US 11,993,773 B2
Methods for extending polynucleotides
Xavier Godron, Paris (FR); Adrian Horgan, Paris (FR); Sylvain Gariel, Paris (FR); Jeffrey Jeddeloh, Verona, WI (US); Robert Nicol, Cambridge, MA (US); and Thomas Ybert, Paris (FR)
Assigned to DNA Script SAS, Le Kremlin-Bicêtre (FR)
Filed by DNA Script SAS, Le Kremlin-Bicêtre (FR)
Filed on Mar. 4, 2021, as Appl. No. 17/192,578.
Application 17/192,578 is a division of application No. 16/970,590, granted, now 11,268,091, issued on Mar. 8, 2022, previously published as PCT/EP2019/084347, filed on Dec. 10, 2019.
Claims priority of application No. 18306687 (EP), filed on Dec. 13, 2018; and application No. 19305219 (EP), filed on Feb. 25, 2019.
Prior Publication US 2021/0198661 A1, Jul. 1, 2021
Int. Cl. C12N 15/10 (2006.01); C12N 15/11 (2006.01); C12Q 1/44 (2006.01); C40B 50/06 (2006.01); C40B 50/08 (2006.01); C40B 50/14 (2006.01); C40B 70/00 (2006.01)
CPC C12N 15/1096 (2013.01) [C12N 15/111 (2013.01); C12Q 1/44 (2013.01); C40B 50/06 (2013.01); C40B 50/08 (2013.01); C40B 50/14 (2013.01); C40B 70/00 (2013.01); C12N 2310/319 (2013.01); C12N 2310/3515 (2013.01); C12N 2330/31 (2013.01)] 7 Claims
OG exemplary drawing
 
1. A method of generating cDNA libraries each having an oligonucleotide label, the method comprising the steps of:
(a) capturing an mRNA by hybridizing the mRNA to capture oligonucleotides attached to one or more solid supports, wherein
the capture oligonucleotides are complementary to segments of the mRNA, and the capture oligonucleotides are attached to the one or more solid supports by 5′-ends and have 3′-ends with free 3′-O-hydroxyls;
(b) extending the 3′-ends of the capture oligonucleotides with a reverse transcriptase using the captured mRNAs as templates to form the cDNA libraries on the one or more solid supports; and
(c) synthesizing oligonucleotide labels on cDNAs of the one or more solid supports by template-free enzymatic synthesis;
wherein:
said step (a) of capturing includes capturing the mRNA of a single cell or single tissue on the one or more solid supports to form said cDNA libraries that are cell-specific or tissue-specific cDNA libraries, respectively;
said oligonucleotide labels are unique cell-specific or tissue-specific oligonucleotide barcodes; and
said one or more solid supports include binding compounds attached thereto for capturing predetermined non-nucleic acid ligands.