Methods and apparatus for sequential amplification reactions

摘要

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.

主权项

1. A method for performing sequential amplification reactions to determine the presence or absence of a plurality of target polynucleotides in a sample, the method comprising the steps of:
(a) providing a reaction chamber suitable for optical interrogation, wherein the reaction chamber is selectably in fluid communication with a waste reservoir, a sample reservoir containing a sample, and a plurality of reactant reservoirs each containing amplification reagents for detecting a different target; (b) loading into the reaction chamber a portion of the sample and amplification reagents from at least one of said reactant reservoirs to form a reaction mixture comprising an amplification indicator, wherein the amplification indicator is capable of generating an optical signal related to a quantity of an amplicon of the target polynucleotide in an amplification reaction; (c) subjecting the reaction mixture to amplification conditions, and monitoring the optical signal of the indicator in the reaction mixture; and (d) whenever the optical signal reaches or exceeds a predetermined level, automatically fluidicly transferring the reaction mixture from the reaction chamber to the waste reservoir, and repeating step (b) through (d), until the optical signal in step (c) fails to exceed such a predetermined level, wherein for each cycle of steps (b) through (d), a different aliquot of the sample and a different set of amplification reagents are loaded into the reaction chamber to detect a different target polynucleotide in a sequential manner.