Isothermal amplification in nucleic acid analysis

A method for detecting at least one target nucleic acid in a sample employing as reagents a stem/loop probe having a long strand complementary with at least a portion of said target nucleic acid, a short strand having a sequence within 0 to 3 bases of the 3′-end of said stem/loop probe that is complementary with a portion of said long strand and a linker joining said long and short strands, and a hybridizing reagent which has a hybridizing region for complementary with the 3′-end of said short strand, said method comprising:

• contacting said stem/loop probe with said sample and said hybridizing reagent and a phosphate bond forming reagent capable of linking said 3′-end to a nucleotide through said phosphate bond, wherein said linking results in attaching an active promoter to said stem/loop probe or, when said hybridizing reagent is circular, extending said 3′-end to form a concatenated DNA sequence;
• when said linking results in the attaching of an active promoter, adding NTPs and RNA polymerase to produce RNA transcripts from said promoter;
• and
• detecting the presence of said RNA transcripts or concatenated DNA sequence as indicating the presence of said target nucleic acid.

A method for detecting at least one target nucleic acid in a sample, said method employing as reagents a stem/loop probe having a long strand complementary with at least a portion of said target nucleic acid, a short strand having a sequence within 0-3 bases of the 3′-end of said stem/loop probe that is complementary with a portion of said long strand and a linker joining said long and short strands as a loop, and a hybridizing reagent which has a hybridizing region complementary with the 3′-end of said short strand, wherein said hybridizing reagent comprises a promoter sequence 5′ of said hybridizing region, said method comprising:

• contacting said stem/loop probe with said sample and said hybridizing reagent, wherein said hybridizing reagent hybridizes with said short strand when said target nucleic acid binds to said stem/loop probe to form a complex of said stem/loop probe and said hybridizing reagent;
• combining said complex with an enzyme and dNTPs for producing an extension of said stem/loop probe along said hybridizing reagent to form a promoter construct;
• combining said promoter construct with RNA polymerase and NTPs, whereby at least a portion of said stem/loop probe of said promoter construct is transcribed by said RNA polymerase to form RNA transcripts;
• and
• detecting the presence of said RNA transcripts as indicating the presence of said target nucleic acid.

A method for detecting at least one target nucleic acid in a sample, said method employing as reagents a stem/loop probe having a long strand complementary with at least a portion of said target nucleic acid, a short strand having a sequence within 1 to 3 bases of the 3′-end of said stem/loop probe that is complementary with a portion of said long strand wherein said 3′-end is mismatched with said long strand, and a linker joining said long and short strands as a loop, and a hybridizing reagent complementary with the 3′ -end of said short strand, wherein said hybridizing reagent comprises a promoter sequence 5′ of said hybridizable region, said method comprising:

• contacting said stem/loop probe with said sample, said hybridizing reagent, DNA polymerase, and dNTPs, whereby said 31-end is extended when target nucleic acid is bound to said stem/loop probe to form a promoter construct;
• combining said promoter construct, RNA polymerase and NTPs, whereby at least a portion of said stem/loop probe is transcribed to form RNA transcripts;
• and
• detecting the presence of said RNA transcripts as indicating the presence of said target nucleic acid.

A method for detecting a target nucleic acid in a sample employing as reagents a stem/loop probe having a long strand complementary with at least a portion of said target nucleic acid, a short strand having a sequence within 0 to 3 bases of the 3′-end of said stem/loop probe that is complementary with a portion of said long strand, and a linker joining said long and short strands as a loop, said linker comprising a restriction enzyme consensus sequence resistant to restriction enzyme cleavage, and a hybridizing reagent capable of hybridizing at its 3′-end to at least a portion of said short strand, said method comprising:

• contacting said stem/loop probe with said sample, said hybridizing reagent, DNA polymerase, dNTPs, and a restriction enzyme specific for said consensus sequence whereby stem/loop probe bound to said target nucleic acid binds to said hybridizing reagent providing a template on which said hybridizing reagent extends by means of said DNA polymerase to form a DNA strand complementary to said stem/loop probe comprising a restriction site, cleaving said DNA strand complementary to said stem/loop probe at said restriction site with said restriction enzyme to provide a cleaved strand comprising a new DNA extension initiation site, and extending said DNA extension initiation site to provide a new DNA strand complementary to said stem/loop probe, resulting in amplification of said DNA strand complementary to said stem/loop probe;
• and
• detecting said DNA strand complementary to said stem/loop probe as indicative of the presence of said target nucleic acid.

A method for detecting at least one target nucleic acid in a sample, said method employing as reagents a stem/loop probe having a long strand complementary with at least a portion of said target nucleic acid, a short strand having a sequence starting within 0 to 3 bases of the 3′-end of said stem/loop probe that is complementary with a portion of said long strand, and a linker joining said long and short strands as a loop, and a hybridizing reagent which is complementary with the 3′-end of said short strand, said method comprising:

• contacting said stem/loop probe with said sample and said hybridizing reagent wherein said stem/loop probe hybridizes with said hybridizing reagent when said target nucleic acid is present in said sample;
• combining said hybridized stem/loop probe with DNA polymerase and dNTPs to cause extension of said stem/loop probe along said hybridizing reagent;
• and
• detecting said extension as indicating the presence of said target nucleic acid.