US 12,286,621 B2
Efficient combinatorial bead barcoding
Eduardo da Veiga Beltrame, Pasadena, CA (US); Jase Gehring, Pasadena, CA (US); Akshay Tambe, Hayward, CA (US); Lior S. Pachter, Pasadena, CA (US); and Taleen Dilanyan, Pasadena, CA (US)
Assigned to California Institute of Technology, Pasadena, CA (US)
Filed by California Institute of Technology, Pasadena, CA (US)
Filed on Sep. 27, 2019, as Appl. No. 16/586,592.
Claims priority of provisional application 62/738,127, filed on Sep. 28, 2018.
Prior Publication US 2020/0102556 A1, Apr. 2, 2020
Int. Cl. C12N 15/10 (2006.01); C12N 15/11 (2006.01); C12N 15/66 (2006.01); C40B 20/04 (2006.01); C40B 50/14 (2006.01); C40B 70/00 (2006.01)
CPC C12N 15/1065 (2013.01) [C12N 15/1096 (2013.01); C12N 15/11 (2013.01); C40B 20/04 (2013.01); C40B 50/14 (2013.01); C40B 70/00 (2013.01)] 18 Claims
 
1. A method of combinatorially barcoding particle-associated oligonucleotides, comprising steps in the following order:
(a) providing a particle comprising a plurality of oligonucleotides each comprising an input primer, wherein the input primer is releasably covalently attached to the particle;
(b) contacting the particle with a solution comprising a first hairpin molecule and a polymerase having strand displacement activity, wherein the first hairpin molecule comprises:
(i) an unpaired first 3′ toehold domain complementary to the input primer,
(ii) a first paired stem domain formed by intramolecular nucleotide base pairing between a 3′ subdomain of the first hairpin molecule and a 5′ subdomain of the first hairpin molecule, wherein the first paired stem domain comprises a first coupling segment, a first barcode segment, and complements thereof, wherein the first barcode segment is 5′ of the first coupling segment and comprises at least 3 nucleotides,
(iii) a first hairpin loop domain, and
(iv) a polymerase stopper capable of terminating polymerization located (1) in the first hairpin loop domain, or (2) between the first paired stem domain and the first hairpin loop domain;
(c) extending the input primer hybridized to the first 3′ toehold domain through the first paired stem domain of the first hairpin molecule, thereby displacing the 5′ subdomain of the first hairpin molecule and forming a first extended oligonucleotide comprising the input primer, the first barcode segment and the first coupling segment.