Hybridization assay using self-quenching fluorescence probe

• US 6,821,727 B1
• Filed: 07/28/2000
• Issued: 11/23/2004
• Est. Priority Date: 11/15/1993
• Status: Expired due to Fees

• Current Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Original Assignee: Applera Corporation (Thermo Fisher Scientific Incorporated)
• Inventors: Mullah, Khairuzzaman Bashar, Flood, Susan J. A., Mamoro, Jeffrey, Livak, Kenneth J.
• Primary Examiner(s): Riley, Jezia
• Application Number:
  US09/627,753
• Time in Patent Office: 1,579 Days
• Field of Search: 435/6, 435/91.1, 435/91.2, 536/22.1, 536/23.1, 536/24.3, 536/24.32, 536/24.33, 536/25.3, 536/25.32, 536/26.6
• US Class Current: 435/6.11
• CPC Class Codes: C12Q 1/6818 involving interaction of two or more labels, e.g. resonant energy transfer
• CPC Class Codes: C12Q 1/6834 Enzymatic or biochemical coupling of nucleic acids to a solid phase
• CPC Class Codes: C12Q 2525/301 Hairpin oligonucleotides
• CPC Class Codes: C12Q 2561/101 Taqman
• CPC Class Codes: C12Q 2565/1015 labels being on the same oligonucleotide
• CPC Class Codes: C12Q 2565/107 Alteration in the property of hybridised versus free label oligonucleotides
• CPC Class Codes: Y10S 977/924 using nanostructure as support of dna analysis

Abstract

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.

Claims

1. A method for detecting nucleic acid target sequences in a sample comprising:

contacting a sample of nucleic acids with an oligonucleotide probe attached to a solid support under conditions favorable for hybridization, the oligonucleotide probe having a sequence at least partially complementary to a target nucleic acid sequence to be detected, the oligonucleotide probe including a fluorescent reporter molecule and a fluorescent quencher molecule capable of quenching the fluorescence of said reporter molecule, said oligonucleotide probe existing in at least one single-stranded confirmation when unhybridized where said quencher molecule quenches the fluorescence of said reporter molecule, said oligonucleotide probe existing in at least one confirmation when hybridized to said target polynucleotide where the fluorescence of said reporter molecule is unquenched, the ratio of the fluorescence intensities of said reporter molecule to said quencher molecule when said probe is hybridized to said target polynucleotide is greater than the ratio of the fluorescence intensities of said reporter molecule to said quencher molecule when said probe is single stranded; and

monitoring the fluorescence of said reporter molecule, an increase in the fluorescence intensity of the reporter molecule indicating the presence of the target sequence.

2. The method according to claim 1 wherein the ratio of the fluorescence intensities of said reporter molecule to said quencher molecule when said probe is hybridized to said target polynucleofide is at least about a factor of 6 greater than the ratio of the fluorescence intensities of said reporter molecule to said quencher molecule when said probe is single-stranded.