IP Library › Granted Patent US 11,293,048
Granted Patent B2
US 11,293,048 · App. 15/938,178 · Granted Apr 5, 2022

Attenuators

Inventors: Joanne M. Yeakley (Encinitas, CA); Bruce Seligmann (Tucson, AZ); Joel McComb (Rancho Santa Fe, CA)
Assignee: Bio Spyder Technologies, Inc.
C12Q1/6806C12Q1/6816C12Q1/6841
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Quick Facts
Patent No.
US 11,293,048
App. No.
15/938,178
Granted
Apr 5, 2022
Kind
B2
Abstract

Methods for detecting nucleic acid sequences, where attenuator oligonucleotides are provided to reduce the number of detection products resulting from highly abundant sequences.

Claims (28)

1. A set of attenuator oligonucleotides for a sample type that has a plurality of relatively high abundance target sequences (HATs), each HAT having a downstream region (DR) and an adjacent upstream region (UR),

wherein the set comprises for each HAT:

(a) a downstream detector oligo (DDO) comprising a P 1 primer sequence and a region complementary to the downstream region of the HAT target sequence (DR′), and

(b) an upstream detector oligo (UDO) comprising a region complementary to the upstream region of the HAT target sequence (UR′) and a P 2 ′ primer hybridization sequence,

wherein both the downstream detector and the upstream detector are capable of hybridizing simultaneously to the HAT target sequence under predetermined hybridization conditions, and;

(c) an attenuator oligo comprising a portion of the DR′ or the UR′ of sufficient length to specifically bind to a corresponding portion of the DR or UR of the HAT target sequence,

and further comprising a sequence X that is different from P 1 or a sequence Y that is different from P 2 ′.

2. The set of attenuator oligonucleotides of claim 1 , further comprising a pool of primer oligos comprising a P 1 sequence and a pool of primer oligos comprising a P 2 sequence.

3. The set of attenuator oligonucleotides of claim 1 , wherein an attenuator comprises a portion at least partially complementary to one of the DR or UR of a HAT, and further comprises a portion at least partially complementary to the other region of the HAT.

4. The set of attenuator oligonucleotides of claim 1 , wherein an attenuator comprises a portion that is complementary to at least a portion of the DR of a HAT and another portion that is complementary to at least a portion of the UR of the HAT.

5. The set of attenuator oligonucleotides of claim 1 , wherein an attenuator comprises at least one mismatched base relative to the DR or UR of a HAT.

6. The set of attenuator oligonucleotides of claim 1 , wherein an attenuator comprises a portion partially complementary to the DR of a HAT, and comprises 1, 2, or 3 mismatched bases at any of the positions at the 3′ end or at 1 or 2 positions from the 3′ end.

7. The set of attenuator oligonucleotides of claim 1 , wherein an attenuator comprises a portion partially complementary to the UR of a HAT, and comprises 1, 2, or 3 mismatched bases at any of the positions at the 5′ end or at 1 or 2 positions from the 5′ end.

8. The set of attenuator oligonucleotides of claim 1 , wherein, under the hybridization conditions in (b), the sequence X does not specifically bind to the P 1 sequence or its complement; or the sequence Y does not specifically bind to the P 2 sequence or its complement.

9. The set of attenuator oligonucleotides of claim 1 , wherein an attenuator comprises a nonamplifiable segment comprising a reverse complementary sequence.

10. The set of attenuator oligonucleotides of claim 1 , wherein the DR of a HAT is connected to the UR of the HAT by a flexible linker.

11. The set of attenuator oligonucleotides of claim 1 , wherein an attenuator is phosphorylated.

12. The set of attenuator oligonucleotides of claim 1 , wherein an attenuator is not phosphorylated.

13. The set of attenuator oligonucleotides of claim 1 , wherein an attenuator further comprises a portion that is complementary to a region adjacent to the DR or UR of a HAT.

14. The set of attenuator oligonucleotides of claim 1 , wherein the DR and UR of a HAT are separated by one or more nucleotides, and an attenuator has a portion that is complementary to the one or more nucleotides.

15. The set of attenuator oligonucleotides of claim 1 , wherein an attenuator comprises a selectively cleavable site.

16. The set of attenuator oligonucleotides of claim 1 , wherein an attenuator further comprises a nonextendable or nonligatable blocking group.

17. The set of attenuator oligonucleotides of claim 1 , wherein an attenuator is detectably labeled.

18. The set of attenuator oligonucleotides of claim 1 , wherein an attenuator comprises a barcode sequence.

19. The set of attenuator oligonucleotides of claim 18 , wherein the barcode sequence is specific to a HAT.

20. The set of attenuator oligonucleotides of claim 1 , wherein a HAT comprises a sequence partially encoding a product selected from the group consisting of cytochrome c, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), ribosomal protein L7 (RPL7), ribosomal protein S6 (rpS6), snRNA RNUs, phosphoglycerokinase (PGK), tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein zeta (YWHAZ), beta-actin, and beta-tubulin.

21. The set of attenuator oligonucleotides of claim 1 , wherein the attenuator comprises a portion of the DR′ and a portion of the UR′.

22. The set of attenuator oligonucleotides of claim 9 , wherein the reverse complementary sequence forms a hairpin loop under the hybridization conditions of (b).

Assignments (2)
LICENSE Recorded Dec 5, 2025
From: BIOSPYDER TECHNOLOGIES, INC.
To: NATIONAL INSTITUTES OF HEALTH
Reel/Frame 073869/0721 →
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Jul 5, 2018
From: YEAKLEY, JOANNE M.; SELIGMANN, BRUCE; MCCOMB, JOEL
To: BIOSPYDER TECHNOLOGIES, INC.
Reel/Frame 046501/0016 →
Continuity (2)
Division 14480525 · Sep 8, 2014
Related Publication 20180216166A1 · Aug 2, 2018
Cited By (1)
US 12,448,645