Granted Patent B2

US 12,337,287 · App. 15/480,739 · Granted Jun 24, 2025

Inventors: Darren Roy Link (Lexington, MA); Michael Weiner (Guilford, CT); David Marran (Durham, CT); Jonathan M. Rothberg (Guilford, CT)

Assignee: Bio-Rad Laboratories, Inc.

B01F23/41B01F33/3011B01J19/0093B01L3/502784B01L3/565B03C5/005B03C5/026C12N15/1068C12N15/1086C12Q1/00C12Q1/6806C12Q1/6837C12Q1/6844C12Q1/6855C12Q1/6869C12Q1/6874G01N15/147G01N21/05G01N21/64G01N27/3275G01N35/08B01L3/502715B01L3/502746B01L7/525B01L9/527B01L2200/027B01L2200/0636B01L2200/0647B01L2200/0673B01L2200/10B01L2300/0636B01L2300/0816B01L2300/0861B01L2300/0864B01L2300/0867B01L2300/165B01L2400/0415B01L2400/0424B01L2400/0487B01L2400/086C12Q2565/628C12Q2565/629G01N2021/0346G01N2035/00237G01N2035/00326G01N2201/024Y10T137/87571Y10T137/87619Y10T137/87652

Abstract

Claims (29)

1. A method for droplet formation for sample preparation for sequencing, the method comprising:

providing, via at least one inlet channel on a microfluidic substrate, an aqueous sample fluid comprising nucleic acid and at least one primer;

providing an immiscible first carrier fluid in a main channel on the microfluidic substrate;

producing a plurality of droplets comprising the aqueous sample fluid at a junction between the main channel and the at least one inlet channel;

copying at least a portion of the nucleic acid within one droplet of the plurality of droplets by extending the at least one primer to generate an extension product; and

breaking the plurality of droplets by introducing a droplet-destabilizing second fluid comprising a destabilizing surfactant, thereby recovering the aqueous sample fluid including the extension product from the plurality of droplets.

2. The method of claim 1 , wherein the aqueous sample fluid comprises one or more cells.

3. The method of claim 1 , further comprising subjecting the extension product to an amplification reaction.

4. The method of claim 3 , wherein the amplification reaction is a PCR reaction.

5. The method of claim 1 , wherein at least one oil supply line provides a constant flow of the first carrier fluid into a nozzle while the plurality of droplets are formed.

6. The method of claim 1 , wherein the first carrier fluid comprises an oil.

7. The method of claim 6 , wherein the oil comprises a fluorocarbon oil or a silicone oil.

8. The method of claim 6 , wherein the oil comprises a surfactant.

9. The method of claim 8 , wherein the surfactant is a fluorosurfactant.

10. The method of claim 1 , wherein the microfluidic substrate is a microfluidic chip.

11. The method of claim 1 , wherein the aqueous sample fluid comprises one or more beads.

12. The method of claim 1 , further comprising centrifuging the plurality of droplets and the droplet-destabilizing second fluid to promote complete coalescence of the plurality of droplets.

13. The method of claim 1 , wherein the destabilizing surfactant comprises a perfluorinated alcohol.

14. The method of claim 13 , wherein the perfluorinated alcohol comprises 1H,1H,2H,2H-Perfluoro-1-octanol.

15. The method of claim 1 , wherein the copying step uses a reverse transcriptase to produce complementary DNA.

16. A sample preparation method comprising:

flowing, via an inlet channel on a microfluidic substrate, an aqueous sample fluid comprising nucleic acid to a junction between the inlet channel and a main channel having an immiscible carrier fluid therein;

producing a plurality of droplets comprising the aqueous sample fluid surrounded by the immiscible carrier fluid in the main channel;

copying at least a portion of the nucleic acid within one droplet of the plurality of droplets by extending a primer to generate an extension product; and

recovering the aqueous sample fluid including the extension product from the plurality of droplets by introducing a droplet-destabilizing second fluid comprising a perfluorinated alcohol.

17. The method of claim 16 , wherein the perfluorinated alcohol comprises 1H,1H,2H,2H-Perfluoro-1-octanol.

18. The method of claim 16 , wherein the copying step uses a reverse transcriptase to produce complementary DNA.

19. The method of claim 16 , wherein the aqueous fluid sample comprises one or more cells.

20. The method of claim 16 , wherein the aqueous fluid sample comprises one or more beads.

Assignments (2)

Continuity (29)

References Cited (400)