Homogeneous nucleotide amplification and assay
First Claim
1. A process for amplifying a target nucleic acid sequence comprising the steps of:
- a) providing a nucleotide amplification reaction mixture containing the target nucleic acid sequence configured to amplify the target nucleic acid sequence;
b) within the nucleotide amplification reaction mixture, contacting the target nucleic acid sequence with a first probe that is complementary to the target nucleic acid sequence and comprises at least one releasable probe portion, wherein the first probe and the target nucleic acid sequence form a duplex having an enzymatically cleavable region wherein enzymatic cleavage of the first probe results in the release of a releasable probe portion; and
c) contacting the first probe and target nucleic acid sequence duplex with an enzyme capable of cleaving the enzymatically cleavable region to release a releasable probe portion and d) making sequential measurements of the release of the releasable probe portion to kinetically characterize the target DNA sequence wherein steps b)-d) are performed in a single reaction mixture.
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Abstract
The invention comprises homogeneous nucleotide amplification strategies and assays. The methods involve amplification of a target nucleic acid sequence that includes the use of a probe that forms a duplex with a target nucleic acid sequence having an enzymatically cleavable region. The probe may anneal to other nucleic acid sequences but only forms an enzymatically cleavable region if the nucleic acid sequence is complementary to the probe. In other embodiments, the probe is configured to act as a primer for the amplification reaction if it anneals to a target nucleic acid sequence and is enzymatically cleaved. The target nucleic acid sequences amplified by the methods of this invention may be assayed by labeling the probe, by employing a second probe having features of the invention or by other suitable methods. Preferably, the probes comprise an RNA portion that forms an RNase H cleavable duplex with DNA.
161 Citations
16 Claims
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1. A process for amplifying a target nucleic acid sequence comprising the steps of:
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a) providing a nucleotide amplification reaction mixture containing the target nucleic acid sequence configured to amplify the target nucleic acid sequence;
b) within the nucleotide amplification reaction mixture, contacting the target nucleic acid sequence with a first probe that is complementary to the target nucleic acid sequence and comprises at least one releasable probe portion, wherein the first probe and the target nucleic acid sequence form a duplex having an enzymatically cleavable region wherein enzymatic cleavage of the first probe results in the release of a releasable probe portion; and
c) contacting the first probe and target nucleic acid sequence duplex with an enzyme capable of cleaving the enzymatically cleavable region to release a releasable probe portion and d) making sequential measurements of the release of the releasable probe portion to kinetically characterize the target DNA sequence wherein steps b)-d) are performed in a single reaction mixture. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15)
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16. A method for the detection of a target nucleic acid sequence in a sample comprising the steps of:
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a) amplifying the target DNA sequence with a polymerase chain reaction amplification reaction;
b) concurrently with step a), contacting and annealing a probe comprising a ribo-oligonucleotide attached to a first label with a sample containing a target nucleic acid sequence having a region complementary to the probe;
c) cleaving at least one of the ribonucleotides of the annealed probe with a ribo-nucleic acid nuclease capable of hydrolyzing ribonucleotides in a double stranded RNA;
DNA duplex to release labeled ribo-oligonucleotide fragments; and
d) generating a signal from the released labeled ribo-oligonucleotide fragments that characterizes the target nucleic acid sequence;
wherein steps a)-d) are performed in a single reaction mixture.
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Specification