NUCLEIC ACID AMPLIFICATION WITH ALLELE-SPECIFIC SUPPRESSION OF SEQUENCE VARIANTS
First Claim
1. A method of selective amplification of a desired variant of a target sequence, which target sequence exists in the form of more than one variant, the method comprising the steps of:
- a) providing a sample possibly comprising at least one variant of the target sequence in a reaction mixture;
b) providing a first oligonucleotide, capable of hybridizing to more than one variant of the target sequence;
c) providing a second oligonucleotide, capable of hybridizing to more than one variant of the target sequence, wherein at least a fraction of said second oligonucleotide contains a modified base in one or more nucleotides at or near the 3′
-terminus;
d) providing a third oligonucleotide, capable of hybridizing to the desired variant of the target sequence with lesser affinity than to the undesired variants of the target sequence and designed to hybridize to the same strand and between 0 and 60 nucleotides downstream of said second oligonucleotide;
e) providing a nucleic acid polymerase substantially lacking 5′
-3′
nuclease activity;
f) subjecting said reaction mixture to polymerase chain reaction,wherein said third oligonucleotide substantially inhibits extension of said second oligonucleotide by said nucleic acid polymerase when said third oligonucleotide is hybridized to the undesired variant of the target sequence, but does not substantially inhibit extension of said second oligonucleotide by said nucleic acid polymerase when said third oligonucleotide is hybridized to the desired variant of the target sequence.
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Accused Products
Abstract
The present invention is an improved method of selective amplification of certain variants of the target sequence, enhanced by allele-specific suppression of amplification of one or more of the other variants of the target sequence. The improvement is accomplished by providing an oligonucleotide, capable of hybridizing to the desired variant of the target sequence with the lesser affinity than to the undesired variants of the target sequence and optionally, by providing chemically modified primers and hot-start conditions.
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Citations
28 Claims
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1. A method of selective amplification of a desired variant of a target sequence, which target sequence exists in the form of more than one variant, the method comprising the steps of:
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a) providing a sample possibly comprising at least one variant of the target sequence in a reaction mixture; b) providing a first oligonucleotide, capable of hybridizing to more than one variant of the target sequence; c) providing a second oligonucleotide, capable of hybridizing to more than one variant of the target sequence, wherein at least a fraction of said second oligonucleotide contains a modified base in one or more nucleotides at or near the 3′
-terminus;d) providing a third oligonucleotide, capable of hybridizing to the desired variant of the target sequence with lesser affinity than to the undesired variants of the target sequence and designed to hybridize to the same strand and between 0 and 60 nucleotides downstream of said second oligonucleotide; e) providing a nucleic acid polymerase substantially lacking 5′
-3′
nuclease activity;f) subjecting said reaction mixture to polymerase chain reaction, wherein said third oligonucleotide substantially inhibits extension of said second oligonucleotide by said nucleic acid polymerase when said third oligonucleotide is hybridized to the undesired variant of the target sequence, but does not substantially inhibit extension of said second oligonucleotide by said nucleic acid polymerase when said third oligonucleotide is hybridized to the desired variant of the target sequence. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9)
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10. A reaction mixture for selective amplification of a desired variant of a target sequence, which target sequence exists in the form of more than one variant, the mixture comprising:
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a) a first oligonucleotide, capable of hybridizing to more than one variant of the target sequence; b) a second oligonucleotide, capable of hybridizing to more than one variant of the target sequence, wherein at least a fraction of said second oligonucleotide contains a modified base in one or more nucleotides at or near the 3′
-terminus;c) a third oligonucleotide, capable of hybridizing to the desired variant of the target sequence with the lesser affinity than to the undesired variants of the target sequence; d) a nucleic acid polymerase substantially lacking 5′
-3′
nuclease activity;wherein said third oligonucleotide is capable of detectably inhibiting amplification of said undesired variants of the target sequence but not the amplification of said desired variant of the target sequence. - View Dependent Claims (11, 12, 13, 14, 15, 16, 17, 18)
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19. A kit for selective amplification of a desired variant of a target sequence, which target sequence exists in the form of more than one variant, the kit comprising:
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a) a first oligonucleotide, capable of hybridizing to more than one variant of the target sequence; b) a second oligonucleotide, capable of hybridizing to more than one variant of the target sequence, wherein at least a fraction of said second oligonucleotide contains a modified base in one or more nucleotides at or near the 3′
-terminus;c) a third oligonucleotide, capable of hybridizing to the desired variant of the target sequence with the lesser affinity than to the undesired variants of the target sequence; d) a nucleic acid polymerase substantially lacking 5′
-3′
nuclease activity;e) the reagents and solutions necessary for amplification of nucleic acids; wherein said third oligonucleotide is capable of detectably inhibiting amplification of said undesired variants of the target sequence but not the amplification of said desired variant of the target sequence. - View Dependent Claims (20, 21, 22, 23, 24, 25, 26, 27, 28)
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Specification