Amplification method for polynucleotide assays

Claim 1

A method of producing multiple copies of a primary polynucleotide sequence located at the 3′ terminus of a polynucleotide, which comprises: (a) forming in the presence of nucleoside triphosphates and template-dependent polynucleotide polymerase an extension of a primary polynucleotide sequence hybridized with at template sequence of a single stranded pattern polynucleotide comprising two or more template sequences each containing one or more cleavable sites, (b) cleaving into fragments said extension at cleavable sites in the presence of means for specifically cleaving said cleavable sites when said extension is hybridized with said template sequence, (c) dissociating said fragments, comprising a primary polynucleotide sequence, (d) hybridizing said fragments with said single stranded pattern polynucleotide, and repeating steps (a)-(d) above wherein steps (a)-(d) are conducted simultaneously or wholly or partially sequentially.

Claim 2

A method of producing multiple copies of a primary polynucleotide sequence located at the 3′ terminus of a polynucleotide, which comprises: (a) forming in the presence of nucleoside triphosphates and template-dependent polynucleotide polymerase an extension of a primary polynucleotide sequence hybridized with at template sequence of a single stranded pattern polynucleotide comprising two or more template sequences each containing one or more cleavable sites, (b) cleaving into fragments said extension at cleavable sites in the presence of means for specifically cleaving said cleavable sites when said extension is hybridized with said template sequence, (c) dissociating said fragments, comprising a primary polynucleotide sequence, (d) hybridizing said fragments with said single stranded pattern polynucleotide, and repeating steps (a)-(d) above wherein steps (a)-(d) are conducted simultaneously or wholly or partially sequentially.

Claim 4

A method of determining the presence of a polynucleotide analyte in a sample suspected of containing said analyte, which comprises: (a) forming in the presence of nucleoside triphosphates and template-dependent polynucleotide polymerase an extension polynucleotide analyte having a 3′ hydroxy nucleotide, a part thereof including said 3′ hydroxy nucleotide being hybridized with a binding polynucleotide sequence of a single stranded pattern polynucleotide comprising said binding polynucleotide sequence connected at its 5′ end to two or more template sequences each containing one or more site specific cleavage sequences. (b) cleaving into fragments said extension at cleavable polynucleotide sequences in the presence of means for specifically cleaving said cleavable polynucleotide sequences when said extension is hybridized with said site specific cleavage sequences, (c) dissociating said fragments, (d) hybridizing said fragments with said single stranded pattern polynucleotide, (e) forming in the presence of said nucleoside triphosphates and said template dependent polynucleotide polymerase an extension of said fragments hybridized with said single stranded pattern polynucleotide, (f) repeating steps (b)-(e) above wherein steps (b)-(e) are conducted simultaneously or wholly or partially sequentially, and (g) detecting said fragments or fragments complementary thereto, the presence thereof indicating the presence of said polynucleotide analyte in said sample.

Claim 5

A method of determining the presence of a polynucleotide analyte in a sample suspected of containing said analyte, which comprises: (a) forming in the presence of nucleoside triphosphates and template-dependent polynucleotide polymerase an extension polynucleotide analyte having a 3′ hydroxy nucleotide, a part thereof including said 3′ hydroxy nucleotide being hybridized with a binding polynucleotide sequence of a single stranded pattern polynucleotide comprising said binding polynucleotide sequence connected at its 5′ end to two or more template sequences each containing one or more site specific cleavage sequences. (b) cleaving into fragments said extension at cleavable polynucleotide sequences in the presence of means for specifically cleaving said cleavable polynucleotide sequences when said extension is hybridized with said site specific cleavage sequences, (c) dissociating said fragments, (d) hybridizing said fragments with said single stranded pattern polynucleotide, (e) forming in the presence of said nucleoside triphosphates and said template dependent polynucleotide polymerase an extension of said fragments hybridized with said single stranded pattern polynucleotide, (f) repeating steps (b)-(e) above wherein steps (b)-(e) are conducted simultaneously or wholly or partially sequentially, and (g) detecting said fragments or fragments complementary thereto, the presence thereof indicating the presence of said polynucleotide analyte in said sample.

Claim 7

A method of producing multiple copies of a primary polynucleotide sequence as the result of the presence of a target polynucleotide sequence located at the 3′ terminus of a polynucleotide, which comprises: (a) forming in the presence of nucleoside triphosphates and template-dependent polynucleotide polymerase an extension of a target polynucleotide sequence hybridized with a binding polynucleotide sequence of a single stranded pattern polynucleotide comprising said binding polynucleotide sequence and two or more copies of a template sequence each containing one or more site specific cleavage sequences, (b) cleaving into fragments said extension at cleavable polynucleotide sequences in the presence of means for specifically cleaving said cleavable polynucleotide sequences when said extension is hybridized with said site specific cleavage sequences, (c) dissociating said fragments, wherein said fragments comprise said primary polynucleotide sequence (d) hybridizing said fragments with said single stranded pattern polynucleotide, (e) forming in the presence of said nucleoside triphosphates and said template dependent polynucleotide polymerase an extension of said fragments hybridized with said single stranded pattern polynucleotide, and (f) repeating steps (b)-(e) above wherein steps (b)-(e) are conducted simultaneously or wholly or partially sequentially.

