Thermal reaction device and method for using the same
A method for carrying out nucleic acid amplification reactions using a microfluidic device is described. Amplification primers and other amplification reagents are deposited at a plurality of reaction sites in the device, a sample solution containing amplifiable polynucleotides is introduced into the reaction sites, and amplification is carried out.
1. A method for carrying out nucleic acid amplification reactions to detect the quantity, presence, or absence of a plurality of target polynucleotide sequences in a sample, the method comprising:
(a) providing a microfluidic device comprising
(i) an inlet,
(ii) a first flow channel in fluid communication with the inlet, and
(iii) a plurality of second channels in fluid communication with the first flow channel, wherein each second channel comprises an entrance and a dead end,
wherein amplification primers, and optionally other amplification reagents, are non-covalently deposited at reaction sites at the dead ends of each second channel, and amplification primers deposited in different second channels amplify different target polynucleotide sequences,
(b) introducing a sample solution comprising the sample through the inlet such that the sample solution flows through the first flow channel and into the plurality of second channels, wherein the sample comprises a plurality of polynucleotides, and wherein introduction of the sample solution into the second channels causes the deposited amplification primers to be dissolved into the sample solution, and
(c) isolating the reaction sites from the first flow channel and from each other,
(d) conducting polynucleotide amplification reactions in the reaction sites, and
(e) detecting the quantity, presence or absence of polynucleotide amplification products in the reaction sites to determine the quantity, presence, or absence of said target polynucleotide sequences in the sample, wherein in Step (c) an elastomeric membrane is deflected into the plurality of second channels to block flow from said reaction sites to said first channel, thereby isolating the reaction sites from the first flow channel and from each other.
2. The method of claim 1 , wherein the microfluidic device comprises at least 100 reaction sites.
3. The method of claim 2 , wherein the microfluidic device comprises 100 to 1,000 reaction sites.
4. The method of claim 1 , wherein said target polynucleotide sequences comprise viral or bacterial sequences.
5. The method of claim 1 , wherein the target polynucleotide sequences are cDNA sequences.
6. The method of claim 1 , wherein the quantity, presence or absence of polynucleotide amplification products is detected using a DNA intercalation dye.
7. The method of claim 1 wherein primers suitable for multiplex amplifications are deposited in at least one of said reaction sites.
8. The method of claim 1 , wherein the polynucleotide amplification reactions are polymerase chain reactions (PCR).
9. The method of claim 8 , wherein the polynucleotide amplification reactions are real time quantitative polymerase chain reactions.
10. A method for carrying out nucleic acid amplification reactions to detect the presence or absence of a plurality of different target viral or bacterial polynucleotide sequences in a biological sample obtained from a human patient, the method comprising:
(a) providing a microfluidic device comprising
(i) an inlet,
(ii) a first flow channel in fluid communication with the inlet, and
(iii) a plurality of second channels in fluid communication with the first flow channel, wherein each second channel comprises an entrance and a dead end,
wherein amplification primers, and optionally other amplification reagents, are non-covalently deposited at a reaction site at the dead-ends of each second channel, and amplification primers deposited in different second channels amplify different target polynucleotide sequences; and
wherein the different target polynucleotide sequences are viral or bacterial sequences,
(b) introducing a sample solution comprising the sample through the inlet such that the sample solution flows through the first flow channel and into the plurality of second channels, wherein the sample comprises a plurality of polynucleotides, and wherein introduction of the sample solution into the second channels causes the deposited amplification primers to be dissolved into the sample solution,
(c) isolating the reaction sites from the first flow channel and from each other,
(d) conducting polynucleotide amplification reactions in the reaction sites, and
(e) detecting the presence or absence of a polynucleotide amplification products in the reaction sites to determine the presence or absence of a viral or bacterial target polynucleotide sequence in the biological sample obtained from the human patient, wherein in Step (c) an elastomeric membrane is deflected into the plurality of second channels to block flow from said reaction sites to said first channel, thereby isolating the reaction sites from the first flow channel and from each other.