Amplification of nucleic acid sequences using oligonucleotides of random sequence as primers
DCFirst Claim
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1. A process for substantially amplifying template nucleic acid sequences in a sample in a randomly primed and template dependent manner, comprising the steps of:
- (a) priming template nucleic acid strands with an excess of random oligonucleotide primers; and
(b) incubating said template nucleic acid strands and said excess random oligonucleotide primers in the presence of an excess of an inducing agent, a strand displacement agent, and an excess of triphosphate substrates to randomly amplify nucleic acid strands.
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Abstract
According to this invention, a process for substantially amplifying template nucleic acid present in a sample is described, wherein said amplification may be performed without prior knowledge of specific sequences, which process comprises apposition of random oligonucleotide primers to said template nucleic acid under conditions such that extension products of said primers are synthesized which are complementary to said template nucleic acid.
407 Citations
50 Claims
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1. A process for substantially amplifying template nucleic acid sequences in a sample in a randomly primed and template dependent manner, comprising the steps of:
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(a) priming template nucleic acid strands with an excess of random oligonucleotide primers; and (b) incubating said template nucleic acid strands and said excess random oligonucleotide primers in the presence of an excess of an inducing agent, a strand displacement agent, and an excess of triphosphate substrates to randomly amplify nucleic acid strands. - 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)
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29. A process for substantially amplifying template nucleic acid sequences in a sample in a randomly primed and template dependent manner, comprising the steps of:
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(a) priming template nucleic acid strands with an excess of random oligonucleotide primers wherein said primers consist of 6-mers to 10 mers; and (b) incubating said template nucleic acid strands and said excess random oligonucleotide primers in the presence of an excess of the Klenow fragment of DNA Polymerase I and an excess of triphosphate substrates to randomly amplify template nucleic acid strands. - View Dependent Claims (30, 31, 32, 33, 34, 35)
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36. A process for detecting a papilloma virus in a sample comprising:
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(a) substantial amplification of said papilloma virus nucleic acid sequences in said sample wherein said substantial amplification comprises a randomly primed but template dependent synthesis of papilloma virus DNA; and (b) detecting said papilloma virus. - View Dependent Claims (37, 38, 39, 40, 41, 42, 43, 44)
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45. A kit for substantially amplifying nucleic acid sequences in a sample in a randomly primed and template dependent manner, comprising a carrier being compartmentalized to receive in close confinement therein one or more containers wherein:
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(a) a first container or series of containers contains random oligonucleotide primers; (b) a second container contains an inducing agent; (c) a third container or series of containers contains triphosphate substrates; and (d) a fourth container or series of containers contains buffer for reconstituting or diluting components of said kit. - View Dependent Claims (46, 47, 48, 49, 50)
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Specification