Claim 8

A method of producing multiple copies of a primary polynucleotide sequence as the result of the presence of a target polynucleotide sequence located at the 3′ terminus of a polynucleotide, which comprises: (a) forming in the presence of nucleoside triphosphates and template-dependent polynucleotide polymerase an extension of a target polynucleotide sequence hybridized with a binding polynucleotide sequence of a single stranded pattern polynucleotide comprising said binding polynucleotide sequence and two or more copies of a template sequence each containing one or more site specific cleavage sequences, (b) cleaving into fragments said extension at cleavable polynucleotide sequences in the presence of means for specifically cleaving said cleavable polynucleotide sequences when said extension is hybridized with said site specific cleavage sequences, (c) dissociating said fragments, wherein said fragments comprise said primary polynucleotide sequence (d) hybridizing said fragments with said single stranded pattern polynucleotide, (e) forming in the presence of said nucleoside triphosphates and said template dependent polynucleotide polymerase an extension of said fragments hybridized with said single stranded pattern polynucleotide, and (f) repeating steps (b)-(e) above wherein steps (b)-(e) are conducted simultaneously or wholly or partially sequentially.

Claim 9

A method for detecting the presence of a polynucleotide analyte in a sample suspected of containing said polynucleotide analyte which comprises: combining either simultaneously or wholly or partially sequentially (1) said sample, (2) means for causing said polynucleotide analyte to be terminated in a 3′-OH group when said analyte is not terminated in a 3′-OH group, (3) a single stranded pattern polynucleotide comprising a binding polynucleotide sequence hybridizable with said polynucleotide analyte and connected to the 3′ end of a strand of two or more template sequences connected in tandem and containing site specific cleavage sequences, (4) nucleoside triphosphates, (5) template-dependent polynucleotide polymerase, and (6) means for specifically cleaving cleavable polynucleotide sequences complementary to said site specific cleavage sequences when said cleavable polynucleotide sequences are hybridized with said site specific cleavage sequences and incubating said combination under conditions for either simultaneously or wholly or partially sequentially (a) causing said polynucleotide analyte to be terminated in a 3′-OH group when said analyte is not terminated in a 3′-OH group, (b) hybridizing said polynucleotide analyte with said single stranded pattern polynucleotide, (c) forming an extension of said polynucleotide analyte comprising a sequence complementary to said template sequence and to said site specific cleavage sequences, (d) cleaving said extension into fragments at said cleavable polynucleotide sequences, (e) dissociating said fragments, (f) hybridizing said dissociated fragments with single stranded pattern polynucleotide, and (g) forming an extension of said hybridized fragments along said pattern polynucleotide, and repeating steps (d)-(g), and determining the presence of said fragments, or fragments complementary thereto, the presence thereof indicating the presence of said polynucleotide analyte in said sample.

Claim 10

A method for detecting the presence of a polynucleotide analyte in a sample suspected of containing said polynucleotide analyte which comprises: combining either simultaneously or wholly or partially sequentially (1) said sample, (2) means for causing said polynucleotide analyte to be terminated in a 3′-OH group when said analyte is not terminated in a 3′-OH group, (3) a single stranded pattern polynucleotide comprising a binding polynucleotide sequence hybridizable with said polynucleotide analyte and connected to the 3′ end of a strand of two or more template sequences connected in tandem and containing site specific cleavage sequences, (4) nucleoside triphosphates, (5) template-dependent polynucleotide polymerase, and (6) means for specifically cleaving cleavable polynucleotide sequences complementary to said site specific cleavage sequences when said cleavable polynucleotide sequences are hybridized with said site specific cleavage sequences and incubating said combination under conditions for either simultaneously or wholly or partially sequentially (a) causing said polynucleotide analyte to be terminated in a 3′-OH group when said analyte is not terminated in a 3′-OH group, (b) hybridizing said polynucleotide analyte with said single stranded pattern polynucleotide, (c) forming an extension of said polynucleotide analyte comprising a sequence complementary to said template sequence and to said site specific cleavage sequences, (d) cleaving said extension into fragments at said cleavable polynucleotide sequences, (e) dissociating said fragments, (f) hybridizing said dissociated fragments with single stranded pattern polynucleotide, and (g) forming an extension of said hybridized fragments along said pattern polynucleotide, and repeating steps (d)-(g), and determining the presence of said fragments, or fragments complementary thereto, the presence thereof indicating the presence of said polynucleotide analyte in said sample.

