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 a template sequence of a single stranded pattern polynucleotide comprising two or more identical 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, 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 a template sequence of a single stranded pattern polynucleotide comprising two or more identical 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, 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 12

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 a template sequence of a single stranded pattern polynucleotide comprising 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, 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 and wherein the number of said copies of said primary polynucleotide sequence formed for each of said extension is increased by at least a factor of three.

Claim 13

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 a template sequence of a single stranded pattern polynucleotide comprising 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, 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 and wherein the number of said copies of said primary polynucleotide sequence formed for each of said extension is increased by at least a factor of three.

Claim 20

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 21

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 33

A method of producing multiple polynucleotide molecules as a function of the presence of a target sequence of nucleotides 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) 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 identical template sequences each containing site specific cleavage sequence, (4) nucleoside triphosphates, (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 polynuceotide, and (g) extension of said hybridized fragments by template dependent polymerization to produce a duplex, and repeating steps (d)-(g).

Claim 34

A method of producing multiple polynucleotide molecules as a function of the presence of a target sequence of nucleotides 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) 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 identical template sequences each containing site specific cleavage sequence, (4) nucleoside triphosphates, (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 polynuceotide, and (g) extension of said hybridized fragments by template dependent polymerization to produce a duplex, and repeating steps (d)-(g).

Claim 55

A method of producing multiple copies of a primary polynucleotide sequence located at the 3' terminus of a polynucleotide which comprises: providing in combination either wholly or partially sequentially or concomitantly (1) said primary polynucleotide sequence hybridized with single stranded pattern polynucleotide, (2) single stranded pattern polynucleotide comprising two or more identical template sequences each containing site specific cleavage sequences, (3) nucleoside triphosphates, (4) template-dependent polynucleotide polymerase, and (5) means for specifically cleaving cleavable polynucleotide sequences complementary to 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 wholly or partially sequentially or concomitantly (a) forming an extension of said primary polynucleotide sequence comprising one or more copies thereof, (b) cleaving said extension at said cleavable polynucleotide sequences into primary polynucleotide sequences, (c) dissociating said hybridized extension, (d) hybridizing said primary polynucleotide sequences with said single stranded pattern polynucleotide, and repeating at least steps (a) and (b) above.

Claim 56

A method of producing multiple copies of a primary polynucleotide sequence located at the 3' terminus of a polynucleotide which comprises: providing in combination either wholly or partially sequentially or concomitantly (1) said primary polynucleotide sequence hybridized with single stranded pattern polynucleotide, (2) single stranded pattern polynucleotide comprising two or more identical template sequences each containing site specific cleavage sequences, (3) nucleoside triphosphates, (4) template-dependent polynucleotide polymerase, and (5) means for specifically cleaving cleavable polynucleotide sequences complementary to 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 wholly or partially sequentially or concomitantly (a) forming an extension of said primary polynucleotide sequence comprising one or more copies thereof, (b) cleaving said extension at said cleavable polynucleotide sequences into primary polynucleotide sequences, (c) dissociating said hybridized extension, (d) hybridizing said primary polynucleotide sequences with said single stranded pattern polynucleotide, and repeating at least steps (a) and (b) above.

Claim 57

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: providing in combination either wholly or partially sequentially or concomitantly (1) said target polynucleotide sequence hybridized to a single stranded pattern polynucleotide comprising a binding sequence complementary to said target polynucleotide sequence and one or more copies of a template sequence complementary to said primary polynucleotide sequence each containing two or more site specific cleavage sequences, (2) single stranded pattern polynucleotide, (3) nucleoside triphosphates, (4) template-dependent polynucleotide polymerase, and (5) 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 wholly or partially sequentially or concomitantly (a) forming an extension of said target sequence comprising one or more of said copies of said primary polynucleotide sequence connected through said cleavable polynucleotide sequences, (b) cleaving said extension at said cleavable polynucleotide sequences into said target sequence and primary polynucleotide sequences, (c) dissociating said hybridized extension, (d) hybridizing said primary polynucleotide sequence with said single stranded pattern polynucleotide, and (e) forming an extension of said primary polynucleotide sequence comprising one or more copies of said primary polynucleotide sequence, and repeating steps (b)-(e).

