The Perkin-Elmer Corporation

Applera Corporation

Susan J A Flood

Kenneth J Livak

Khairuzzaman Bashar Mullah

Jeffrey Mamoro

A hybridization assay is provided which uses an oligonucleotide probe which includes a fluorescent reporter molecule and a quencher molecule capable of quenching the fluorescence of the reporter molecule. The oligonucleotide probe is constructed such that the probe exists in at least one single-stranded confirmation when unhybridized where the quencher molecule is near enough to the reporter molecule to quench the fluorescence of the reporter molecule. The oligonucleotide probe also exists in at least one conformation when hybridized to a target polynucleotide where the quencher molecule is not positioned close enough to the reporter molecule to quench the fluorescence of the reporter molecule. By adopting these hybridized and unhybridized conformations, the reporter molecule and quencher molecule on the probe exhibits different fluorescence signal intensities when the probe is hybridized and unhybridized. As a result, it is possible to determine whether the probe is hybridized or unhybridized based on a change in the fluorescence intensity of the reporter molecule, the quencher molecule, or a combination thereof. In addition, because the probe can be designed such that the quencher molecule quenches the reporter molecule when the probe is not hybridized, the probe can be designed such that the reporter molecule exhibits limited fluorescence until the probe is either hybridized or digested.

Hybridization assay using self-quenching fluorescence probe

Provided is a method of nucleic acid amplification. In one embodiment, the method comprises performing nucleic acid amplification on a target polynucleotide using a nucleic acid polymerase having 5&prime; to 3&prime; nuclease activity, a primer capable of hybridizing to the target polynucleotide, and an oligonucleotide probe under amplification conditions such that the probe hybridizes to the target polynucleotide 3&prime; relative to the primer and the probe does not hybridize with itself to form a hairpin structure. The oligonucleotide probe has at one end a fluorescent reporter and at the other end a quencher that quenches the fluorescence of the reporter molecule when both the fluorescent reporter and quencher are attached to the probe. Digestion of the oligonucleotide probe by the polymerase during amplification is effective to separate the reporter from the quencher, whereby a fluorescence signal of the reporter is increased.

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Fremont, CA, United States of America

Susan J A Flood