Nucleic acid amplification using single primer

Claim 3

The method of Claim 1 wherein the last 10 nucleotides of the 3′-end of said polynucleotide primer are in the same sequence as said second flanking sequence.

Claim 4

The method of Claim 1 and 2 wherein the product formed after repeating step (a) contains an inverted repeat.

Claim 5

The method of Claim 1, 2 or 3 wherein said hybridizable sequence of said polynucleotide primer is 15 to 100 nucleotides in length.

Claim 6

The method of Claim 1, 2, 3 or 4 wherein said template-dependent polynucleotide polymerase is a DNA polymerase and said nucleoside triphosphates are dATP, dGTP, dCTP, and dTTP.

Claim 8

The method of Claim 6 wherein said first and second sequences are complementary.

Claim 11

The method of Claim 8 wherein said first and second flanking sequences are comprised of complementary sequences of at least 10 bases.

Claim 12

A method according to Claim 8 or 9 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.

Claim 13

The method of Claim 10 wherein a portion of said polynucleotide primer is labeled with one reporter group and a portion is labeled with a second reporter group.

Claim 15

The method of Claim 12 wherein said polynucleotide primer is labeled with a reporter group selected from the group consisting of, fluorescers, chemiluminescers, catalysts, co-enzymes, radioactive substances, amplifiable polynucleotide sequences, and small organic molecules.

Claim 16

The method of Claim 12 or 13 wherein said first and second polynucleotide probes when hybridized with said analyte are at least one nucleotide apart and conditions for ligating said first and second polynucleotide probes include (a) adding nucleotide triphosphates and a polynucleotide polymerase to render the hybridized portions contiguous and (b) adding ligase to ligate said hybridized portions.

Claim 17

A method according to Claim 8 for detecting the presence of a polynucleotide analyte in a sample suspected of containing said analyte, which comprises the steps of: (a) ligating third and fourth sequences of a polynucleotide probe, said third sequence having a free 3′-end and said fourth sequence having a free 5′-end and each being hybridizable to separate portions of said analyte and ligatable, or capable of being rendered ligatable, only in the presence of said analyte, said polynucleotide probe further comprising at least partially complementary first and second flanking sequences, wherein said first sequence is 5′ of said third sequence, said second sequence is 5′ of said first sequence and said fourth sequence is 5′ of said second sequence, (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 said first flanking sequence of said polynucleotide probe, wherein steps (a) and (b) are performed wholly or partially sequentially or concomitantly and, detecting extended polynucleotide primer and/or a sequence identical to and/or complementary with at least that portion of said ligated third or fourth sequences complementary to said separate portions of said analyte.

Claim 18

The method of Claim 15 wherein said probe contains an operator sequence.

Claim 19

A method according to Claim 8, 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 first and second polynucleotide probes, said first probe comprising a first flanking sequence at least partially complementary with a second flanking sequence in said second probe and attached at its 3′-end to a sequence hybridizable with a first portion of one strand of said analyte wherein said second flanking sequence is attached at its 5′-end to a sequence hybridizable with a second portion of said 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 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 second flanking sequence 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 on partially sequentially or concomitantly followed by step (e), and (f) detecting said extended primer.

Claim 20

The method of Claim 17 wherein separate molecules of polynucleotide primer are labelled with a group capable of becoming bound to a surface.

Claim 21

The method of Claim 17 or 18 wherein said extending of said polynucleotide primer along said ligated first and second probes forms at least part of an operator and said detecting includes: (a) adding a repressor bound to a solid support and (b) detecting a sequence bound to said solid support that is identical or complementary to a sequence in said first polynucleotide probe.

Claim 22

A method according to Claim 8 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 (1) a first flanking sequence complementary to a second flanking sequence in said second probe and (2) a sequence complementary to a portion of said analyte other than a portion complementary to said second probe 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, (b) 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 first or said second probe, wherein steps (a) and (b) are carried out wholly or partially sequentially or concomitantly and detecting said extended polynucleotide primer and/or ligated first and second polynucleotide probes.

Claim 23

A kit for use in determining a polynucleotide analyte, which comprises in packaged combination: a first polynucleotide probe having a first nucleotide sequence capable of hybridizing to a first portion of said polynucleotide analyte, a second polynucleotide probe having a second nucleotide sequence capable of hybridizing to a second portion of the same strand of said polynucleotide analyte other than with the portion recognized by said first nucleotide sequence of said first polynucleotide probe, means for covalently attaching said first and second polynucleotide sequences when said first and second polynucleotide probes are hybridized with said polynucleotide analyte, and a polynucleotide primer capable of hybridizing with said second polynucleotide probe and of being extended along said covalently attached probes in the direction of said first polynucleotide probe.

Claim 24

A kit for use in determining a polynucleotide analyte, which comprises in packaged combination: a first polynucleotide probe having a first nucleotide sequence capable of hybridizing to a first portion of said polynucleotide analyte, a second polynucleotide probe having a second nucleotide sequence capable of hybridizing to a second portion of the same strand of said polynucleotide analyte other than with the portion recognized by said first nucleotide sequence of said first polynucleotide probe, means for covalently attaching said first and second polynucleotide sequences when said first and second polynucleotide probes are hybridized with said polynucleotide analyte, and a polynucleotide primer capable of hybridizing with said second polynucleotide probe and of being extended along said covalently attached probes in the direction of said first polynucleotide probe.

Claim 25

A method of detecting the presence of a polynucleotide analyte in a sample suspected of containing said analyte, according to Claim 8, which comprises as step (a) of Claim 8 the steps of: (i) forming in the presence of nucleoside triphosphates and template dependent polynucleotide polymerase along a first single stranded polynucleotide an extension of said analyte or a fragment thereof (extended polynucleotide analyte) to form double-stranded polynucleotide 1, (ii) dissociating single stranded extended polynucleotide analyte from said polynucleotide 1, (iii) forming in the presence of nucleoside triphosphates and template dependent polynucleoside polymerase along said single stranded extended polynucleotide analyte an extension of a first polynucleotide primer at least the 3′-end of which has at least a 10 base sequence hybridizable with at least a portion of said single stranded extended polynucleotide analyte to form double stranded polynucleotide 2, (iv) dissociating single stranded extended first polynucleotide primer from said polynucleotide 2, (v) forming in the presence of nucleoside triphosphates and template dependent polynucleotide polymerase along a second polynucleotide primer an extension of said single stranded extended first polynucleotide primer wherein at least the 3′-end of said second polynucleotide primer has at least a 10 base sequence hybridizable with at least a portion of said single stranded extended first polynucleotide primer to form double stranded polynucleotide 3, wherein the 3′-end and the 5′-end of single stranded twice extended first polynucleotide primer each have sequences of 10 bases which sequences are at least partially complementary, and (vi) dissociating said single stranded twice extended first polynucleotide primer from said polynucleotide 3; and as step (b) of Claim 8, the steps of: (i)forming in the presence of nucleoside triphosphates and template dependent polynucleotide polymerase along said single stranded twice extended first polynucleotide primer an extension of said first polynucleotide primer to form double stranded polynucleotide 4, (ii) dissociating single stranded extended first polynucleotide primer from polynucleotide 4, and iii) repeating step (i).