Nucleic acid amplification using single primer

A method for determining the presence of a polynucleotide analyte in a sample suspected of containing said analyte, which comprises the steps of:

• (a) forming as a result of the presence of said analyte a single stranded polynucleotide flanked by at least 80% complementary first and second flanking sequences, each comprised of at least 15 bases,
• (b) forming in the presence of nucleoside triphosphates and template dependent polynucleotide polymerase an extension of a polynucleotide primer at least the 3'-end of which can hybridize with the flanking sequence at the 3'-end of said single stranded polynucleotide sequence,
• wherein steps (a) and (b) can be performed wholly or partially sequentially or concomitantly, and
• (c) detecting extended polynucleotide primer containing a sequence identical to and/or complementary with said polynucleotide sequence, the presence thereof indicating the presence of said analyte.

A method for determining the presence of a polynucleotide analyte in a sample suspected of containing said analyte, which comprises;

• (a) contacting said sample with first and second polynucleotide probes, said first probe comprising (i) a target polynucleotide binding sequence at its 3'-end complementary to a first portion of one strand of said analyte and (ii) a first flanking sequence and said second probe comprising (i) a target polynucleotide binding sequence at its 5' end complementary to a second portion of the same strand of said analyte and (ii) a second flanking sequence wherein said first and second flanking sequences are at least 65% complementary, under conditions for binding of said first and second probes with said analyte,
• (b) providing conditions for ligating said first and second polynucleotide probes to one another only when bound to said analyte,
• (c) forming in the presence of nucleoside triphosphates and template dependent polynucleotide polymerase an extension of a polynucleotide primer at least the 3'-end of which can hybridize with said second flanking sequence, and
• (d) detecting extended polynucleotide primer containing a sequence complementary to said first probe, the presence thereof indicating the presence of said analyte.

A method for detecting the presence of a polynucleotide analyte in a sample suspected of containing said analyte, which comprises:

• (a) hybridizing to said analyte a first polynucleotide probe and a second polynucleotide probe, said first polynucleotide probe having (i) a sequence S1 that is at least 10 nucleotides in length and at least 50% complementary to a sequence S2 of said second polynucleotide probe and (ii) a sequence S3 that is 3' of said S1 and having a free 3'-end, said second polynucleotide probe having a sequence S4 that is 5' of said S2, said S4 having a free 5'-end, wherein the 3'-end of said S3 and the 5'-end of said S4 are ligatable, said first polynucleotide probe hybridizing to said analyte at a portion thereof that is 3' of the portion to which said second polynucleotide probe hybridizes to said analyte,
• (b) ligating said S3 and said S4 to form ligated first and second polynucleotide probes,
• (c) forming in the presence of nucleoside triphosphates and template dependent polynucleotide polymerase an extension of a polynucleotide primer at least the 3'-end of which hybridizes with said S2 of said ligated first and second polynucleotide probes,
• wherein steps (a) and (b) and (c) are performed wholly or partially sequentially of concomitantly and,
• (d) detecting extended polynucleotide primer and/or a sequence identical to and/or complementary with at least that portion of said ligated first and second polynucleotide probes, the presence thereof indicating the presence of said analyte.

A method for determining the presence of a polynucleotide analyte in a sample suspected of containing said analyte, which comprises;

• (a) combining in an assay medium said sample with a first polynucleotide probe and a second polynucleotide probe, said first probe comprising a sequence S1 at least 10 nucleotides in lenath and at least 80% complementary with a sequence S2 in said second probe, said first probe being attached at its 3'-end to a sequence having a portion S3 that is hybridizable with a first portion of a single strand of said analyte wherein said S2 is attached at is 5'-end to a sequence having a portion S4 that is hybridizable with a second portion of said single strand of said analyte,
• (b) incubating said assay medium under conditions for binding of said first and second probes with said analyte, said first and second probes being ligatable, or capable of being rendered ligatable, to one another only when bound to said analyte,
• (c) ligating said first and second probes to form ligated first and second probes with the proviso that, if necessary, where they are not otherwise ligatable, said first and second probes are rendered ligatable,
• (d) combining said assay medium with a polynucleotide primer having a 3' terminal sequence complementary to said S2 in said second probe,
• (e) incubating said assay medium under conditions for either wholly or partially sequentially or concomitantly (1) dissociating any internally hybridized ligated first and second probes, (2) hybridizing said polynucleotide primer with said ligated first and second probes, (3) extending said polynucleotide primer along said ligated first and second probes, (4) dissociating said hybridized extended polynucleotide primer and said ligated first and second probes, (5) hybridizing said extended polynucleotide primer with said polynucleotide primer, (6) extending said polynucleotide primer along said extended polynucleotide primer, (7) dissociating said hybridized extended primer, and (8) repeating steps (5)-(7) above,
• wherein steps (a)-(d) are carried out wholly or partially sequentially or concomitantly followed by step (e), and
• (f) detecting said extended primer, the presence thereof indicating the presence of said analyte.

A method for determining the presence of a polynucleotide analyte in a sample suspected of containing said analyte, which comprises:

• (a) contacting said sample with a first polynucleotide probe and a second polynucleotide probe, said first probe comprising (1) a first sequence S1 that is at least 10 nucleotides in length and at least 80% complementary to a second sequence S2 in said second probe and (2) a sequence S3 complementary to a portion of said analyte other than a portion complementary to a sequence S4 of said second probe under conditions for binding of said first and second probes with said analyte,
• (b) ligating said first and second probes to one another to form ligated first and second probes,
• (c) forming in the presence of nucleoside triphosphates and template dependent polynucleotide polymerase an extension of a polynucleotide primer at least a portion of which is hybridized with said S1 or said S2 of said ligated first and second probes,
• wherein steps (a) and (b) and (c) are carried out wholly or partially sequentially or concomitantly and
• (d) detecting said extended polynucleotide primer and/or said ligated first and second probes, the presence thereof indicating the presence of said analyte.