Closed-system multi-stage nucleic acid amplification reactions
The invention is directed to systems, methods, and apparatus for carrying out multi-stage amplification reactions, especially under fluidly closed conditions. In one aspect, methods of the invention are carried out in a fluidly closed reaction system that permits the isolation of a portion of a first (or prior) reaction mixture and its use as a sample or specimen in a second (or subsequent) reaction mixture, thereby substantially avoiding interfering effects that first reaction components may have in the second reaction if both reaction mixtures were simply combined together. In this aspect, systems, methods, and apparatus of the invention may be used with any amplification reaction that permits multiple stages of amplification based on the use of nested primers.
1. A method of detecting the presence or absence of one or more target polynucleotides in a sample, the method comprising the steps of:
amplifying in a reaction chamber of a reaction system one or more target polynucleotides from the sample using first-stage amplification reagents in a first reaction mixture to form one or more first amplicons, the first-stage amplification reagents including initial primers for each target polynucleotide, wherein the one or more first amplicons are amplified in the presence of a fluorescent indicator capable of generating an optical signal related to a quantity of an amplicon in the first-stage amplification reaction, and wherein the reaction chamber is selectively in fluid communication with a waste reservoir;
monitoring the optical signal of the fluorescent indicator in the first-stage amplification reaction in the reaction chamber;
automatically withholding in the reaction chamber an effective portion of the first reaction mixture and fluidly transferring remainder of the first reaction mixture to the waste reservoir when the optical signal reaches or exceeds a predetermined level;
introducing second-stage amplification reagents into the effective portion of the first reaction mixture to produce a second reaction mixture in the reaction chamber;
amplifying in the reaction chamber the one or more first amplicons in the second reaction mixture, the second-stage amplification reagents including at least one secondary primer for each of the one or more first amplicons, such that each secondary primer is nested in such first amplicon relative to an initial primer of such first amplicon; and
detecting the one or more second amplicons to determine the presence or absence of the one or more target polynucleotides in the sample.
2. The method of claim 1 wherein the reaction chamber is a fluidly closed reaction chamber.
3. The method of claim 1 wherein said effective portion is a volume of said first reaction mixture sufficient to contain at least one molecule of said amplicon.
4. The method of claim 3 wherein said effective portion is a volume of said first reaction mixture sufficient to contain at least 100 molecules of said amplicon.
5. The method of claim 4 wherein said first reaction mixture has a volume and wherein said effective portion is a percentage of the volume of said first reaction mixture, the percentage being selected from the range of 0.5 to 10 percent.
6. The method of claim 1 wherein said predetermined level is a multiple of a baseline signal level, the multiple being selected from the range of 1.5 to 25.
7. The method of claim 1 wherein said first-stage amplification reagents and said second-stage amplification reagents include reagents for performing a polymerase chain reaction or a NASBA reaction.
8. The method of claim 7 wherein said fluorescent indicator is selected an intercalating dye that specifically binds to double stranded DNA or comprises an oligonucleotide moiety that specifically binds to an amplification product in said first reaction mixture.
9. The method of claim 8 wherein said one of said one or more amplicons is produced by amplification of a reference sequence in said first reaction mixture.
10. The method of claim 7 wherein said reaction system is a microfluidics device.
11. The method of claim 7 wherein said reaction system comprises:
the reaction chamber selectably in fluid communication with a sample reservoir containing said sample, a first reactant reservoir containing first-stage amplification reagents, and a second reactant reservoir containing second-stage amplification reagents, each of said reservoirs being fluidly closed; and
a pump operationally associated with a rotary valve for fluidly transferring said sample and said first-stage amplification reagents to the reaction chamber, for automatically separating said effective portion of said first reaction mixture whenever said optical signal equals or is greater than said predetermined level; and for fluidly transferring said second-stage amplification reagents to the reaction chamber for amplifying said one or more first amplicons in said effective portion.