US 12,378,548 B2
Methods and compositions for cell-free cloning
Matthew Hill, Los Altos, CA (US)
Assigned to Elegen Corporation, Menlo Park, CA (US)
Filed by Elegen Corporation, Los Altos, CA (US)
Filed on Feb. 12, 2024, as Appl. No. 18/439,353.
Application 18/439,353 is a continuation of application No. 18/336,283, filed on Jun. 16, 2023, granted, now 11,993,771.
Application 18/336,283 is a continuation of application No. PCT/US2022/071707, filed on Apr. 13, 2022.
Claims priority of provisional application 63/174,192, filed on Apr. 13, 2021.
Prior Publication US 2024/0229016 A1, Jul. 11, 2024
Int. Cl. C12N 15/10 (2006.01)
CPC C12N 15/1065 (2013.01) 30 Claims
OG exemplary drawing
 
1. A method of generating a population of polynucleotides, wherein the method comprises:
(a) separately in each of a set of initial sources of nucleic acid molecules comprising the nucleic acid molecules and a set of between 3 to 1000 unique, non-degenerate barcodes, performing a tagging reaction to produce a set of source samples each comprising at least 1×106 tagged candidate nucleic acid molecules,
wherein each of the tagged candidate nucleic acid molecules comprises a tag comprising one or more of the non-degenerate barcodes;
(b) diluting a subvolume of each source sample of the set of source samples to form a set of diluted samples each having a target number of the tagged candidate nucleic acid molecules isolated from each source sample in the set of source samples, wherein the target number of the tagged candidate nucleic acid molecules isolated from each source sample is between 10 and 2000 nucleic acid molecules;
(c) combining a portion of each diluted sample in the set of diluted samples to form a combined diluted sample, wherein the combined diluted sample comprises tagged candidate nucleic acid species derived from one or more of the tagged candidate nucleic acid molecules from each diluted sample of the set of diluted samples, wherein the nucleic acid molecules of each tagged candidate nucleic acid species have an identical nucleic acid sequence comprising the nucleic acid sequence of the one or more tagged candidate nucleic acid molecules from which it was derived, and wherein at least one of the tagged candidate nucleic acid species from each diluted sample in the combined diluted sample is uniquely tagged;
(d) determining the sequence of at least some of the tagged candidate nucleic acid species from each of the diluted samples in the combined diluted, wherein at least 1 of the tagged candidate nucleic acid species from each diluted sample in the combined diluted sample is a desired uniquely tagged nucleic acid species, wherein the desired uniquely tagged nucleic acid species comprise a sequence-perfect desired nucleic acid sequence; and
(e) enriching the desired uniquely tagged nucleic acid species from each diluted sample of the set of diluted samples by amplifying in each diluted sample one or more tagged candidate nucleic acid molecules of the desired uniquely tagged nucleic acid species using one or more primers that bind one or more of the barcodes on the tagged candidate nucleic acid molecules of the desired uniquely tagged nucleic acid species to generate a set of populations of product polynucleotides.