Claim 11

A method of producing multiple polynucleotide molecules as a function of the presence of a target sequence of nucleotide present in a polynucleotide sample, which method comprises: combining either wholly or partially sequentially or simultaneously (1) said polynucleotide sample containing said target sequence, (2) means to cause said target sequence to terminate in a free 3′-OH group when said target sequence does not already terminate in a free 3′-OH group, (3) a single stranded pattern polynucleotide comprising a binding polynucleotide sequence complementary to substantially all of said target sequence, joined at its 5′ end to two or more template sequences each containing site specific cleavage sequence, (4) nucleoside triphophates, (5) template-dependent polynucleotide polymerase, and (6) means for cleaving a sequence complementary to said site specific cleavage sequences when hybridized with said site specific cleavage sequence, said combining being carried out under conditions which promote either wholly or partially sequentially or simultaneously (a) denaturation of said target sequence when said target sequence is double stranded, (b) hybridization of said target sequence with said binding polynucleotide sequence, (c) extension of said target sequence by template dependent polymerization to produce a duplex, (d) cleavage of said extension in said duplex into fragments, (e) reversible denaturation of said fragments, (f) hybridization of said fragments with single stranded pattern polynucleotide, and (g) extension of said hybridized fragments by template dependent polymerization to produce a duplex, and repeating steps (d)-(g).

Claim 12

A method of producing multiple polynucleotide molecules as a function of the presence of a target sequence of nucleotide present in a polynucleotide sample, which method comprises: combining either wholly or partially sequentially or simultaneously (1) said polynucleotide sample containing said target sequence, (2) means to cause said target sequence to terminate in a free 3′-OH group when said target sequence does not already terminate in a free 3′-OH group, (3) a single stranded pattern polynucleotide comprising a binding polynucleotide sequence complementary to substantially all of said target sequence, joined at its 5′ end to two or more template sequences each containing site specific cleavage sequence, (4) nucleoside triphophates, (5) template-dependent polynucleotide polymerase, and (6) means for cleaving a sequence complementary to said site specific cleavage sequences when hybridized with said site specific cleavage sequence, said combining being carried out under conditions which promote either wholly or partially sequentially or simultaneously (a) denaturation of said target sequence when said target sequence is double stranded, (b) hybridization of said target sequence with said binding polynucleotide sequence, (c) extension of said target sequence by template dependent polymerization to produce a duplex, (d) cleavage of said extension in said duplex into fragments, (e) reversible denaturation of said fragments, (f) hybridization of said fragments with single stranded pattern polynucleotide, and (g) extension of said hybridized fragments by template dependent polymerization to produce a duplex, and repeating steps (d)-(g).

Claim 13

The method of any one of Claims 1-7, wherein said single stranded pattern polynucleotide comprises an oligomer of at least three of an identical template sequence.

Claim 15

The method of any one of Claims 2-9, wherein said site specific cleavage sequence is a restriction endonuclease site and said means for specifically cleaving said cleavable polynucleotide sequences comprises a restriction endonuclease.

Claim 16

The method of any one of Claims 1-10, wherein said 3′ end of said template sequences in said single stranded pattern polynucleotide is connected to a polynucleotide sequence complementary to a polynucleotide analyte in a sample suspected of containing said polynucleotide analyte.

Claim 17

The method of any one of Claims 1-11, wherein said single stranded pattern polynucleotide is at least part of a cyclic polynucleotide.

Claim 18

The method of any one of Claims 1-12, wherein said template sequences and the sequences connecting said template sequences consist of a continuous sequence of nucleotide selecte from three members of the group consisting of A and dA, U and dT, C and dC, and G and dG and derivatives thereof.

Claim 22

A method comprising the preparation of a single stranded DNA oligomer bonded at its 3′ end to a single stranded nucleic acid binding sequence consisting of at least about 16 nucleotides, said oligomer consisting of from about 3-100 oligonucleotide units each consisting of an identical oligonucleotide template sequence having from about 8-100 nucleotides and at least one restriction site, wherein said oligomer is composed of nucleotides selected from three members of the group consisting of dA, dT, dG, and dC and derivatives thereof.

Claim 23

A method comprising the preparation of a single stranded DNA oligomer bonded at its 3′ end to a single stranded nucleic acid binding sequence consisting of at least about 16 nucleotides, said oligomer consisting of from about 3-100 oligonucleotide units each consisting of an identical oligonucleotide template sequence having from about 8-100 nucleotides and at least one restriction site, wherein said oligomer is composed of nucleotides selected from three members of the group consisting of dA, dT, dG, and dC and derivatives thereof.