Methods, kits and reaction mixtures for analyzing single-stranded nucleic acid sequences

Patent metadata

Field	Value
Publication number	US-9353407-B2
Application number	US-201013503324-A
Country	US
Kind code	B2
Filing date	Oct 21, 2010
Priority date	Oct 21, 2009
Publication date	May 31, 2016
Grant date	May 31, 2016

Abstract

Provided herein are fluorescence detection methods for nucleic acid sequences and to kits for performing such methods.

First claim

We claim:

1. A homogeneous assay method for analyzing at least one single-stranded nucleic acid target sequence in a sample, comprising
a) providing a sample containing a nucleic acid comprising said at least one single-stranded nucleic acid target sequence and multiple detectably distinguishable probe sets, each set of the multiple detectably distinguishable probe sets comprising two probes:
i) a probe labeled with a non-fluorescent quencher moiety that hybridizes to a region of one of said at least one single-stranded nucleic acid target sequence, and
ii) a probe labeled with a fluorescent moiety that hybridizes to a different region of one of said at least one single-stranded nucleic acid target sequence adjacent to the region of said one of said at least one single-stranded nucleic acid target sequence that the probe labeled with the non-fluorescent quencher moiety hybridizes to,
wherein if said probe labeled with a fluorescent moiety hybridizes to said one of said at least one single-stranded nucleic acid target sequence in said sample in the absence of said probe labeled with a non-fluorescent quencher moiety, said fluorescent moiety emits a fluorescent signal, wherein, if both the probe labeled with a non-fluorescent quencher moiety and the probe labeled with a fluorescent moiety hybridize to said one of said at least one single-stranded nucleic acid target sequence, the non-fluorescent quencher moiety quenches the fluorescent signal from the fluorescent moiety;

b) hybridizing said at least one single-stranded nucleic acid target sequence to the multiple detectably distinguishable probe sets so that the multiple detectably distinguishable probe sets hybridize to one of said at least one single-stranded nucleic acid target sequence and, after the hybridization, the multiple detectably distinguishable probe sets are arranged adjacent to each other on one of said at least one single-stranded nucleic acid target sequence, and there is no space between each of the multiple detectably distinguishable probe sets; and
c) analyzing the hybridization of the multiple detectably distinguishable probe sets to said one of said at least one single-stranded nucleic acid target sequence by assessing a fluorescent signal produced in step b) as a function of temperature.

2. The method of claim 1 wherein one probe is a part of two probe sets of the multiple detectably distinguishable probe sets.

3. The method of claim 2 wherein two sets of the multiple detectably distinguishable probe sets collectively comprise:
(A) a quencher probe comprising a non-fluorescent quencher moiety on its one end, which is the probe labeled with a non-fluorescent quencher moiety of a first set of the two sets of the multiple detectably distinguishable probe sets;
(B) a first signaling probe comprising a fluorescent moiety on its first end and a non-fluorescent quencher moiety on its second end, which is the probe labeled with a fluorescent moiety of the first set of the two sets of the multiple detectably distinguishable probe sets or is the probe labeled with a non-fluorescent quencher moiety of a second set of the two sets of the multiple detectably distinguishable probe sets; and
(C) a second signaling probe comprising a fluorescent moiety on its first end and a non-fluorescent quencher moiety on its second end, which is the probe labeled with a fluorescent moiety of the second set of the two sets of the multiple detectably distinguishable probe sets;
wherein, in step b), the non-fluorescent quencher moiety of the quencher probe interacts with the fluorescent moiety of the first signaling probe and the non-fluorescent quencher moiety of the first signaling probe interacts with the fluorescent moiety of the second signaling probe.

4. The method of claim 1 wherein the probe labeled with a fluorescent moiety in each set of the multiple detectably distinguishable probe sets is also labeled with a non-fluorescent quencher moiety.

5. The method of claim 1 wherein two sets of the multiple detectably distinguishable probe sets collectively comprise:
(A) a quencher probe comprising a non-fluorescent quencher moiety on its one end, which is the probe labeled with a non-fluorescent quencher moiety of a first set of the two sets of the multiple detectably distinguishable probe sets;
(B) a first signaling probe comprising a fluorescent moiety on its first end and a non-fluorescent quencher moiety on its second end, which is the probe labeled with a fluorescent moiety of the first set of the two sets of the multiple detectably distinguishable probe sets or is the probe labeled with a non-fluorescent quencher moiety of a second set of the two sets of the multiple detectably distinguishable probe sets; and
(C) a second signaling probe comprising a fluorescent moiety on its first end and a non-fluorescent quencher moiety on its second end, which is the probe labeled with a fluorescent moiety of the second set of the two sets of the multiple detectably distinguishable probe sets;
wherein, in step b), the non-fluorescent quencher moiety of the quencher probe interacts with the fluorescent moiety of the first signaling probe and the non-fluorescent quencher moiety of the first signaling probe interacts with the fluorescent moiety of the second signaling probe.

6. The method of claim 1 wherein the melting temperature of the probe labeled with a fluorescent moiety in at least one set of the multiple detectably distinguishable probe sets is higher than the melting temperature of its corresponding probe labeled with a non-fluorescent quencher moiety in the same set of said at least one set of the multiple detectably distinguishable probe sets.

7. The method of claim 1 wherein the concentration of said at least one single-stranded nucleic acid target sequence is lower than the concentration of at least one probe in said multiple detectably distinguishable probe sets.

8. The method of claim 1 wherein the concentration of the probe labeled with a fluorescent moiety of at least one set of the multiple detectably distinguishable probe sets is lower than the concentration of its corresponding probe labeled with a non-fluorescent quencher moiety in the same set of said at least one set of the multiple detectably distinguishable probe sets.

9. The method of claim 1 wherein, when the probe labeled with a non-fluorescent quencher moiety and the probe labeled with a fluorescent moiety in one set of the multiple detectably distinguishable probe sets hybridize to one of said at least one single-stranded nucleic acid target sequence, and said fluorescent moiety and said non-fluorescent quencher moiety in the one set of the multiple detectably distinguishable probe sets interact with each other by Fluorescent Resonance Energy Transfer (FRET).

10. The method of claim 1 wherein said fluorescent moiety and said non-fluorescent quencher moiety in at least one set of the multiple detectably distinguishable probe sets interact with each other by contact quenching.

11. The method of claim 1 wherein step c) further comprises generating at least one melting curve or at least one annealing curve.

12. The method of claim 1 wherein said providing the sample containing said at least one single-stranded nucleic acid target sequence comprises amplifying said at least one single-stranded nucleic acid target sequence.

13. The method of claim 12 wherein said amplifying said at least one single-stranded nucleic acid target sequence is by a PCR amplification reaction.

14. The method of claim 13 wherein the melting temperatures of the probes in the multiple detectably distinguishable probe sets are below the an

Assignees

• Wangh Lawrence
• Rice John
• Sanchez J Aquiles
• Reis Arthur
• Univ Brandeis

Inventors

• Wangh Lawrence
• Rice John
• Sanchez J Aquiles
• Reis Arthur

Classifications

• C12Q2600/156

  Polymorphic or mutational markers · CPC title

• C12Q1/6818

  involving interaction of two or more labels, e.g. resonant energy transfer · CPC title

• C12Q1/6858

  Allele-specific amplification · CPC title

• C12Q1/6886

  for cancer (immunoassay for cancer G01N33/575) · CPC title

• C12Q1/6827 Primary

  for detection of mutation or polymorphism · CPC title