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Nucleic acid assays

  • US 20040248087A1
  • Filed: 07/09/2001
  • Published: 12/09/2004
  • Est. Priority Date: 05/02/1997
  • Status: Active Grant
First Claim
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1. A method for the automated isothermal amplification and detection of a specific nucleic acid in a test sample to be tested comprising:

  • a) combining a test sample to be tested with a buffer, a mixture of free nucleotides, specific oligonucleotide primers, and optionally thermostable nucleic acid polymerization enzyme, in a first reaction vessel and placing the reaction vessel in an automated apparatus such that;

    b) the automated apparatus heats the first reaction vessel to a temperature, and for a time sufficient to denature the nucleic acid in the sample to be tested;

    c) the automated apparatus cools the first reaction vessel to a temperature such that oligonucleotide primers can specifically anneal to the target nucleic acid;

    d) the automated apparatus transfers the reaction mixture from the first reaction vessel to a second reaction vessel, and brings the reaction mixture in contact with isothermal nucleic acid amplification enzyme;

    e) the automated apparatus maintains the temperature of the second reaction vessel at a temperature which allows primer mediated amplification of the nucleic acid;

    f) the automated apparatus contacts the amplified nucleic acid in the second reaction vessel with a capture nucleic acid specific for the nucleic acid to be tested such that they form a specifically-bound nucleic acid-capture probe complex;

    g) the automated apparatus optionally washes the specifically captured amplified nucleic acid such that non-specifically bound nucleic acid is washed away from the specifically-bound nucleic acid-capture probe complex;

    h) the automated apparatus contacts the specifically-bound nucleic acid-capture probe complex with a labeled nucleic acid probe specific for the amplified nucleic acid such that a complex is formed between the specifically amplified nucleic acid and the labeled nucleic acid probe;

    i) the automated apparatus washes the specifically-bound nucleic acid-capture probe-labeled probe complex such that non-specifically bound labeled probe nucleic acid is washed away from the specifically bound complex;

    j) the automated apparatus contacts the specifically bound complex with a solution wherein an enzymatic reaction between the labeled nucleic acid probe is effected between the solution and the label attached to the nucleic acid such that a detectable signal is generated from the sample in proportion the amount of specifically-bound amplified nucleic acid in the sample;

    wherein each of steps h, i and j can be performed sequentially or together;

    k) the automated apparatus detects the signal and optionally displays a value for the signal, or optionally records a value for the signal.

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