Methods for detecting nucleic acid sequence variations
OwnerSha-Sha Wang, Keith Thornton +2inventors
InventorsSha-Sha Wang, Keith Thornton, James G. Nadeau, Tobin J. Hellyer
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Abstract
The invention employs an unlabeled signal primer comprising a 5′ adapter sequence for detection of variations in nucleic acid target sequences. The detection system further comprises a reporter probe, the 3′ end of which hybridizes to the complement of the 5′ adapter sequence of the signal primer to produce a 5′ overhang. Polymerase is used to fill in the overhang and synthesize the complement of the 5′ overhang of the reporter probe. Synthesis of the reporter probe complement is detected, either directly or indirectly, as an indication of the presence of the target.
Claims23
A method for detecting a presence or absence of at least one nucleotide variation in one or more target nucleic acids contained in a sample, comprising:
- selecting one or more target binding sequences for one or more signal primers, wherein each of said one or more target binding sequences is specific for a specific nucleotide variation of a target nucleic acid;
- forming a complement of said one or more signal primers;
- hybridizing one or more reporter probes specific for said complement of the one or more signal primers to said complement;
- and producing a signal indicating that a corresponding at least one nucleotide variation is present.
A composition for detecting a nucleotide variation in one or more target nucleic acids, comprising:
- two or more signal primers, and at least two reporter probes, wherein a first of said reporter probes is labeled with a first label and is specific for a complement of a first signal primer and a second of said reporter probes is labeled with a second label and is specific for a complement of a second signal primer.
A method for detecting at least one nucleotide variation in a target nucleic acid, comprising:
- in an amplification reaction:
- a) hybridizing a signal primer comprising an adapter sequence to the target nucleic acid;
- b) extending the signal primer on the target nucleic acid to produce an extension product;
- c) hybridizing an amplification primer to the extension product and extending the amplification primer to synthesize a complement of the adapter sequence;
- d) hybridizing to the complement of the adapter sequence a reporter probe comprising a reporter moiety, whereby a double-stranded reporter moiety is produced;
- e) detecting the double-stranded reporter moiety as an indication of a presence of said at least one nucleotide variation in said target nucleic acid.
A method for detecting at least one nucleotide variation in a target nucleic acid, comprising:
- a) hybridizing a signal primer comprising a 3′ target binding sequence and a 5′ adapter sequence to the target nucleic acid such that the adapter sequence produces a 5′ overhang;
- b) synthesizing a complement of the adapter sequence by extension;
- c) hybridizing a reporter probe comprising a reporter moiety to the complement of the adapter sequence, whereby a double-stranded reporter moiety is produced, and d) detecting the double-stranded reporter moiety as an indication of a presence of the one or more single nucleotide variations.
A set of oligonucleotides for detecting at least one nucleotide variation in a target nucleic acid, comprising:
- a) an unlabeled signal primer comprising a 3′ target binding sequence and a 5′ adapter sequence, and b) a reporter probe comprising a 5′ reporter moiety and 3′ sequence which is substantially identical to the 5′ adapter sequence.
A kit for detecting a presence or absence of at least one nucleotide variation in one or more target nucleic acids contained in a sample, comprising:
- two or more primers, wherein each of said two or more primers comprises a diagnostic nucleotide;
- and at least two reporter probes, wherein a first of said reporter probes is labeled with a first label and is specific for a complement of a first primer and a second of said reporter probes is labeled with a second label and is specific for a complement of a second primer.
A method for genotyping a biological sample, comprising:
- collecting a sample;
- and detecting a presence or an absence of at least one nucleotide variation of a target nucleic acid contained in said sample, said detecting comprising selecting one or more target binding sequences for one or more signal primers, wherein each of said one or more target binding sequences is specific for a specific nucleotide variation of a target nucleic acid, wherein only those signal primers which comprise a corresponding target nucleic acid hybridize to said corresponding target nucleic acid.
A method for detecting a presence or absence of at least one nucleotide variation in one or more target nucleic acids contained in a sample, comprising:
- selecting one or more target binding sequences for one or more primers, wherein each of said one or more target binding sequences is specific for a specific nucleotide variation of a target nucleic acid, wherein said one or more primers comprises at least one pair of primers, each of said at least one pair of primers comprising a first primer and a second primer, wherein said first primer hybridizes to a first strand of said target nucleic acid, and wherein said second primer hybridizes to a second strand of said target nucleic acid;
- forming a complement of said one or more primers;
- hybridizing one or more reporter probes specific for said complement of the one or more primers to said complement;
- and producing a signal indicating that a corresponding at least one nucleotide variation is present.
Dependencies are inferred from the claim text.