Probes and methods for detection of nucleic acids
OwnerJames Nadeau, Tobin Hellyerinventors
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Abstract
The invention employs an unlabeled signal primer comprising a 5′ adapter sequence for detection of 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.
Claims60
A method for detecting a nucleic acid target sequence comprising:
- a) hybridizing a signal primer comprising an adapter sequence to the target sequence, whereby a complement of the adapter sequence is produced;
- b) hybridizing a reporter probe comprising a reporter moiety to the complement of the adapter sequence, whereby a double-stranded reporter moiety is produced, and;
- c) detecting synthesis of the complement of the reporter moiety as an indication of the presence of the target sequence.
A method for detecting amplification of a target sequence comprising, in an amplification reaction:
- a) hybridizing a signal primer comprising an adapter sequence to the target sequence;
- b) extending the signal primer on the target sequence 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 amplification of the target sequence.
A method for detecting a nucleic acid target sequence comprising:
- a) hybridizing a signal primer comprising an adapter sequence to the target sequence such that the adapter sequence produces a 5′ overhang;
- b) synthesizing a complement of the adapter sequence by extension of the hybridized target sequence;
- 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 the presence of the target sequence.
A set of oligonucleotides for detecting a target sequence comprising:
- a) first unlabeled signal primer comprising a single oligonucleotide having a 3′ target binding sequence and a 5′ adapter sequence,
- b) a second signal primer having an adapter sequence which is substantially identical to an adapter sequence of the first signal primer:
- and;
- c) a reporter probe comprising a 5′ reporter moiety and 3′ sequence which is substantially identical to the adapter sequence.
An oligonucleotide comprising a reporter moiety and nucleotides 15-37 of SEQ ID NO:2, nucleotides 10-34 of SEQ ID NO:15, nucleotides 16-40 of SEQ ID NO:16, nucleotides 16-35 of SEQ ID NO:17, nucleotides 16-30 of SEQ ID NO:
- 18, nucleotides 16-40 of SEQ ID NO:
- 19 or nucleotides 19-43 of SEQ ID NO:20.
An oligonucleotide comprising the target binding sequence of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13 or SEQ ID NO:14 and an adapter sequence.
A set of oligonucleotides for detecting a target sequence comprising:
- a) an unlabeled signal primer comprising a single oligonucleotide having a 3′ target binding sequence and a 5′ adapter sequence,
- b) a second signal primer having an adapter sequence which is different from an adapter sequence of a first signal primer;
- and
- b) a reporter probe comprising a 5′ reporter moiety and 3′ sequence which is substantially identical to the adapter sequence.
Dependencies are inferred from the claim text.