Methods of using microfluidic positional encoding devices
The invention relates to methods and compositions useful for routing and tracking multiple mobile units within a microfluidic device. Mobile units may be routed through a plurality of chemical environments, and the mobile units may be tracked to determine the path and/or environments that the mobile units have routed through. Mobile units may be routed in accordance with a predetermined algorithm. Mobile units may be routed through microfluidic devices in ordered flow. Mobile units routed through the microfluidic device can be used to perform various chemical reactions uniquely associated to the units, including without limitation peptide synthesis, enzymatic gene synthesis and gene assembly.
1 . A method of synthesizing oligomers associated with mobile units, the method comprising:
(a) routing k mobile units through a first channel of a microfluidic device in a first order;
(b) distributing at least a subset of the k mobile units into at least z branch channels; and
(c) routing the at least a subset of the k mobile units from the at least z branch channels into the first channel in a second order;
wherein at least a subset of the k mobile units are functionalized with a group suitable to synthesize an oligomer; wherein at least a subset of the k mobile units are mappable to a path comprising a specific one of the z branch channels; wherein at least a subset of the k mobile units are subjected to reaction conditions comprising conditions for a step of a synthesis reaction inside the z branch channels;
and wherein steps a-c are repeated for n cycles, wherein n is at least 2 and z is at least 2.
2 . The method of claim 1 , wherein the synthesis reaction comprises a nucleic acid synthesis reaction or a peptide synthesis reaction.
3 . The method of claim 2 , wherein the nucleic acid synthesis reaction is a template independent nucleic acid synthesis reaction.
4 . The method of claim 1 , wherein the reaction conditions comprise an enzyme.
5 . The method of claim 4 , wherein the enzyme is selected from a terminal deoxynucleotidyl transferase, a thermostable DNA polymerase, a DNA polymerase theta, a Poly (A) polymerase, and a DNA polymerase encoded by a variant of the 9°N DNA Polymerase gene from Thermococcus species 9°N-7.
6 . The method of claim 5 , wherein the variant of the 9°N DNA Polymerase gene comprises the 9°N (D141A/E143A/A485L) DNA Polymerase gene or the 9°N (E143D) DNA Polymerase gene.
7 . The method of claim 4 , wherein the enzyme is conjugated to a nucleotide or a nucleotide analog.
8 . The method of claim 1 , wherein the reaction conditions comprise a nucleotide or a nucleotide analog.
9 . The method of claim 1 , wherein at least a subset of the k mobile units are functionalized with an initiator nucleic acid or a nascent oligonucleotide.
10 . The method of claim 1 , wherein the synthesis reaction inside the z branch channels comprises performing a coupling reaction by catalyzing formation of a covalent bond between a terminal nucleotide of initiator nucleic acids or nascent oligonucleotides associated with at least a subset of the k mobile units and a new nucleotide or nucleotide analog in the presence of a transferase enzyme.
11 . The method of claim 10 , wherein the new nucleotide or nucleotide analog comprises a blocking moiety.
12 . The method of claim 11 , further comprising performing a deblocking reaction thereby removing the blocking moiety from the newly incorporated nucleotide or nucleotide analog.
13 . The method of claim 10 , further comprising one or more steps selected from the group consisting of a washing step, a modification step, a cleaving step, and a capping step.
14 . The method of claim 13 , wherein two or more of the steps selected from the group consisting of the coupling reaction, the deblocking reaction, the washing step, the modification step, the cleaving step, and the capping step are performed in different cycles.
15 . The method of claim 1 , wherein the oligomers are oligonucleotides and wherein the method further comprises assembling the oligonucleotides into genes.
16 . The method of claim 1 , wherein the reaction conditions comprise one or more of reagents selected from the group consisting of an amino acid, a dipeptide, a polypeptide, and a carbodiimide.
17 . The method of claim 16 , further comprising performing a coupling reaction by catalyzing the formation of a covalent bond between the terminal end of nascent peptides associated with at least a subset of the k mobile units and a new amino acid, dipeptide or polypeptide.
18 . The method of claim 17 , further comprising performing one or more step selected from the group consisting of a capping step, a washing step, and a deprotecting step.
19 . The method of claim 18 , wherein two or more of the steps selected from the group consisting of the coupling reaction, capping step, washing step and the deprotecting step are performed in different cycles.
20 . The method of claim 1 , wherein the same z branch channels are used in at least two of the n cycles.
21 . The method of claim 1 , wherein k is at least 2.
22 . The method of claim 1 , wherein
(a) the mobile units are selected from the group consisting of beads, droplets, cells, bubbles, slugs, immiscible volumes, glass beads, polymer beads, cross-linked beads, cross-linked polymer beads, divinylbenzene cross-linked polymer beads, and divinylbenzene cross-linked polystyrene beads; and/or
(b) the first order is different in at least two of the n cycles; and/or
(c) the second order is different in at least two of the n cycles.