METHODS AND APPARATUS FOR SEQUENTIAL AMPLIFICATION REACTIONS
First Claim
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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.
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Abstract
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.
18 Citations
29 Claims
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1. A method of controlling a plurality of sequential amplification reactions, the method comprising the steps of:
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(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. - View Dependent Claims (2, 3, 4, 5, 6)
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7. A method of determining the presence or absence of a plurality target polynucleotides in a sample, the method comprising the steps of:
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(a) providing a reaction chamber in a fluidly closed reaction system; (b) mixing a portion of the sample with amplification reagents to form a reaction mixture; (c) amplifying in the reaction chamber one or more target polynucleotides in the reaction mixture to form one or more amplicons; (d) detecting the one or more amplicons to determine the presence or absence of the one or more target polynucleotides in the sample; and (e) repeating steps (b) through (d) until the presence or absence of the plurality of target polynucleotides is determined. - View Dependent Claims (8, 9, 10, 11, 12, 13, 14, 15)
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16. A method of determining the presence or absence of a plurality of target polynucleotides in a sample, the method comprising the steps of:
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providing a reaction chamber selectably in fluid communication with a waste reservoir, a sample reservoir containing a sample, a first reactant reservoir containing first amplification reagents, and a second reactant reservoir containing second amplification reagents, each of the reservoirs being fluidly closed; combining a first portion of the sample and the first amplification reagents to form a first reaction mixture; subjecting the first reaction mixture to amplification reaction conditions in the reaction chamber to produce a first amplicon of one or more target polynucleotides whenever such polynucleotides are present in the sample; fluidly transferring the first reaction mixture to the waste reservoir; combining a second portion of the sample and the second amplification reagents to form a second reaction mixture; and subjecting the second reaction mixture to amplification reaction conditions in the reaction chamber to produce a second amplicon of one or more target polynucleotides whenever such polynucleotides are present in the sample, wherein detection of the first and second amplicons determines the presence or absence of the plurality of target polynucleotides in the sample. - View Dependent Claims (17, 18, 19, 20, 21, 22, 23)
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24. A fluidly closed reaction system for performing multiple sequential amplification reactions, the system comprising:
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a reaction chamber selectably in fluid communication with a sample reservoir, a waste reservoir, and a plurality of reactant reservoirs containing different amplification reagents, each of the reservoirs being fluidly closed; and a pump operationally associated with a rotary valve for performing repeated cycles of; (a) fluidly transferring a portion of a sample from the waste reservoir and amplification reagents from at least one of the reactant reservoirs to the reaction chamber, wherein an amplification reaction occurs to form one or more amplicons in a reaction mixture; (b) removing the reaction mixture from the reaction chamber and fluidly transferring it to the waste reservoir after the one or more amplicons are detected. - View Dependent Claims (25, 26, 27)
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28. A computer-readable product embodying a program for execution by a computer to control the performance of a plurality of sequential amplification reactions, the program comprising instructions for:
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(a) reading values of an optical signal from an amplification reaction in a reaction chamber, the optical signal being monotonically related to a concentration of an amplicon in the amplification reaction, and the values of the optical signal having a most recent value; (b) determining a baseline signal level from the values of the optical signal; (c) computing a predetermined value from the values of the optical signal; (d) comparing the predetermined value with the most recent value of the optical signal; and (e) repeating step (d) until the most recent value of the optical signal is equal to or greater than the predetermined level, where upon a subsequent amplification reaction is initiated.
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29. A method of determining the presence or absence of a microorganism by a plurality of amplification reactions carried out in sequence, the method comprising the steps of:
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(a) amplifying one or more target polynucleotides from a portion of a sample 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, 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 transferring to the reaction chamber with a subsequent reaction mixture when the optical signal reaches or exceeds a predetermined level, otherwise automatically terminating the sequence of amplification reactions; and (d) repeating steps (a) through (c) until the sequence of amplification reactions has been carried out or the sequence of amplification reactions has been terminated.
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Specification