Claim 58

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: providing in combination either wholly or partially sequentially or concomitantly (1) said target polynucleotide sequence hybridized to a single stranded pattern polynucleotide comprising a binding sequence complementary to said target polynucleotide sequence and one or more copies of a template sequence complementary to said primary polynucleotide sequence each containing two or more site specific cleavage sequences, (2) single stranded pattern polynucleotide, (3) nucleoside triphosphates, (4) template-dependent polynucleotide polymerase, and (5) 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 wholly or partially sequentially or concomitantly (a) forming an extension of said target sequence comprising one or more of said copies of said primary polynucleotide sequence connected through said cleavable polynucleotide sequences, (b) cleaving said extension at said cleavable polynucleotide sequences into said target sequence and primary polynucleotide sequences, (c) dissociating said hybridized extension, (d) hybridizing said primary polynucleotide sequence with said single stranded pattern polynucleotide, and (e) forming an extension of said primary polynucleotide sequence comprising one or more copies of said primary polynucleotide sequence, and repeating steps (b)-(e).

Claim 59

A method for producing multiple copies of a primary polydeoxynucleotide sequence complementary to a template sequence, which comprises: providing in an aqueous medium either simultaneously or wholly or partially sequentially a mixture comprising (1) said primary polydeoxynucleotide sequence; (2) a cyclic single stranded pattern polydeoxynucleotide comprising an oligomer of at least three identical template sequences each containing one or more restriction endonuclease sites, said oligomer consisting of only three independent nucleotides selected from the group consisting of deoxynucleotides or corresponding derivatives thereof; (3) the three deoxynucleotide triphosphates complementary to said deoxynucleotides corresponding to the bases in said primary polydeoxynucleotide sequence; (4) DNA dependent DNA polymerase; and (5) one or more restriction endonucleases capable of cleaving a duplex consisting of said single stranded pattern polydeoxynucleotide and single stranded DNA complementary to said pattern polydeoxynucleotide, said cleaving occurring at said restriction sites and only when said single stranded pattern polydeoxynucleotide is bound to said complementary single stranded DNA; and incubating said mixture under conditions for either simultaneously or wholly or partially sequentially hybridizing said primary polydeoxynucleotide with said single stranded pattern polydeoxynucleotide, extending said primary polydeoxynucleotide along said single stranded pattern polynucleotide to form said duplex, cleaving said duplex at said restriction sites, dissociating said primary polydeoxynucleotide from said template sequence, and rehybridizing said primary polydeoxynucleotide with said single stranded pattern polydeoxynucleotide.

Claim 60

A method for producing multiple copies of a primary polydeoxynucleotide sequence complementary to a template sequence, which comprises: providing in an aqueous medium either simultaneously or wholly or partially sequentially a mixture comprising (1) said primary polydeoxynucleotide sequence; (2) a cyclic single stranded pattern polydeoxynucleotide comprising an oligomer of at least three identical template sequences each containing one or more restriction endonuclease sites, said oligomer consisting of only three independent nucleotides selected from the group consisting of deoxynucleotides or corresponding derivatives thereof; (3) the three deoxynucleotide triphosphates complementary to said deoxynucleotides corresponding to the bases in said primary polydeoxynucleotide sequence; (4) DNA dependent DNA polymerase; and (5) one or more restriction endonucleases capable of cleaving a duplex consisting of said single stranded pattern polydeoxynucleotide and single stranded DNA complementary to said pattern polydeoxynucleotide, said cleaving occurring at said restriction sites and only when said single stranded pattern polydeoxynucleotide is bound to said complementary single stranded DNA; and incubating said mixture under conditions for either simultaneously or wholly or partially sequentially hybridizing said primary polydeoxynucleotide with said single stranded pattern polydeoxynucleotide, extending said primary polydeoxynucleotide along said single stranded pattern polynucleotide to form said duplex, cleaving said duplex at said restriction sites, dissociating said primary polydeoxynucleotide from said template sequence, and rehybridizing said primary polydeoxynucleotide with said single stranded pattern polydeoxynucleotide.

Claim 67

A method of producing multiple copies of a primary polydeoxynucleotide sequence complementary to a template sequence which comprises: (a) forming in the presence of three members selected from the group consisting of deoxynucleoside triphosphates or corresponding derivatives thereof and DNA-dependent DNA polymerase an extension of a primary polydeoxynucleotide sequence hybridized with a template sequence of a cyclic single stranded pattern polydeoxynucleotide comprising an oligomer having at least three identical template sequences each containing one or more restriction endonuclease sites and a sequence of nucleotides complementary to said three members and derivatives thereof, (b) cleaving into fragments said extension at said restriction endonuclease sites in the presence of one or more restriction endonucleases capable of cleaving at said restriction endonuclease sites only when said extension is hybridized with said single stranded pattern polynucleotide, (c) dissociating said fragments, (d) hybridizing said fragments with said single stranded pattern polydeoxynucleotide, and repeating steps (a)-(d) above wherein steps (a)-(d) are conducted simultaneously or wholly or partially sequentially.

