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 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.
2. The method of claim 1 wherein at least one step (d) includes rinsing the reaction chamber with a wash solution and fluidly transferring the wash solution to the waste reservoir, prior to repeating steps (b) and (d).
3. The method of claim 2 , further comprising after transferring the wash solution to the waste reservoir the step of transferring air into the reaction chamber and heating the reaction chamber to a temperature at or above a DNA denaturation temperature.
4. The method of claim 3 , wherein the DNA denaturation temperature is in the range of from about 90° C. to 99° C.
5. The method of claim 1 , wherein the sample comprises a microorganism.
6. The method of claim 1 wherein the step of amplifying is carried out with a polymerase chain reaction.
7. The method of claim 1 , wherein each of the amplification reactions is a linear polymerase reaction.
8. The method of claim 1 , wherein the amplification reaction is a ligase chain reaction.
9. The method of claim 1 , wherein the amplification reaction is a strand displacement reaction.
10. The method of claim 1 , wherein the amplification reaction is a nucleic acid sequenced based amplification.
11. The method of claim 1 , wherein the amplification reaction is a rolling circle amplification reaction.
12. The method of claim 1 , wherein the optical signal is selected from the group consisting of a fluorescent signal, a chemiluminescent signal, an electrochemiluminescent signal, and a colorimetric signal.
13. The method of claim 12 , wherein the optical signal is a fluorescent optical signal generated by a fluorescent indicator.
14. The method of claim 13 , wherein the fluorescent indicator is a non-specific intercalating dye that binds to double-stranded DNA products.
15. The method of claim 13 wherein the fluorescent indicator is a target sequence specific probe selected from the group consisting of a TAQMAN probe, a SCORPION probe, and a MOLECULAR BEACON.