US 11,739,316 B2
Oligonucleotide-tethered nucleotides
Arvydas Lubys, Vilnius (LT); Inga Cikotiene, Vilnius (LT); Zana Kapustina, Vilnius (LT); Arturas Berezniakovas, Vilnius (LT); Justina Medziune, Vilnius (LT); Simona Zeimyte, Vilnius (LT); Mark Andersen, Carlsbad, CA (US); Michael Allen, San Marcos, CA (US); and Sihong Chen, Vista, CA (US)
Assigned to Thermo Fisher Scientific Baltics UAB, Vilnius (LT); and Life Technologies Corporation, Carlsbad, CA (US)
Filed by THERMO FISHER SCIENTIFIC BALTICS UAB, Vilnius (LT); and LIFE TECHNOLOGIES CORPORATION, Carlsbad, CA (US)
Filed on Jun. 22, 2020, as Appl. No. 16/908,567.
Claims priority of provisional application 63/032,297, filed on May 29, 2020.
Claims priority of provisional application 62/864,589, filed on Jun. 21, 2019.
Prior Publication US 2020/0399633 A1, Dec. 24, 2020
Int. Cl. C12N 15/10 (2006.01); C12N 15/115 (2010.01); C12Q 1/6806 (2018.01)
CPC C12N 15/1065 (2013.01) [C12N 15/115 (2013.01); C12Q 1/6806 (2013.01); C12N 2310/3517 (2013.01)] 11 Claims
 
1. A method for generating a library of nucleic acids from a sample comprising one or more nucleic acids, optionally wherein the sample comprises a plurality of cells or cell nuclei, comprising:
a. annealing a first primer which is at least partially complementary to the one or more nucleic acids,
b. contacting the one or more nucleic acids with a first nucleic acid polymerase, at least one nucleotide not tethered to an oligonucleotide, and at least one oligonucleotide-tethered dideoxynucleotide to form a plurality of first extension products comprising the oligonucleotide-tethered dideoxynucleotide at the 3′ end;
c. annealing a splint oligonucleotide which is at least partially complementary to the tethered oligonucleotide of the first extension products, and
d. contacting the first extension products with a nucleic acid polymerase and one or more nucleotides to allow the polymerase to extend across the annealed splint from the 3′ hydroxyl of the tethered oligonucleotide to produce a second extension product, thereby producing a library of nucleic acids,
wherein if the sample comprises a plurality of cells or cell nuclei, the cells or cell nuclei are fixed and permeabilized prior to annealing the first primer.