Hybridization assay using self-quenching fluorescence probe

• US 6,030,787 A
• Filed: 12/07/1998
• Issued: 02/29/2000
• Est. Priority Date: 11/16/1994
• Status: Expired due to Term

• Current Assignee: Perkinelmer Incorporated, Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Original Assignee: PE Corp.
• Inventors: Livak, Kenneth J., Mullah, Khairuzzaman Bashar, Flood, Susan J. A., Mamoro, Jeffrey
• Primary Examiner(s): Riley, Jezia
• Application Number:
  US09/207,170
• Time in Patent Office: 449 Days
• Field of Search: 435/5, 435/6, 435/91.1, 435/91.2, 536/24.3, 536/24.33, 536/24.32, 536/26.6, 536/25.3, 536/25.32
• 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 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 2525/185 incorporating bases where the precise position of the bases in the nucleic acid string is important
• CPC Class Codes: C12Q 2525/301 Hairpin oligonucleotides
• CPC Class Codes: C12Q 2561/101 Taqman
• CPC Class Codes: C12Q 2563/107 fluorescence
• 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: C12Q 2565/519 characterised by the capture moiety being a single stranded oligonucleotide

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 conformation 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 a target polynucleotide in a sample comprising:

contacting said sample of nucleic acids with an oligonucleotide probe under conditions where said oligonucleotide probe selectively hybridizes to said target polynucleotide, said oligonucleotide probe including a fluorescent reporter molecule and a quencher molecule capable of quenching the fluorescence of said reporter molecule which are attached to said oligonucleotide probe such that said oligonucleotide probe is capable of adopting at least one single-stranded conformation when not hybridized to said target polynucleotide where said quencher molecule is fluorescent and quenches the fluorescence of said reporter molecule and is capable of adopting at least one conformation when hybridized to said target polynucleotide, where the fluorescence of said reporter molecule is unquenched such that the fluorescence intensity of said reporter molecule is greater than the fluorescence intensity of said quencher molecule when said oligonucleotide probe is hybridized to said target polynucleotide and said oligonucleotide probe is not hybridized with itself in the form of a hairpin structure; and

monitoring the fluorescence of said reporter molecule under conditions where said oligonucleotide probe does not hybridize with itself to form a hairpin structure in order to detect the hybridization of said target polynucleotide to said oligonucleotide probe.

2. The method according to claim 1 wherein the fluorescence intensity of said reporter molecule is at least about a factor of 3.5 greater than the fluorescence intensity of said quencher molecule when said probe is hybridized to said target polynucleotide.

3. The method according to claim 1 wherein said reporter molecule is separated from said quencher molecule by at least 15 nucleotides.

4. The method according to claim 1 wherein said reporter molecule is separated from said quencher molecule by between 15 and 60 nucleotides.

5. The method according to claim 1 wherein said reporter molecule is separated from said quencher molecule by at least 18 nucleotides.

6. The method according to claim 1 wherein said reporter molecule is separated from said quencher molecule by between 18 and 30 nucleotides.

7. A method for detecting a target polynucleotide in a sample comprising:

contacting said sample of nucleic acids with an oligonucleotide probe attached to a solid support under conditions favorable for hybridization of said oligonucleotide probe to said target polynucleotide, said oligonucleotide probe including a fluorescent reporter molecule and a quencher molecule capable of quenching the fluorescence of said reporter molecule which are attached to said oligonucleotide probe such that said oligonucleotide probe is capable of adopting at least one single-stranded conformation when not hybridized to said target polynucleotide where said quencher molecule is fluorescent and quenches the fluorescence of said reporter molecule and is capable of adopting at least one conformation when hybridized to said target polynucleotide, where the fluorescence of said reporter molecule is unquenched such that the fluorescence intensity of said reporter molecule is greater than the fluorescence intensity of said quencher molecule when said oligonucleotide probe is hybridized to said target polynucleotide and said oligonucleotide probe is not hybridized with itself in the form of a hairpin structure; and

monitoring the fluorescence of said reporter molecule under conditions where said oligonucleotide probe does not hybridize with itself to form a hairpin structure in order to detect the hybridization of said target polynucleotide to said oligonucleotide probe.

8. The method according to claim 7 wherein the fluorescence intensity of said reporter molecule is at least about a factor of 3.5 greater than the fluorescence intensity of said quencher molecule when said probe is hybridized to said target polynucleotide.

9. The method according to claim 8 wherein said reporter molecule is separated from said quencher molecule by at least 15 nucleotides.

10. The method according to claim 8 wherein said reporter molecule is separated from said quencher molecule by between 15 and 60 nucleotides.

11. The method according to claim 8 wherein said reporter molecule is separated from said quencher molecule by at least 18 nucleotides.

12. The method according to claim 8 wherein said reporter molecule is separated from said quencher molecule by between 18 and 30 nucleotides.