Rapid amplification and detection of nucleic acids

Details Rapid amplification and detection of nucleic acids Rapid amplification and detection of nucleic acids

C - Chemistry, Metallurgy

12

Q

C12Q 1/70 (2006.01) C07H 21/04 (2006.01) C12N 15/10 (2006.01)

Patent

CA 2075083

ABSTRACT Disclosed herein are methods, primers, probes, and kits for the rapid amplification and detection of nucleic acids. In a preferred embodiment, a target nucleic acid sequence in a sample is amplified by: 1) adding nucleoside triphosphates, primer pairs comprising two oligonucleotide primers, and a nucleic acid polymerase to the sample; 2) denaturing the target nucleic acid sequence to form separate strands; and 3) maintaining a reaction temperature in a range from 68°C to 80°C and appropriate reaction conditions wherein the following cycle occurs: the primers hybridize to the target strands, primer extension products are formed, the extension products separate to become templates for the primers, and new extension products are formed. In an alternative embodiment, the amplification occurs at two different temperature ranges. Primer extension products are formed in a temperature range from 68°C to 82°C, and the products are separated by raising the temperature to range of 88°C to 96°C. Detection of the amplified sequences occurs by using some biotin-labeled nucleoside triphosphates, which produces nucleotide sequences that are copies of the target sequence and that contain one or more biotin-labeled nucleotides. The amplified sequences are detected by contacting the sample with immobilized probes and then detecting the presence of the biotin. A two-stage amplification process is also provided wherein: 1) the probe-target sequence complexes are contacted with a first moiety that binds to biotin; 2) a second moiety, comprising biotin bound to a detectable moiety is added, wherein the biotin in the second moiety binds to the first moiety; and 3) detecting or measuring the detectable moiety. In a particularly preferred embodiment, primer pairs and a probe are disclosed for use when the target nucleic acid is HIV-1 DNA.

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