INTEGRATED MICROFLUIDIC DEVICE FOR GENE SYNTHESIS

• US 20110124049A1
• Filed: 07/31/2008
• Published: 05/26/2011
• Est. Priority Date: 08/07/2007
• Status: Abandoned Application

• Current Assignee: Agency For Science Technology and Research (Government of Singapore)
• Original Assignee: Agency For Science Technology and Research (Government of Singapore)
• Inventors: Huang, Mo-Chao, Pin Ng, Sean, Li, Mo-Huang, Ying, Jackie Y.
• Application Number:
  US12/672,673
• Publication Number:
  US 20110124049A1
• Time in Patent Office: Days
• US Class Current: 435/91.2
• CPC Class Codes: C12N 15/1031 mutagenesis by gene assembly, e.g. assembly by oligonucleotide extension PCR
• CPC Class Codes: C12N 15/1093 General methods of preparing gene libraries, not provided for in other subgroups

Abstract

We report making an integrated micro-fluidic device for synthesizing double stranded DNA from short oligo-nucleotides. We demonstrate successful synthesis of a 760 bp gene segment from a pool of 39 oligonucleotides on a micro-fluidic device using both the one-step and two-step synthesis processes. We also describe purifying the double stranded DNA PCR product and filtering out sequence errors in the double stranded DNA product, all on the same device.

Claims

1. A method for synthesizing double-stranded DNA in a microfluidic device, the device comprising a PCR-assembly (PCA) chamber in controllable fluid communication with a polymerase chain reaction (PCR) chamber, the method comprising the steps of:

(a) applying a time-varying thermal field to the PCA chamber containing a plurality of different oligonucleotides and polymerase, wherein each oligonucleotide has partial base complementarity with at least one other oligonucleotide, thereby assembling the oligonucleotides into templates for PCR in the absence of terminal PCR primers;

(b) loading the templates produced in step (a) into the PCR chamber in the presence of a PCR precursor mix comprising the terminal PCR primers, dNTPs and polymerase; and

(c) applying a time-varying thermal field to the PCR chamber, thereby obtaining a PCR product mixture comprising the double-stranded DNA.

2. The method of claim 1 wherein the device further comprises a purification chamber in controllable fluid communication with the PCR chamber, the method further comprising the step of:

(d) loading the PCR product mixture into the purification chamber to immobilize the double-stranded DNA, thereby separating the double-stranded DNA from free dNTPs, primers and unpolymerized oligonucleotides.

3. The method of claim 2 wherein the double-stranded DNA is immobilized on magnetic beads.

4. The method of claim 3 further comprising the step of extracting the double-stranded DNA from the magnetic beads by subjecting the bead-immobilized DNA to heatshock conditions of 60°

C. for 3 minutes.

5. The method of claim 2 wherein the device further comprises an error filtration chamber in controllable fluid communication with the purification chamber, the method further comprising the step of:

(e) loading the double-stranded DNA produced in step (d) into the error filter chamber to remove double-stranded DNA that contain base-pair mismatches.

6. The method of claim 1 wherein the device further comprises a purification chamber in controllable fluid communication with the PCA chamber, the method further comprising the step of:

(d) loading the templates produced in step (a) into the purification chamber to immobilize the templates, thereby separating the templates from free dNTPs and unpolymerized oligonucleotides; and

then proceeding to step (b).

7. The method of claim 6 wherein the templates are immobilized on magnetic beads.

8. The method of claim 7 further comprising the step of extracting the templates from the magnetic beads by subjecting the bead-immobilized templates to heatshock conditions of 60°

C. for 3 minutes.

9. The method of claim 6 wherein the device further comprises an error filtration chamber in controllable fluid communication with the purification chamber, the method further comprising the step of:

(e) loading the template produced in step (d) into the error filter chamber to remove templates that contain base-pair mismatches; and

then proceeding to step (b).

10. The method of claim 1 wherein the device further comprises a micro-mixer, the method further comprising the step of:

in step (b), mixing the PCR precursor mix with the templates produced in step (a); and

/or

in step (d), mixing the PCR product mixture with DNA-adsorbing solid phase media.

16. The method of claim 1 wherein the device is operably linked to a fluid-flow actuator.

17. The method of claim 16 wherein the fluid-flow actuator is a pump or a centrifuge.

18. The method of claim 1 wherein the chambers are in controllable fluid communication with one another via channels comprising valves.

19. The method of claim 18 wherein the valves are responsive to temperature changes and wherein the valves that control sealing of the PCR chamber are able to withstand at least 6.8 psi of pressure.

20. The method of claim 1 wherein the device is operably linked to a heating element, a cooling element, a temperature-sensor, and a temperature controller.

11. A method for synthesizing double-stranded DNA in a microfluidic device, the device comprising a synthesis chamber in controllable fluid communication with a purification chamber, the method comprising the steps of:

(a) applying a time-varying thermal field to the synthesis chamber containing terminal PCR primers, polymerase, dNTPs and a plurality of different oligonucleotides wherein each oligonucleotide has partial base complementarity with at least one other oligonucleotide, thereby obtaining a PCR product mixture comprising the double-stranded DNA; and

(b) loading the PCR product mixture into the purification chamber to immobilize the double-stranded DNA, thereby separating the double-stranded DNA from free dNTPs, primers and unpolymerized oligonucleotides.

12. The method of claim 11 wherein the double-stranded DNA in step (b) is immobilized on magnetic beads.

13. The method of claim 12 further comprising the step of extracting the double-stranded DNA from the magnetic beads by subjecting the bead-immobilized DNA to heatshock conditions of 60°

C. for 3 minutes.

14. The method of claim 11 wherein the device further comprises an error filtration chamber in controllable fluid communication with the purification chamber, the method further comprising the step of:

(c) loading the double-stranded DNA produced in step (b) into the error filter chamber to remove double-stranded DNA that contain base-pair mismatches.

15. The method of claim 11 wherein the device further comprises a micro-mixer, the method further comprising the step of mixing the PCR product mixture in step (b) with DNA-adsorbing solid phase media.