Allele-Specific Amplification
First Claim
1. A method of allele-specific amplification of a variant of a target sequence, which exists in the form of several variant sequences, comprising(a) providing a sample, possibly containing at least one variant of a target sequence;
- (b) providing a first oligonucleotide, at least partially complementary to one or more variants of the target sequence;
(c) providing a second oligonucleotide, at least partially complementary to one or more variants of the target sequence, having a 3′
-terminal nucleotide complementary to only one variant of the target sequence;
wherein said second oligonucleotide incorporates at least one nucleotide with a base covalently modified at the exocyclic amino group;
(d) providing conditions suitable for the hybridization of said first and second oligonucleotides to at least one variant of the target sequence; and
(e) providing conditions suitable for the oligonucleotide extension by a nucleotide-incorporating biocatalyst;
wherein said biocatalyst is capable of extending said second oligonucleotide when it is hybridized to the variant of the target sequence for which it has said complementary 3′
-terminal nucleotide, and substantially less when said second oligonucleotide is hybridized to the variant of the target sequence for which it has a non-complementary 3′
-terminal nucleotide.
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Accused Products
Abstract
The present invention includes a method of allele-specific amplification, utilizing an allele-specific oligonucleotide, at least partially complementary to more than one variant of the target sequence, but having a 3′-terminal nucleotide complementary to only one variant of the target sequence and having at least one nucleotide with a base covalently modified at the exocyclic amino group, wherein the allele-specific oligonucleotide is extended by a nucleotide-incorporating biocatalyst predominantly when hybridized to the variant of the target sequence for which it has said complementary 3′-terminal nucleotide.
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Citations
27 Claims
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1. A method of allele-specific amplification of a variant of a target sequence, which exists in the form of several variant sequences, comprising
(a) providing a sample, possibly containing at least one variant of a target sequence; -
(b) providing a first oligonucleotide, at least partially complementary to one or more variants of the target sequence; (c) providing a second oligonucleotide, at least partially complementary to one or more variants of the target sequence, having a 3′
-terminal nucleotide complementary to only one variant of the target sequence;
wherein said second oligonucleotide incorporates at least one nucleotide with a base covalently modified at the exocyclic amino group;(d) providing conditions suitable for the hybridization of said first and second oligonucleotides to at least one variant of the target sequence; and (e) providing conditions suitable for the oligonucleotide extension by a nucleotide-incorporating biocatalyst;
wherein said biocatalyst is capable of extending said second oligonucleotide when it is hybridized to the variant of the target sequence for which it has said complementary 3′
-terminal nucleotide, and substantially less when said second oligonucleotide is hybridized to the variant of the target sequence for which it has a non-complementary 3′
-terminal nucleotide. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12)
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13. A method of detecting a variant of a target sequence in a sample, which exists in the form of several variant sequences comprising
(a) hybridizing a first and second oligonucleotides to at least one variant of the target sequence; - wherein the first oligonucleotide is at least partially complementary to one or more variant of the target sequence and the second oligonucleotide is at least partially complementary to one or more variant of the target sequence and has a 3′
-terminal nucleotide complementary to only one variant of the target sequence, said second oligonucleotide incorporating at least one nucleotide with a base covalently modified at the exocyclic amino group;(b) extending the second oligonucleotide with a nucleotide-incorporating biocatalyst;
wherein said biocatalyst is capable of detectably extending only the oligonucleotide, hybridized to the variant of the target sequence for which it has said complementary 3′
-terminal nucleotide; and(c) detecting the products of said second oligonucleotide extension, wherein the extension signifies the presence of the variant of a target sequence to which the oligonucleotide has a complementary 3′
-terminal nucleotide.
- wherein the first oligonucleotide is at least partially complementary to one or more variant of the target sequence and the second oligonucleotide is at least partially complementary to one or more variant of the target sequence and has a 3′
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14. A kit for allele-specific amplification of a target sequence, which exists in the form of several variant sequences, comprising
(a) a first oligonucleotide, at least partially complementary to one or more variant of the target sequence; - and
(b) a second oligonucleotide, at least partially complementary to one or more variant of the target sequence and having a 3′
-terminal nucleotide complementary to only one variant of the target sequence;
wherein said second oligonucleotide incorporates at least one nucleotide with a base covalently modified at the exocyclic amino group. - View Dependent Claims (15)
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16. An oligonucleotide for performing an allele-specific amplification of a target sequence, which exists in the form of several variant sequences, comprising
a sequence at least partially complementary to a portion of one or more variants of said target sequence; -
a 3′
-terminal nucleotide which is complementary to only one variant of said target sequence andat least one nucleotide with a base covalently modified at the exocyclic amino group. - View Dependent Claims (17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27)
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Specification