Covered sequence conversion dna and detection methods

Abstract

Disclosed are methods for detecting a target nucleic acid in sample, which include contacting the sample with an oligonucleotide having a hairpin structure, where the oligonucleotide includes, in the 5′ to 3′ direction, an arbitrary sequence, an endonuclease recognition site for a nicking reaction, a sequence complementary to the arbitrary sequence, and a sequence complementary to the target nucleic acid; a polymerase; and an endonuclease capable of nicking the endonuclease recognition site. The disclosure also provides compositions and kits comprising an oligonucleotide having a hairpin structure, where the oligonucleotide includes, in the 5′ to 3′ direction an arbitrary sequence, an endonuclease recognition site for a nicking reaction, a sequence complementary to the arbitrary sequence, and a sequence complementary to a target nucleic acid.

1 . A method for detecting a target nucleic acid in a sample, said method comprising contacting said sample with:
 a hairpin oligonucleotide comprising, in the 5′ to 3′ direction, a first arbitrary sequence, an endonuclease recognition site, a sequence complementary to said first arbitrary sequence, a sequence complementary to the 3′ end of a target nucleic acid, and a 3′-end modification;   a polymerase; and   an endonuclease for a nicking reaction.   
     
     
         2 . The method of  claim 1  further comprising determining the presence or absence of a signal DNA, wherein the presence of the signal DNA indicates the presence of the target nucleic acid in the sample. 
     
     
         3 . The method of  claim 1  wherein said method is performed at a substantially constant temperature. 
     
     
         4 . The method of  claim 1  wherein said method is performed at a temperature of from about 20° C. to about 42° C. The method of  claim 1  wherein said method is performed at a temperature of about 37° C. 
     
     
         6 . The method of  claim 1  wherein said target is a micro-RNA. 
     
     
         7 . The method of  claim 1  wherein said target is a micro-RNA selected from the group consisting of hsa-miR-24, hsa-miR-107, hsa-miR-221, hsa-miR-21, hsa-miR-500 and hsa-miR-106a. 
     
     
         8 . The method of  claim 1  wherein said target originates from an infectious agent. 
     
     
         9 . The method of  claim 1  wherein said target DNA originates from an infectious agent selected from the group consisting of hepatitis B virus, hepatitis C virus, human immunodeficiency virus, Chlamydia trachomatis, Neisseria gonorrhoeae, influenza A virus, influenza B virus, or respiratory syncytial virus. 
     
     
         10 . The method of  claim 1  wherein said target nucleic acid comprises a sequence within any of SEQ ID NO: 6, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO.: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, or SEQ ID NO: 30. 
     
     
         11 . The method of  claim 1  wherein the oligonucleotide further comprises a modification at the 5′ end, at the endonuclease recognition site, or at both the 5′ end and the endonuclease recognition site. 
     
     
         12 . A composition for detecting a target nucleic acid in a sample, said composition comprising:
 an oligonucleotide having a hairpin structure, wherein the oligonucleotide comprises, in the 5′ to 3′ direction, a first arbitrary sequence, an endonuclease recognition site, a sequence complementary to said first arbitrary sequence, a sequence complementary to the 3′ end of a target nucleic acid, and a 3′-end modification, wherein at least a portion of said first arbitrary sequence and at least a portion of said sequence complementary to said first arbitrary sequence hybridize to form said hairpin structure;   a polymerase; and   an endonuclease for a nicking reaction.   
     
     
         13 . The composition of  claim 12  wherein said sequence complementary to said target nucleic acid binds to a micro-RNA. 
     
     
         14 . The composition of  claim 12  wherein said sequence complementary to said target nucleic acid binds to a micro-RNA selected from the group consisting of hsa-miR-24, hsa-miR-107, hsa-miR-221, hsa-miR-21, hsa-miR-500 and hsa-miR-106a. 
     
     
         15 . The composition of  claim 12  wherein said sequence complementary to said target nucleic acid binds a target nucleic acid comprising a sequence within any of SEQ ID NO: 6, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO.: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, or SEQ ID NO: 30. 
     
     
         16 . The composition of  claim 12  wherein the oligonucleotide further comprises a modification at the 5′ end, at the endonuclease recognition site, or at both the 5′ end and the endonuclease recognition site. 
     
     
         17 . A kit for detecting a target nucleic acid in a sample, said kit comprising:
 an oligonucleotide having a hairpin structure, wherein the oligonucleotide comprises, in the 5′ to 3′ direction, a first arbitrary sequence, an endonuclease recognition site, a sequence complementary to said first arbitrary sequence, a sequence complementary to the 3′ end of a target nucleic acid, and a 3′-end modification, wherein at least a portion of said first arbitrary sequence and at least a portion of said sequence complementary to said first arbitrary sequence hybridize to form said hairpin structure;   a polymerase; and   an endonuclease for a nicking reaction.   
     
     
         18 . The kit of  claim 17  wherein said sequence complementary to said target nucleic acid binds to a micro-RNA. 
     
     
         19 . The kit of  claim 17  wherein said sequence complementary to said target nucleic acid binds to a micro-RNA selected from the group consisting of hsa-miR-24, hsa-miR-107, hsa-miR-221, hsa-miR-21, hsa-miR-500 and hsa-miR-106a. 
     
     
         20 . The kit of  claim 17  wherein said sequence complementary to said target nucleic acid binds a target nucleic acid comprising a sequence within any of SEQ ID NO: 6, SEQ ID NO:
 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO.: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, or SEQ ID NO: 30. 
 
     
     
         21 . The kit of  claim 17  wherein the oligonucleotide further comprises a modification at the 5′ end, at the endonuclease recognition site, or at both the 5′ end and the endonuclease recognition site. 
     
     
         22 . The method of  claim 1 , wherein said target nucleic acid binds to said hairpin oligonucleotide at said sequence complementary to the 3′ end of said target nucleic acid, and initiates strand displacement replication thereby generating a double stranded endonuclease recognition site and a double stranded first arbitrary sequence. 
     
     
         23 . The method of  claim 22 , wherein said endonuclease nicks said double stranded endonuclease recognition site, and strand displacement replication is initiated.