Becton Dickinson and Company Limited

Beckon, Dickson and Company

C Preston Linn

J Bruce Pitner

James G Nadeau

James L Schram

G Terrance Walker

Glenn P Vonk

Helen V Hsieh

Cheryl H Dean

Pat D Mize

Signal primers are employed for detection of nucleic acid target sequences by fluorescence quenching mechanisms. The signal primer comprises a first and a second oligonucleotide and is partially single-stranded and partially double-stranded. In the presence of target, the second oligonucleotide of the signal primer is displaced from the first and a conformational change in a reporter probe occurs which changes the distance between the members of a donor/quencher dye pair linked to the reporter probe. The change in proximity between the dyes causes an increase or a decrease in fluorescence quenching, which is detected as an indication of the presence of the target sequence.

Universal probes and methods for detection of nucleic acids

Detector nucleic acids are employed for detection of nucleic acid target sequences by fluorescence quenching mechanisms. The detector nucleic acid comprises at least two oligonucleotides and is partially single-stranded and partially double-stranded. One of the two dyes of a donor/acceptor dye pair is linked to the first oligonucleotide and the other is linked to a second oligonucleotide such that they are in close spatial proximity when the first and second oligonucleotides are base-paired and donor fluorescence is quenched. A single second oligonucleotide may be hybridized to the first oligonucleotide or multiple second oligonucleotides may be hybridized to the first oligonucleotide and to each other, forming a junction structure comprising multiple donor/acceptor dye pairs. The detector oligonucleotide retains its partially single-stranded and partially double-stranded conformation in the absence of target. In the presence of target, however, the second oligonucleotide(s) of the detector nucleic acid is/are completely or partially displaced from the first, increasing the distance between the donor and acceptor dyes and causing a change in fluorescence which may be detected as an indication of the presence of the target sequence.

Detection of nucleic acids by strand displacement

Detection of nucleic acids by fluorescence quenching

The present invention provides labelled phosphoramidite compositions, methods for making these labelled phosphoramidite compositions, and methods for using these labelled phosphoramidite compositions for labelling oligonucleotides. Even more particularly, the present invention provides compositions and methods for labeling the 5' end of oligonucleotides during synthesis of the oligonucleotides.

Synthesis and use of labelled phosphoramidite compositions

Single-stranded signal primers are modified by linkage to two dyes which form a donor/acceptor dye pair. The two dyes are positioned in sufficiently close spatial proximity on the signal primer that the fluorescence of the first dye is quenched by the second dye. The signal primer may further comprise a restriction endonuclease recognition site (RERS) between the two dyes. As the signal primer is initially single-stranded and remains single-stranded in the absence of target, the restriction endonuclease recognition site is not cleavable or nickable by the restriction endonuclease. In the presence of target, however, signal primer and the restriction endonuclease recognition site are rendered double-stranded and cleavable or nickable by the restriction endonuclease. Cleavage or nicking separates the two dyes and a change in fluorescence due to decreased quenching is detected as an indication of the presence of the target sequence or of target sequence amplification.

A detector oligonucleotide having a sequence which forms an intramolecularly base-paired secondary structure is described for use in detecting nucleic acid target sequences and target sequence amplification. The detector oligonucleotide is further modified by linkage to two dyes which form a donor/acceptor dye pair. The two dyes are positioned on the detector oligonucleotide such that they are in close spatial proximity in the base-paired, folded secondary structure, thereby causing quenching of donor fluorescence. The detector oligonucleotide may optionally further comprise a restriction endonuclease recognition site (RERS) which remains partially or entirely single-stranded in the base-paired secondary structure. The RERS is flanked by the two dyes. In the presence of target, the base-paired secondary structure is unfolded or linearized, increasing the distance between the donor and acceptor dyes and causing a change in fluorescence of the donor and/or the acceptor. If an RERS is present, it is rendered double-stranded in the presence of target, allowing cleavage or nicking by a restriction endonuclease and separation of the two dyes onto separate nucleic acid fragments. This may further contribute to the magnitude of the change in fluorescence.

The present invention relates to conjugates of a cyanine dye and an oligonucleotide. When these conjugates hybridize or bind to a target, a detectable increase in fluorescence intensity or change in fluorescence polarization is observed.

Covalent cyanine dye oligonucleotide conjugates

Fluorescence polarization methods for detection of nucleic acid amplification. A fluorescently labelled oligodeoxynucleotide probe is converted from single- to double-stranded form in a target dependent manner during amplification of the target sequence. This conformational change is accompanied by an increase in fluorescence polarization values. The increase in fluorescence polarization can be measured on a transient-state fluorometer in real-time during the amplification reaction without any physical manipulation of the sample. The inventive methods therefore provide a closed, homogeneous system for both amplification of target sequences and detection of amplification. Alternatively, amplification may be detected in the fluorometer after the amplification reaction is completed.

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Durham, NC, United States of America

C Preston Linn