US 12,258,620 B2
Allele-specific design of cooperative primers for improved nucleic acid variant genotyping
Jana Kent, Salt Lake City, UT (US); Masen Chad Christensen, Salt Lake City, UT (US); and Brent Coleman Satterfield, Wichita Falls, TX (US)
Assigned to CO-DIAGNOSTICS, INC., Salt Lake City, UT (US)
Appl. No. 17/279,825
Filed by CO-DIAGNOSTICS, INC., Salt Lake City, UT (US)
PCT Filed Sep. 25, 2019, PCT No. PCT/US2019/052957
§ 371(c)(1), (2) Date Mar. 25, 2021,
PCT Pub. No. WO2020/068983, PCT Pub. Date Apr. 2, 2020.
Claims priority of provisional application 62/736,094, filed on Sep. 25, 2018.
Prior Publication US 2021/0395800 A1, Dec. 23, 2021
Int. Cl. C12Q 1/6827 (2018.01)
CPC C12Q 1/6827 (2013.01) 15 Claims
OG exemplary drawing
 
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.