Claim 68

A method of producing multiple copies of a primary polydeoxynucleotide sequence complementary to a template sequence which comprises: (a) forming in the presence of three members selected from the group consisting of deoxynucleoside triphosphates or corresponding derivatives thereof and DNA-dependent DNA polymerase an extension of a primary polydeoxynucleotide sequence hybridized with a template sequence of a cyclic single stranded pattern polydeoxynucleotide comprising an oligomer having at least three identical template sequences each containing one or more restriction endonuclease sites and a sequence of nucleotides complementary to said three members and derivatives thereof, (b) cleaving into fragments said extension at said restriction endonuclease sites in the presence of one or more restriction endonucleases capable of cleaving at said restriction endonuclease sites only when said extension is hybridized with said single stranded pattern polynucleotide, (c) dissociating said fragments, (d) hybridizing said fragments with said single stranded pattern polydeoxynucleotide, and repeating steps (a)-(d) above wherein steps (a)-(d) are conducted simultaneously or wholly or partially sequentially.

Claim 75

A method for producing multiple copies of a primary polynucleotide sequence as the result of the presence of a target polynucleotide sequence, which method comprises: (a) combining (1) a single stranded pattern polynucleotide comprising a binding sequence substantially complementary to said target polynucleotide sequence and two or more identical template sequences each containing one or more restriction sites with (2) said target polynucleotide sequence, (b) incubating said combination at a temperature that causes said target polynucleotide sequence to anneal selectively with said binding sequence, (c) adding to said combination template-dependent polynucleotide polymerase, a restriction endonuclease for at least one of said restriction sites in said template sequence and, if not already present, nucleoside triphosphates, (d) incubating said combination at a temperature sufficient to achieve chain extension of said target polynucleotide sequence and cleavage of said single stranded polynucleotide into fragments, (e) denaturing said fragments, said denatured fragments including said primary polynucleotide sequence, (f) incubating said combination at a temperature sufficient to anneal said primary polynucleotide sequence to said single stranded pattern polynucleotide, and (g) repeating steps (c)-(f) above.

Claim 76

A method for producing multiple copies of a primary polynucleotide sequence as the result of the presence of a target polynucleotide sequence, which method comprises: (a) combining (1) a single stranded pattern polynucleotide comprising a binding sequence substantially complementary to said target polynucleotide sequence and two or more identical template sequences each containing one or more restriction sites with (2) said target polynucleotide sequence, (b) incubating said combination at a temperature that causes said target polynucleotide sequence to anneal selectively with said binding sequence, (c) adding to said combination template-dependent polynucleotide polymerase, a restriction endonuclease for at least one of said restriction sites in said template sequence and, if not already present, nucleoside triphosphates, (d) incubating said combination at a temperature sufficient to achieve chain extension of said target polynucleotide sequence and cleavage of said single stranded polynucleotide into fragments, (e) denaturing said fragments, said denatured fragments including said primary polynucleotide sequence, (f) incubating said combination at a temperature sufficient to anneal said primary polynucleotide sequence to said single stranded pattern polynucleotide, and (g) repeating steps (c)-(f) above.

Claim 77

A method for producing multiple copies of a sequence of nucleotides, which comprises hybridizing a primer polynucleotide sequence to a complementary polynucleotide sequence, said complementary polynucleotide sequence being part of a polynucleotide sequence having multiply repeating units complementary to said sequence of nucleotides to be copied, said multiply repeating units containing only two or three of the nucleotides A and dA or a derivative thereof, C and dC or a derivative thereof, U and dT or a derivative thereof, and G and dG or a derivative thereof, combining the hybridized material with only those nucleoside triphosphates complementary to said two or three nucleotides and with a polynucleotide polymerase, and incubating said combination under conditions for forming an extension of said primer polynucleotide sequence, wherein the above steps are carried out simultaneously or wholly or partially sequentially.

Claim 78

A method for producing multiple copies of a sequence of nucleotides, which comprises hybridizing a primer polynucleotide sequence to a complementary polynucleotide sequence, said complementary polynucleotide sequence being part of a polynucleotide sequence having multiply repeating units complementary to said sequence of nucleotides to be copied, said multiply repeating units containing only two or three of the nucleotides A and dA or a derivative thereof, C and dC or a derivative thereof, U and dT or a derivative thereof, and G and dG or a derivative thereof, combining the hybridized material with only those nucleoside triphosphates complementary to said two or three nucleotides and with a polynucleotide polymerase, and incubating said combination under conditions for forming an extension of said primer polynucleotide sequence, wherein the above steps are carried out simultaneously or wholly or partially sequentially.