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.
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