Method for performing amplification of nucleic acid on supports
First Claim
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1. A method of detecting the presence or absence of a target nucleic acid sequence in a test sample comprising the steps of:
- a) providing a test sample containing a target nucleic acid sequence;
b) providing an optical fiber having a proximal end and a distal end, said distal end having two or more oligonucleotides immobilized thereon, wherein the nucleotide sequences of the oligonucleotides are complementary to the nucleic acid sequence of the target;
c) contacting the distal end of the fiber with the test sample and maintaining the test sample in contact with the fiber under conditions suitable for the amplification of the target sequence and for time sufficient to obtain a detectable amount of target sequence, thereby producing an amplification product which is immobilized on the optic fiber; and
d) detecting the presence of the target sequence in the test sample by the detection of an optically detectable signal produced by the presence of the amplification product.
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Abstract
This invention features methods, apparatus and kits for performing nucleic acid hybridization and amplification reactions on a support. Such methods and apparatus are useful in diagnostic and therapeutic processes for synthesizing nucleic acid and detecting target nucleic acids in a sample.
204 Citations
58 Claims
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1. A method of detecting the presence or absence of a target nucleic acid sequence in a test sample comprising the steps of:
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a) providing a test sample containing a target nucleic acid sequence; b) providing an optical fiber having a proximal end and a distal end, said distal end having two or more oligonucleotides immobilized thereon, wherein the nucleotide sequences of the oligonucleotides are complementary to the nucleic acid sequence of the target; c) contacting the distal end of the fiber with the test sample and maintaining the test sample in contact with the fiber under conditions suitable for the amplification of the target sequence and for time sufficient to obtain a detectable amount of target sequence, thereby producing an amplification product which is immobilized on the optic fiber; and d) detecting the presence of the target sequence in the test sample by the detection of an optically detectable signal produced by the presence of the amplification product. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36)
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37. A method of detecting the presence or absence of a target nucleic acid sequence in a test sample, comprising the steps of:
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a) providing a test sample containing a target nucleic acid sequence; b) providing an optical fiber having a proximal end and a distal end, said distal end having two or more oligonucleotides immobilized thereon, wherein the nucleotide sequences of the oligonucleotides are complementary to a sequence flanking the target sequence; c) contacting the distal end of said optical fiber with the test sample in the presence of deoxyribonucleotide triphosphates and one or more appropriate enzymes, under conditions suitable for extension of the primer sequence and amplification of the target sequence thereby producing an amplification product; and d) detecting the presence of the amplification product, wherein detection of the amplification product is indicative of the presence of the target nucleic acid sequence. - View Dependent Claims (38, 39, 40, 41, 42, 43, 44)
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45. A method of detecting a target nucleic acid sequence in a test sample, comprising the steps of:
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a) providing a test sample containing a target nucleic acid sequence; b) providing an optical fiber having a proximal end and a distal end, said distal end having a first oligonucleotide immobilized thereon, said nucleotide sequence of the oligonucleotide being complementary to a sequence within the target sequence; c) contacting the distal end of the optical fiber with the test sample in the presence of a second oligonucleotide being complementary to a sequence within the target sequence that is immediately contiguous with the sequence complementary to the first oligonucleotide sequence and at least one ligase enzyme under conditions suitable for annealing of the first and second oligonucleotides to the target sequence, ligation of the annealed oligonucleotides and amplification of the target sequence, thereby producing an amplification product; and d) detecting the presence of the amplification product, wherein detection of the amplification product is indicative of the presence of the target sequence. - View Dependent Claims (46, 47, 48, 49, 50)
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51. A method for determining polynucleotide damage and repair ex vivo in a double stranded oligonucleotide having about 30-500 base pairs and including a lesion, said method comprising:
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a) providing an optical fiber having a proximal end and a distal end, said distal end having a double stranded oligonucleotide immobilized on an optical fiber; b) treating said optical fiber having said oligonucleotide immobilized thereto with a composition comprising, i. a lysate from a candidate cell population and ii. a polynucleotide repair reagent, for a time sufficient for said lysate to act upon said lesion and effect incorporation of said polynucleotide repair reagent into said lesion; and c) measuring a property of the oligonucleotide treated as in step b) while coupled to said optical fiber, the resulting measurement being determinative of the extent of repair of said oligonucleotide. - View Dependent Claims (52, 53)
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54. A method for determining the ability of neoplastic cells to repair lesions formed by therapeutic agents in double stranded oligomeric polynucleotide having about 30-500 base pairs, said method comprising:
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a) providing said double stranded oligonucleotide covalently coupled to an optical fiber; b) treating said optical fiber having said oligonucleotide covalently coupled thereto with a composition comprising, i. a lysate from said neoplastic cells and ii a polynucleotide repair reagent, for a time sufficient for said lysate to act upon said lesion and effect incorporation of said polynucleotide repair reagent into said lesion; and c) measuring a property of the oligonucleotide treated as in step b) while coupled to said optical fiber, the resulting measurement being determinative of the extent of repair of said oligonucleotide. - View Dependent Claims (55, 56, 57, 58)
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Specification