US 12,146,134 B2
Microfluidic devices and methods of use in the formation and control of nanoreactors
Darren Roy Link, Lexington, MA (US); Laurent Boitard, Gullford, CT (US); Jeffrey Branciforte, Cromwel, CT (US); Yves Charles, Waterbury, CT (US); Gilbert Feke, West Simsbury, CT (US); John Q. Lu, Gullford, CT (US); David Marran, Durham, CT (US); Ahmadali Tabatabai, Branford, CT (US); Michael Weiner, Guilford, CT (US); Wolfgang Hinz, Guilford, CT (US); and Jonathan M. Rothberg, Guilford, CT (US)
Assigned to Bio-Rad Laboratories, Inc., Hercules, CA (US)
Filed by Bio-Rad Laboratories, Inc., Hercules, CA (US)
Filed on Jun. 1, 2018, as Appl. No. 15/996,222.
Application 15/331,452 is a division of application No. 14/248,991, filed on Apr. 9, 2014, granted, now 9,534,216, issued on Jan. 3, 2017.
Application 15/996,222 is a continuation of application No. 15/694,108, filed on Sep. 1, 2017, granted, now 10,633,652.
Application 15/694,108 is a continuation of application No. 15/331,452, filed on Oct. 21, 2016, granted, now 9,752,141, issued on Sep. 5, 2017.
Application 14/248,991 is a continuation of application No. 13/759,660, filed on Feb. 5, 2013, granted, now 9,328,344, issued on May 3, 2016.
Application 13/759,660 is a continuation of application No. 12/087,713, abandoned, previously published as PCT/US2006/021280, filed on Jun. 1, 2006.
Claims priority of provisional application 60/771,286, filed on Feb. 7, 2006.
Claims priority of provisional application 60/763,524, filed on Jan. 30, 2006.
Prior Publication US 2018/0355350 A1, Dec. 13, 2018
This patent is subject to a terminal disclaimer.
Int. Cl. C12N 15/10 (2006.01); B01F 33/302 (2022.01); B01F 33/3031 (2022.01); B01J 19/00 (2006.01); B01L 3/00 (2006.01); B82Y 5/00 (2011.01); B82Y 30/00 (2011.01); C07K 1/04 (2006.01); C12Q 1/686 (2018.01); C12Q 1/6874 (2018.01); C12Q 1/6876 (2018.01); C40B 30/04 (2006.01); C40B 40/04 (2006.01); C40B 50/08 (2006.01); C40B 60/08 (2006.01); C40B 60/12 (2006.01); C40B 70/00 (2006.01); G01N 33/536 (2006.01); G01N 33/543 (2006.01); G01N 33/68 (2006.01); B01F 23/41 (2022.01); B01L 7/00 (2006.01); C12Q 1/6804 (2018.01); C12Q 1/6816 (2018.01); C12Q 1/6825 (2018.01); C12Q 1/6834 (2018.01); C12Q 1/6869 (2018.01); G01N 21/64 (2006.01); G01N 33/542 (2006.01)
CPC C12N 15/1075 (2013.01) [B01F 33/3021 (2022.01); B01F 33/3031 (2022.01); B01J 19/0046 (2013.01); B01L 3/502784 (2013.01); B82Y 5/00 (2013.01); B82Y 30/00 (2013.01); C07K 1/047 (2013.01); C12Q 1/686 (2013.01); C12Q 1/6874 (2013.01); C12Q 1/6876 (2013.01); C40B 30/04 (2013.01); C40B 40/04 (2013.01); C40B 50/08 (2013.01); C40B 60/08 (2013.01); C40B 60/12 (2013.01); C40B 70/00 (2013.01); G01N 33/536 (2013.01); G01N 33/543 (2013.01); G01N 33/6845 (2013.01); G01N 33/6854 (2013.01); B01F 23/41 (2022.01); B01J 2219/00286 (2013.01); B01J 2219/00459 (2013.01); B01J 2219/00466 (2013.01); B01J 2219/00468 (2013.01); B01J 2219/00479 (2013.01); B01J 2219/005 (2013.01); B01J 2219/00576 (2013.01); B01J 2219/00599 (2013.01); B01J 2219/00702 (2013.01); B01J 2219/0072 (2013.01); B01J 2219/00743 (2013.01); B01L 3/5027 (2013.01); B01L 3/502715 (2013.01); B01L 3/50273 (2013.01); B01L 3/502746 (2013.01); B01L 3/502761 (2013.01); B01L 3/502792 (2013.01); B01L 3/565 (2013.01); B01L 7/52 (2013.01); B01L 2200/027 (2013.01); B01L 2200/0621 (2013.01); B01L 2200/0647 (2013.01); B01L 2200/0652 (2013.01); B01L 2200/0673 (2013.01); B01L 2300/0816 (2013.01); B01L 2300/0864 (2013.01); B01L 2300/0867 (2013.01); B01L 2300/0896 (2013.01); B01L 2400/0415 (2013.01); B01L 2400/0424 (2013.01); B01L 2400/0487 (2013.01); B01L 2400/084 (2013.01); C12Q 1/6804 (2013.01); C12Q 1/6816 (2013.01); C12Q 1/6825 (2013.01); C12Q 1/6834 (2013.01); C12Q 1/6869 (2013.01); C12Q 2525/161 (2013.01); C12Q 2561/119 (2013.01); C12Q 2563/103 (2013.01); C12Q 2563/107 (2013.01); C12Q 2563/159 (2013.01); C12Q 2565/537 (2013.01); C12Q 2565/629 (2013.01); C12Q 2600/16 (2013.01); G01N 21/6428 (2013.01); G01N 21/6445 (2013.01); G01N 21/6458 (2013.01); G01N 33/542 (2013.01); G01N 2500/10 (2013.01)] 15 Claims
 
1. A method for preparing nucleic acids for sequencing, the method comprising:
providing a first dispersed phase solution comprising primer-bound microbeads and primer pairs and a second dispersed phase solution comprising nucleic acid templates via respective first and second inlet channels in a coalescence module of a microfluidic device;
coalescing by the coalescence module droplets of the first and the second dispersed phase solution to generate nanoreactor droplets, a plurality of the droplets each comprising one of the primer-bound microbeads, at least one of the nucleic acid templates, and a label for amplified DNA;
amplifying the nucleic acids templates with the primer-bound microbeads and the primer pairs to generate bead-bound amplified nucleic acids;
separating using the label the droplets into a first population of the droplets that each contain the amplified DNA and a second population of the droplets that do not contain the amplified DNA;
breaking the first population of the droplets;
depositing the bead-bound amplified nucleic acids onto a solid support for sequencing.