US 11,891,666 B2
Methods and systems for detecting biological components
Adam R. Abate, Daly City, CA (US); Dennis Jay Eastburn, Burlingame, CA (US); and Adam R. Sciambi, San Francisco, CA (US)
Assigned to The Regents of the University of California, Oakland, CA (US)
Filed by The Regents of the University of California, Oakland, CA (US)
Filed on Apr. 6, 2021, as Appl. No. 17/223,565.
Application 17/223,565 is a continuation of application No. 16/382,080, filed on Apr. 11, 2019, granted, now 11,001,896.
Application 16/382,080 is a continuation of application No. 16/164,707, filed on Oct. 18, 2018, granted, now 11,203,787.
Application 16/164,707 is a continuation of application No. 14/420,646, granted, now 10,161,007, issued on Dec. 25, 2018, previously published as PCT/US2013/054517, filed on Aug. 12, 2013.
Claims priority of provisional application 61/784,754, filed on Mar. 14, 2013.
Claims priority of provisional application 61/682,707, filed on Aug. 13, 2012.
Prior Publication US 2021/0388446 A1, Dec. 16, 2021
This patent is subject to a terminal disclaimer.
Int. Cl. C12P 19/34 (2006.01); C12Q 1/6886 (2018.01); C12Q 1/6806 (2018.01); B01L 3/00 (2006.01); B01L 7/00 (2006.01); B01F 23/41 (2022.01); B01F 25/433 (2022.01); B01F 33/3011 (2022.01); B01F 33/3031 (2022.01); C12Q 1/686 (2018.01); C12Q 1/6844 (2018.01)
CPC C12Q 1/6886 (2013.01) [B01F 23/41 (2022.01); B01F 25/4335 (2022.01); B01F 33/3011 (2022.01); B01F 33/3031 (2022.01); B01L 3/502784 (2013.01); B01L 7/52 (2013.01); C12Q 1/686 (2013.01); C12Q 1/6806 (2013.01); C12Q 1/6844 (2013.01); B01L 2200/0652 (2013.01); B01L 2300/0816 (2013.01); B01L 2300/0864 (2013.01); B01L 2300/0867 (2013.01); B01L 2300/0883 (2013.01); B01L 2300/1822 (2013.01); B01L 2400/0415 (2013.01); B01L 2400/0487 (2013.01); C12Q 2600/118 (2013.01); C12Q 2600/158 (2013.01); C12Q 2600/16 (2013.01)] 10 Claims
 
1. A method for genotyping a cell, the method comprising:
(a) encapsulating lysis reagents and the cell containing a target polynucleotide in a droplet, the lysis reagents comprising an enzyme having protease activity, wherein the lysate droplet is surrounded by an immiscible carrier fluid;
(b) incubating the droplet containing the cell and the lysis reagents comprising the enzyme having protease activity for a period of time sufficient to lyse the cell and form a cell lysate comprising the target polynucleotide and for the protease to digest inhibitory proteins;
(c) inactivating the enzyme having protease activity;
(d) introducing amplification reagents to form a droplet containing the cell lysate and the amplification reagents; and
(e) amplifying the target polynucleotide or a complement of the target polynucleotide within the droplet containing the cell lysate and the amplification reagents, wherein the method does not comprise a step of selectively removing reagents from the droplet containing the cell lysate and amplification reagents prior to step (e); and
(f) sequencing the amplified target polynucleotide or amplified complement of the target polynucleotide, thereby detecting a presence or absence of one or more changed nucleotide bases in the target polynucleotide.