences complementary to a sequence on a target capture probe and a target nucleic acid molecule, and a discrimination extender with an phosphorylated 5′
terminus and a sequence complementary to a sequence on a capture probe and a target nucleic acid molecule wherein the nucleotide at the 5′
terminus of discrimination extender is complementary to a single nucleotide polymorphism position of an allele;
c) a capture probe linked to the solid support at the 3′
termini directly or with spacers, one or more target capture probes with blocked 3′
termini and linked to the solid support at the 5′
terminus directly or with spacers, one or more target capture extenders with blocked 3′
termini and sequences complementary to a sequence on a target capture probe and a target nucleic acid molecule, and a discrimination extender with an unblocked 3′
terminus and a sequence complementary to a sequence on a capture probe and a target nucleic acid molecule wherein the nucleotide at the 3′
terminus of the discrimination extender is complementary to a single nucleotide polymorphism position of an allele; and
combination thereof. - View Dependent Claims (22)
-
-
23. A solid support selected from the group consisting of:
-
a) a solid support comprising a discrimination probe linked to the solid support at the 5′
termini directly or with spacers, one or more target capture probes with blocked 3′
termini and linked to the solid support at the 5′
terminus directly or with spacers, wherein a sequence of a target probe is complementary to a sequence on a target nucleic acid molecule and a sequence on the discrimination probe is complementary to a sequence on the target nucleic acid molecule wherein the nucleotide at the 3′
terminus of the discrimination probe is unblocked and complementary to a single nucleotide polymorphism position of an allele;
b) a solid support comprising a discrimination probe linked to the solid support at the 3′
termini directly or with spacers, one or more target capture probes with unphosphorylated 5′
termini and linked to the solid support at the 3′
terminus directly or with spacers, wherein a sequence of a target probe is complementary to a sequence on a target nucleic acid molecule and a sequence on the discrimination probe is complementary to a sequence on the target nucleic acid molecule wherein the nucleotide at the 5′
terminus of the discrimination probe is phosphorylated and complementary to a single nucleotide polymorphism position of an allele; and
combinations thereof. - View Dependent Claims (24)
-
Specification