AMPLIFICATION REPORTER WITH BASE-PAIRING OLIGOMERS

• US 20150148250A1
• Filed: 08/12/2014
• Published: 05/28/2015
• 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, Cooper, Samantha, Hefner, Eli A., Klitgord, Niels, Shelton, Dawne N., Legler, Tina C., Yang, Wei
• Granted Patent: US 9,556,475 B2
• Time in Patent Office: Days
• US Class Current: 506/9
• 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 the reporter;

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;

detecting the photoluminescence of the reporter from 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 property of the target based on the photoluminescence detected.

2. The method of claim 1, wherein the step of amplifying uses the first oligomer as a primer.

3. The method of claim 2, wherein the first oligomer includes a photoluminophore from which the photoluminescence is detected.

4. The method of claim 2, wherein the second oligomer includes a photoluminophore from which the photoluminescence is detected, and wherein the first oligomer includes an energy transfer partner of the photoluminophore.

5. The method of claim 4, wherein the energy transfer partner is a quencher.

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

7. The method of claim 2, wherein the first oligomer forms a hybrid with the target having a higher melting temperature than the melting temperature of the reporter.

8. The method of claim 1, wherein the first oligomer includes a photoluminophore from which the photoluminescence is detected and also includes an energy transfer partner of the photoluminophore, and wherein the second oligomer does not include an energy transfer partner of the photoluminophore.

9. 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.

10. The method of claim 1, wherein the first oligomer includes a photoluminophore from which the photoluminescence is detected and an energy transfer partner of the photoluminophore, and wherein the second oligomer also includes an energy transfer partner of the photoluminophore.

11. The method of claim 1, wherein the step of amplifying causes cleavage of the first oligomer, the second oligomer, or both the first oligomer and the second oligomer, and wherein the cleavage affects the photoluminescence detected.

12. 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.

13. The method of claim 1, wherein the first oligomer has (a) a region dedicated to base-pairing with the target such that the first oligomer functions as a primer included in the one or more primers and (b) another region dedicated to base-pairing with the second oligomer and not the target.

14. A method of analysis, the method comprising:

forming partitions each containing a reporter including a first oligomer having a photoluminophore and also including a second oligomer capable of base-pairing with the first oligomer below a melting temperature of the reporter to decrease, by energy transfer, a photoluminescence detectable from the photoluminophore;

exposing the partitions to a plurality of thermal cycles, to amplify a target sequence at least in part by extending one or more primers at a temperature above the melting temperature of the reporter, wherein the target sequence is present in only a subset of the partitions;

detecting the photoluminescence of the photoluminophore for each partition of a plurality of the partitions while the partition is at a temperature below the melting temperature of the reporter; and

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

15. The method of claim 14, wherein the partitions are maintained continuously above the melting temperature of the reporter throughout the plurality of thermal cycles.

16. The method of claim 14, wherein the first oligomer is a primer included in the one or more primers.

17. The method of claim 16, wherein the photoluminophore is included in the first oligomer.

18. The method of claim 17, wherein a quencher for the photoluminophore is included in the second oligomer.

19. The method of claim 17, wherein a quencher for the photoluminophore is included in the first oligomer.

20. A composition, comprising:

a plurality of partitions containing a target at partial occupancy and amplification reagents to amplify the target and also containing a reporter to detect target amplification, the reporter including a first oligomer having a photoluminophore and a second oligomer that is base-paired with the first oligomer to affect a photoluminescence detectable from the photoluminophore.