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US 7,678,541 B2Granted
Methods and compositions for the detection of a nucleic acid using a non-invasive cleavage reaction
AssigneeHologic, Inc.
InventorsJoseph A. Sorge, Scott Happe, Andrew Firmin
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Abstract
The invention provides methods, compositions and kits for generating a signal indicative of the presence of a target nucleic acid sequence in a sample comprising forming a cleavage structure by incubating a sample comprising a target nucleic acid sequence with upstream and downstream oligonucleotides, and cleaving the cleavage structure with a nuclease to generate a signal. The presence of a detectable signal is indicative of the presence of a target nucleic acid sequence and a non-invasive cleavage structure.
Claims37
A method of detecting a target nucleic acid, comprising
- (a) providing:
- a target nucleic acid, which comprises in 3′ to 5′ order a first region and a second region,
- a template nucleic acid, which comprises in 3′ to 5′ order a first region and a second region,
- a first oligonucleotide that is at least partially complementary to said first region of said target nucleic acid,
- a second oligonucleotide comprising a 5′ region and a 3′ region, wherein said 3′ region is complementary to said second region of said target nucleic acid and wherein said 5′ region is not complementary to said target nucleic acid but is at least partially complementary to said first region of said template nucleic acid,
- a third oligonucleotide comprising a 5′ region and a 3′ region, wherein said 3′ region is at least partially complementary to said second region of said template nucleic acid and said 5′ region is not complementary to said template nucleic acid,
- a cleavage agent, and
- a polymerization agent;
- (b) mixing said target nucleic acid, said template nucleic acid, said first oligonucleotide, said second oligonucleotide, said third oligonucleotide, said cleavage agent and said polymerization agent under reaction conditions, wherein said reaction conditions permit:
- forming a first duplex between said target nucleic acid and each of said fast oligonucleotide and said second oligonucleotide, wherein the 5′ region of the second oligonucleotide forms a first flap of a first non-invasive cleavage structure, wherein the complementary portions of said first oligonucleotide and said second oligonucleotide are separated by at least a nick when the fast duplex is formed,
- cleaving the fast flap by said cleavage agent, thereby permitting release of the first flap,
- forming a second duplex with the released fast flap, said template nucleic acid, and said third oligonucleotide, wherein the 5′ region of the third oligonucleotide forms a second flap of a second non-invasive cleavage structure, wherein the complementary portions of said first flap and said third oligonucleotide are separated by at least a nick when said second duplex is formed, and
- cleaving the second flap by said cleavage agent;
- and
- (c) detecting the non-invasive cleavage of the second flap, wherein detection of said non-invasive cleavage of said second flap is indicative of the presence of said target nucleic acid.
12 more elements
A method of detecting a target nucleic acid, comprising
- (a) providing:
- a target nucleic acid, which comprises in 3′ to 5′ order a first region and a second region,
- a template nucleic acid, which comprises in 3′ to 5′ order a first region and a second region,
- a fast oligonucleotide that is at least partially complementary to said first region of said target nucleic acid,
- a second oligonucleotide comprising a 5′ region and a 3′ region, wherein said 3′ region is complementary to said second region of said target nucleic acid and wherein said 5′ region is not complementary to said target nucleic acid but is at least partially complementary to said first region of said template nucleic acid,
- a third oligonucleotide comprising a 5′ region and a 3′ region, wherein said 3′ region is at least partially complementary to said second region of said template nucleic acid and said 5′ region is not complementary to said template nucleic acid,
- a cleavage agent, and
- a polymerization agent;
- (b) forming a reaction mixture by mixing said target nucleic acid, said first oligonucleotide, said second oligonucleotide, said cleavage agent and said polymerization agent under reaction conditions, wherein said reaction conditions permit:
- forming of a fast duplex between said target nucleic acid and each of said first oligonucleotide and said second oligonucleotide, wherein the 5′ region of the second oligonucleotide forms a first flap of a first non-invasive cleavage structure, and wherein the complementary portions of said fast oligonucleotide and said second oligonucleotide are separated by at least a nick when said fast duplex is formed,
- cleavage of the first flap by said cleavage agent, thereby permitting release of the first flap,
- (c) mixing the released first flap, said third oligonucleotide, and said template nucleic acid in said reaction mixture under reaction conditions, wherein said reaction conditions permit:
- forming of a second duplex with the released first flap, said template nucleic acid, and said third oligonucleotide, wherein the 5′ region of the third oligonucleotide forms a second flap of a second non-invasive cleavage structure, wherein the complementary portions of said first flap and said third oligonucleotide are separated by at least a nick when said second duplex is formed, and
- cleaving of the second flap by said cleavage agent;
- and
- (d) detecting the non-invasive cleavage of the second flap, wherein detection of said non-invasive cleavage of said second flap is indicative of the presence of said target nucleic acid.
13 more elements
A composition comprising:
- a target nucleic acid, which comprises in 3′ to 5′ order a first region and a second region,
- (ii) a template nucleic acid, which comprises in 3′ to 5′ order a first region and a second region,
- (iii) a first oligonucleotide that is at least partially complementary to said first region of a said target nucleic acid;
- (iv) a second oligonucleotide comprising a 5′ flap region and a 3′ region, wherein said 3′ region is at least partially complementary to said second region of said target nucleic acid and wherein said 5′ flap region is non-complementary to said target nucleic acid but is at least partially complementary to said fast region of said template nucleic acid;
- and
- (v) a third oligonucleotide comprising a 5′ flap region and a 3′ region, wherein said 3′ region is at least partially complementary to said second region of said template nucleic acid and said 5′ flap region is non-complementary to said template nucleic acid, and wherein a first member of a pair of interactive labels is operatively coupled to said 5′ flap region and wherein a second member of the pair of interactive labels is operatively coupled to the said 3′ region.
3 more elements
A method of detecting a target nucleic acid, comprising
- (a) providing:
- a target nucleic acid, which comprises in 3′ to 5′ order a first region and a second region,
- a fast oligonucleotide that is at least partially complementary to said fast region of said target nucleic acid,
- a second oligonucleotide comprising a 5′ region and a 3′ region, wherein said 3′ region is complementary to said second region of said target nucleic acid and wherein said 5′ region is not complementary to said target nucleic acid,
- a cleavage agent, and
- a polymerization agent;
- (b) mixing said target nucleic acid, said first oligonucleotide, said second oligonucleotide, said cleavage agent and said polymerization agent under reaction conditions, wherein said reaction conditions permit:
- forming a duplex between said target nucleic acid and each of said first oligonucleotide and said second oligonucleotide, wherein the 5′ region of the second oligonucleotide forms a flap of a non-invasive cleavage structure, wherein the complementary portions of said first oligonucleotide and said second oligonucleotide are separated by at least a nick when said duplex is formed,
- cleaving the flap by said cleavage agent, and
- (c) detecting the non-invasive cleavage of the flap, wherein detection of said non-invasive cleavage of said flap is indicative of the presence of said target nucleic acid.
7 more elements
Dependencies are inferred from the claim text.