Amplification reporter with base-pairing oligomers

• US 9,556,475 B2
• Filed: 08/12/2014
• Issued: 01/31/2017
• Est. Priority Date: 08/12/2013
• Status: Active Grant

• Current Assignee: Bio-Rad Laboratories Incorporated
• Original Assignee: Bio-Rad Laboratories Incorporated
• Inventors: Regan, John F., Maar, Dianna, Shelton, Dawne N., Legler, Tina C., Cooper, Samantha, Yang, Wei, Hefner, Eli A., Klitgord, Niels
• Primary Examiner(s): PRIEST, AARON A
• Application Number:
  US14/457,863
• Publication Number:
  US 20150148250A1
• Time in Patent Office: 903 Days
• Field of Search: None
• US Class Current: 1/1
• CPC Class Codes: C12Q 1/6818 involving interaction of two or more labels, e.g. resonant energy transfer
• CPC Class Codes: C12Q 1/6823 Release of bound markers
• CPC Class Codes: C12Q 1/6848 characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction
• CPC Class Codes: C12Q 2527/107 Temperature of melting, i.e. Tm
• CPC Class Codes: C12Q 2537/1373 Displacement by a nucleic acid
• CPC Class Codes: C12Q 2545/107 with a competitive internal standard/control
• CPC Class Codes: C12Q 2565/101 Interaction between at least two labels
• CPC Class Codes: C12Q 2565/1015 labels being on the same oligonucleotide

Abstract

System, including methods, apparatus, and compositions, for performing amplification assays with an amplification reporter including a first oligomer and a second oligomer capable of base-pairing with one another below a melting temperature of the reporter. The reporter may have a detectable photoluminescence that is affected, such as reduced, by base-pairing of the first and second oligomers with one another. A target, such as a nucleic acid target sequence, may be amplified in at least one volume, such as a plurality of partitions, above the melting temperature, and photoluminescence of the reporter may be detected from the at least one volume below the melting temperature. A property of the target, such as a concentration of the target, may be determined based on the photoluminescence detected.

Claims

1. A method of analysis, the method comprising:

forming at least one volume containing a reporter including a first oligomer and a second oligomer capable of base-pairing with one another below a melting temperature of the reporter to affect a photoluminescence detectable from a photoluminophore of the first oligomer, wherein the first oligomer and the second oligomer are not covalently linked to one another;

amplifying a target in the at least one volume, at least in part by extending one or more primers at a temperature above the melting temperature of the reporter, wherein the step of amplifying causes cleavage of the first oligomer, and wherein the cleavage affects the photoluminescence detectable from the photoluminophore;

detecting photoluminescence from the photoluminophore in the at least one volume while the at least one volume is at a temperature below the melting temperature of the reporter; and

determining a level of the target based on the photoluminescence detected.

2. The method of claim 1, wherein the first oligomer has a longer chain of base-containing units than the second oligomer.

3. The method of claim 1, wherein the first oligomer forms a hybrid with the target having a higher melting temperature than the melting temperature of the reporter.

4. The method of claim 1, wherein the photoluminescence of the reporter decreases when the first oligomer and the second oligomer base-pair with one another.

5. The method of claim 1, wherein the first oligomer includes an energy transfer partner of the photoluminophore, and wherein the second oligomer also includes an energy transfer partner of the photoluminophore.

6. The method of claim 1, wherein the step of forming at least one volume includes a step of forming a plurality of partitions that contain the target at partial occupancy, wherein the step of amplifying includes a step of exposing the plurality of partitions to a plurality of thermal cycles, and wherein the partitions are maintained continuously above the melting temperature of the reporter throughout the plurality of thermal cycles.

7. The method of claim 1, wherein the step of extending one or more primers is performed at a temperature below a melting temperature of the first oligomer hybridized with the target.

8. The method of claim 1, wherein the step of amplifying includes a step of annealing the one or more primers to the target at a temperature above the melting temperature of the reporter.

9. The method of claim 5, wherein each energy transfer partner is a quencher.

10. The method of claim 6, wherein the partitions are droplets.