Becton Dickinson and Company Limited

G Terrance Walker

James G Nadeau

C Preston Linn

J Bruce Pitner

Glenn P Vonk

James L Schram

James A Down

Patricia A Spears

Melinda S Fraiser

Carl Preston Linn

Cheryl H Dean

David J Wright

Maria A Milla

The present invention provides methods for detecting and identifying sequence variations in a nucleic acid sequence of interest using a detector primer. It has been found that the reduced efficiency of primer extension by DNA polymerases when the 3' end of a primer does not hybridize perfectly with the target can be adapted for use as a means for distinguishing or identifying the nucleotide in the target which is at the site where the diagnostic mismatch between the detector primer and the target occurs. The detector primer hybridizes to the sequence of interest and is extended with polymerase. The efficiency of detector primer extension is detected as an indication of the presence and/or identity of the sequence variation in the target. The inventive methods make use of nucleotide mismatches at or near the 3&#x2032; end of the detector primer to discriminate between the nucleotide sequence of interest and a second nucleotide sequence which may occur at that same site in the target. The methods are particularly well suited for detecting and identifying single nucleotide differences between a target sequence of interest (e.g., a mutant allele of a gene) and a second nucleic acid sequence (e.g., a wild type allele for the same gene).

Methods for detecting single nucleotide polymorphisms

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

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.

Detection of nucleic acids by fluorescence quenching

A process for lysing Mycobacteria to liberate nucleic acids such as DNA and RNA comprises heating the Mycobacteria in an aqueous solution to lyse the Mycobacteria and release the nucleic acids, with the aqueous solution containing a chelating agent such as EDTA or EGTA in an amount effective to inhibit degradation of the released nucleic acids. Examples of Mycobacteria which can be lysed include Mycobacterium fortuitum and Mycobacterium tuberculosis. After lysis, the nucleic acid is preferably then amplified and detected. Kits useful for carrying out the present invention are also disclosed.

Process for lysing mycobacteria

Fluorescence polarization methods for detection of nucleic acid amplification at thermophilic temperatures employ a fluorescently labeled oligonucleotide signal primer which is converted from single- to double-stranded form in a target amplification-dependent manner. This conformational change is accompanied by an increase in fluorescence polarization values. The decrease in FP typically observed for the duplex at elevated temperatures is overcome by double-stranded DNA binding proteins which are believed to stabilize the double-stranded structure by reducing the single-strandedness normally associated with higher temperatures. The inventive methods provide a closed, homogeneous system for amplification and detection of amplification in real-time or at an endpoint.

Method for detection of nucleic acid targets by amplification and

The present invention provides methods for detecting amplified or unamplified nucleic acid target sequences at increased temperatures by changes in fluorescence polarization. The decrease in fluorescence polarization associated with hybridization of oligonucleotides at higher, more stringent, temperatures is overcome by including a double-stranded DNA binding protein in the assay. At elevated temperatures, the double-stranded DNA binding protein restores, and often enhances, the magnitude of the change in fluorescence polarization associated with single- to double-stranded conversion of an oligonucleotide probe or primer.

Fluorescence polarization detection of nucleic acids

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.

Fluorerscence polarization detection of nucleic acid amplication

Amplification of a target sequence is detected qualitatively or quantitatively by concurrent generation of secondary amplification products labeled with a lipophilic label. The secondary amplification products are designed such that they are generated and cleaved or nicked in a target amplification-dependent manner. This reduces the number of hydrophilic nucleotides linked to the lipophilic label and allows the cleaved or nicked secondary amplification product comprising the lipophilic label to be transferred from the aqueous reaction phase to an organic phase for detection as an indicator of target amplification.

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Chapel Hill, NC, United States of America

G Terrance Walker