Methods and apparatus for sequential amplification reactions
The invention provides methods and apparatus for carrying out multiple amplification reactions in a single reaction chamber by successive cycles of loading reaction mixture, amplifying, and removing spent reaction mixture in a fluidly closed reaction system. In particular, the present invention allows amplification of a plurality of target polynucleotides from a single sample by carrying out under closed-loop control successive amplifications of different target polynucleotides from different portions of the sample.
1. A method of controlling a plurality of sequential amplification reactions, the method comprising the steps of:
(a) amplifying a target polynucleotide in the presence of an indicator in a reaction mixture, the indicator being capable of generating an optical signal related to a quantity of an amplicon of the target polynucleotide in the amplification reaction, and the reaction mixture being disposed in a reaction chamber;
(b) monitoring the optical signal of the indicator in the reaction mixture;
(c) automatically removing the reaction mixture from the reaction chamber and loading the reaction chamber with a subsequent reaction mixture whenever the optical signal reaches or exceeds a predetermined level; and
(d) repeating steps (a) through (c) until the plurality of amplification reactions has been carried out.
2. The method of claim 1 wherein the plurality of sequential amplification reactions amplify the target polynucleotides from a sample and wherein each reaction mixture of step (a) comprises a portion of the sample and amplification reagents.
3. The method of claim 2 wherein the reaction chamber is in a fluidly closed reaction system and wherein each step (c) includes fluidly transferring the removed reaction mixture to a waste reservoir after the optical signal reaches or exceeds the predetermined level.
4. The method of claim 3 wherein each step (c) includes rinsing the reaction chamber with a wash solution after removing the reaction mixture and fluidly transferring the wash solution to the waste reservoir.
5. The method of claim 1 wherein each step (c) further includes, after the step of removing the reaction mixture from the reaction chamber and prior to the step of loading the subsequent reaction mixture, the steps of transferring air into the reaction chamber and heating the reaction chamber to a temperature at or above a DNA denaturation temperature.
6. The method of claim 5 further comprising the step of cooling the reaction chamber to a DNA annealing temperature after the step of heating the reaction chamber to a temperature at or above a DNA denaturation temperature.