Multi-primer amplification method for barcoding of target nucleic acids
In certain embodiments, the present invention provides amplification methods in which nucleotide tag(s) and, optionally, a barcode nucleotide sequence are added to target nucleotide sequences. In other embodiments, the present invention provides a microfluidic device that includes a plurality of first input lines and a plurality of second input lines. The microfluidic device also includes a plurality of sets of first chambers and a plurality of sets of second chambers. Each set of first chambers is in fluid communication with one of the plurality of first input lines. Each set of second chambers is in fluid communication with one of the plurality of second input lines. The microfluidic device further includes a plurality of first pump elements in fluid communication with a first portion of the plurality of second input lines and a plurality of second pump elements in fluid communication with a second portion of the plurality of second input lines.
1. A method of operating a microfluidic device, said device comprising independent sample or first reagent inputs combined with independent assay or second reagent inputs in an M×N array configuration, the method comprising:
loading M samples or first reagents into sample chambers through sample input lines arranged as columns;
loading N assays or second reagents into assay chambers through assay input lines arranged as rows crossing the columns;
wherein during loading, the sample chambers and the assay chambers are in fluidic isolation;
opening an interface valve between pairs of M sample and N assay chambers to provide fluid communication between M×N pairwise combinations of M samples or first reagents and N assays or second reagents, such that each M sample or first reagent is combined with each N assay or second reagent to produce M×N unique pairwise reactions;
reacting the M×N unique pairwise reactions to form reaction products;
flowing a harvesting reagent from a harvesting port to the sample chambers of each column
to recover reaction products from the microfluidic device in separate reaction pools, each reaction pool containing a given M sample or first reagent reacted with each of the N assays or second reagents.
2. The method of claim 1 further comprising thermocycling the M×N unique pairwise reactions.
3. The method of claim 1 wherein recovering the reaction products from the microfluidic device comprises flowing the reaction products through at least a portion of the sample input lines to sample input ports.
4. The method of claim 3 wherein flowing the reaction products through at least a portion of the sample input lines comprises performing dilation pumping.
5. The method of claim 4 wherein dilation pumping comprises a fluid flow rate of less than or equal to 10 μl per hour.
6. The method of claim 4 wherein performing dilation pumping comprises:
a) closing a first valve disposed between the sample chamber and the sample input port;
b) opening a second valve disposed between the harvesting port and the sample chamber;
c) closing the second valve;
d) opening the first valve; and
repeating steps (a) through (d) a predetermined number of times.
7. The method of claim 5 wherein the fluid flow rate is less than or equal to 5 μl per hour.
8. The method of claim 7 wherein the fluid flow rate is less than or equal to 1 μl per hour.
9. The method of claim 1 wherein production of M×N unique pairwise reactions comprises a free interface diffusion process.
10. The method of claim 1 wherein removing the reaction product comprises a fluid flow rate of less than or equal to 10 microliters per hour.
11. The method of claim 10 wherein the fluid flow rate is less than or equal to 5 microliters per hour.
12. The method of claim 11 wherein the fluid flow rate is less than or equal to 2 microliters per hour.
13. The method of claim 12 wherein the fluid flow rate is less than or equal to 1 microliter per hour.
14. The method of claim 1 wherein recovering the reaction products comprises removing at least 95% of the reaction products from the microfluidic device.