Amplification method for polynucleotide detection assays
First Claim
1. 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 of a 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.
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Abstract
A method is disclosed for producing multiple copies of a primary polynucleotide sequence located at the 3'"'"' terminus of a polynucleotide. The method 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, (d) hybridizing said fragments with single stranded pattern polynucleotide, and repeating steps (a)-(d). Steps (a)-(d) may be conducted simultaneously or wholly or partially sequentially. The method may be applied in the detection of a polynucleotide analyte in a sample suspected of containing such analyte to facilitate such detection. Also disclosed are compositions for conducting the method of the invention.
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Citations
42 Claims
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1. A method of determining the presence of a polynucleotide analyte in a sample suspected of containing said analyte, which comprises:
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(a) forming in the presence of nucleoside triphosphates and template-dependent polynucleotide polymerase an extension of a 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. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9)
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10. A method for detecting the presence of a polynucleotide analyte in a sample suspected of containing said polynucleotide analyte which comprises:
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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. - View Dependent Claims (11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24)
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25. A method for determining the presence of a polynucleotide analyte in a sample suspected of containing said polynucleotide analyte, which method comprises:
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combining in an aqueous medium either simultaneously or wholly or partially sequentially (1) said sample (2) means for obtaining from said polynucleotide analyte a target sequence and for terminating said sequence in a 3'"'"'--OH group when said sequence is not terminated in a 3'"'"'--OH group, (3) single stranded pattern polydeoxynucleotide comprised of a binding polynucleotide sequence complementary to said target sequence joined at its 5'"'"' end to a multiply repeated sequence of at least 10 nucleotides, said repeated sequence, when hybridized to its complementary sequence and incubated with a restriction endonuclease, promoting cleavage of said complementary sequence to form restriction fragments, (4) deoxynucleoside triphosphates, (5) a DNA dependent DNA polymerase for extending said target sequence to provide said complementary sequence, and (6) a restriction endonuclease for forming said restriction fragments, said combining being carried out under conditions for either simultaneously or wholly or partially sequentially (a) obtaining said target sequence from said polynucleotide analyte, (b) hybridizing said target sequence to said single stranded pattern oligodeoxynucleotide, (c) extending said target sequence to form said complementary sequence, (d) cleaving said complementary sequence, (e) denaturing the duplex of said complementary sequence and said repeated sequence, (f) hybridizing said complementary sequence with single stranded pattern polydeoxynucleotide, (g) extending said complementary sequence, and repeating steps (d)-(g) above; and detecting said cleaved complementary sequence, the presence thereof indicating the presence of said polynucleotide analyte in said sample. - View Dependent Claims (26, 27, 28, 29, 30, 31, 32, 33, 34, 35)
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36. A method for determining the presence of a target sequence of nucleotides in a polynucleotide analyte in a sample suspected of containing said polynucleotide analyte, which method comprises:
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combining either simultaneously or wholly or partially sequentially said sample and (1) means capable of causing said target sequence to be terminated in a 3'"'"'--OH group when said target sequence is not already terminated in a 3'"'"'--OH group, (2) single stranded pattern polydeoxynucleotide comprised of a sequence complementary to said target sequence, said sequence bonded at its 5'"'"' end to a multiply repeating sequence of at least 10 nucleotides which, when hybridized to a complementary sequence, promotes cleavage of said complementary sequence to form fragments (3) deoxynucleoside triphosphates, (4) DNA dependent DNA polymerase and (5) a restriction endonuclease for cleaving said complementary sequence when hybridized to said multiply repeating sequence; incubating the individual components and mixtures formed therefrom under conditions which promote either wholly or partially sequential or simultaneous (a) denaturation of said target sequence when said target sequence is double stranded, (b) hybridization of said target sequence with said single stranded pattern polydeoxynucleotide, (c) extension of said target sequence by DNA dependent DNA polymerase to produce a duplex containing said complementary sequence, (d) cleavage of said complementary sequence in said duplex, (e) melting of said duplex, (f) hybridization of said cleaved complementary sequence, (g) extension of said complementary sequence by DNA dependent DNA polymerase to produce a duplex containing said complementary sequence, and repeating steps (d)-(g) above; and detecting said complementary sequence or fragments of said pattern polydeoxynucleotide produced during cleavage of said complementary sequence, the presence thereof indicating the presence of said analyte in said sample. - View Dependent Claims (37, 38, 39, 40, 41, 42)
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Specification