Granted Patent B2

US 12,258,620 · App. 17/279,825 · Granted Mar 25, 2025

Inventors: Jana Kent (Salt Lake City, UT); Masen Chad Christensen (Salt Lake City, UT); Brent Coleman Satterfield (Wichita Falls, TX)

Assignee: CO-DIAGNOSTICS, INC.

C12Q1/6827

Abstract

Claims (20)

1. A method of synthesizing a target nucleic acid preferentially relative to a nucleic acid with one or more nucleotides that differ from the target nucleic acid, the method comprising:

a. exposing a cooperative nucleic acid molecule to a solution suspected of comprising a target nucleic acid and also potentially comprising a nucleic acid with one or more nucleotides that differ from the target nucleic acid (differing nucleic acid), wherein the cooperative nucleic acid molecule comprises, from 3′ to 5′:

i. a first nucleic acid sequence, wherein the first nucleic acid sequence is complementary to a first region of the target nucleic acid, and further wherein a penultimate nucleotide of the 3′ end of the first nucleic acid sequence is complementary to the target nucleic acid, but is not complementary to the differing nucleic acid, and yet further wherein the first nucleic acid is extendable on the 3′ end;

ii. a linker connecting said first nucleic acid sequence and a second nucleic acid sequence in a manner that allows both the said first and second nucleic acid sequences to hybridize to the target nucleic acid at the same time;

iii. the second nucleic acid sequence, wherein the second nucleic acid sequence is complementary to a second region of the target nucleic acid, wherein the complementarity between the second nucleic acid sequence and the second region of the target nucleic acid starts within one nucleotide or less of the first region of the target nucleic acid, such that it hybridizes to the second target nucleic acid downstream from the 3′ end of the first nucleic acid sequence, or overlaps on a 5′ end of the second nucleic acid sequence with the 3′ end of the first nucleic acid sequence;

b. providing conditions appropriate for nucleic acid synthesis wherein a polymerase extends from the 3′ end of the first nucleic acid sequence through the second nucleic acid sequence, thereby synthesizing the target nucleic acid preferentially to the differing nucleic acid if it is present in the solution.

2. The method of claim 1 , wherein the target nucleic acid and differing nucleic acid differ by one nucleotide and wherein the one differing nucleotide comprises a single nucleotide polymorphism (SNP).

3. The method of claim 1 , wherein the target nucleic acid and the differing nucleic acid differ by two nucleotides.

4. The method of claim 1 , wherein the complementarity between the second nucleic acid sequence and the second region of the target nucleic acid is either within one nucleotide of the first region of the target nucleic acid, or is contiguous so that there are no gaps between where the first nucleic acid sequence hybridizes and the second nucleic acid sequence hybridizes, or is overlapping so that there is a negative gap between where the first nucleic acid sequence hybridizes and the second nucleic acid sequence hybridizes, and further wherein the overlap is one nucleotide in length, or more than one nucleotide in length.

5. The method of claim 1 , wherein a second cooperative nucleic acid molecule is exposed simultaneously with the cooperative nucleic acid molecule of claim 1 to a solution comprising the differing nucleic acid and the target nucleic acid, wherein said second cooperative nucleic acid molecule targets the differing nucleic acid for amplification, such that the penultimate nucleotide of the 3′ end of the first sequence of the second cooperative nucleic acid molecule is complementary to the differing nucleic acid, but is not complementary to the target nucleic acid.

6. The method of claim 5 , wherein the first and second cooperative nucleic acid molecules can amplify their intended targets under substantially similar conditions.

7. The method of claim 5 , wherein one or more additional differing nucleic acids are present in the solution.

8. The method of claim 7 , wherein the one or more additional differing nucleic acids differ at a same nucleotide position as the target and first differing nucleic acid or the additional differing nucleic acids differ at different nucleotide positions than the target nucleic acid and a second differing nucleic acid.

9. The method of claim 8 , wherein where the one or more additional differing nucleic acids differ at a same nucleotide position as the target and first differing nucleic acid, the one or more additional differing nucleic acids have different nucleotides at the same nucleotide position as the target nucleic acid and the first differing nucleic acid.

10. The method of claim 7 , wherein three or more cooperative nucleic acid molecules are simultaneously exposed to the target and differing nucleic acids.

11. The method of claim 1 , wherein hybridization of the first nucleic acid sequence of the cooperative nucleic acid molecule is conditional upon hybridization of the second nucleic acid sequence to the target nucleic acid.

12. The method of claim 1 , wherein the cooperative nucleic acid molecule comprises a label.

13. The method of claim 12 , wherein the label is a fluorescent label.

14. The method of claim 13 , wherein the molecule further comprises a quencher.

15. The method of claim 1 , wherein the target nucleic acid and the differing nucleic acid differ due to a deletion, addition, or substitution of a nucleotide.

Assignments (1)

Continuity (2)

References Cited (188)