Oligonucleotide-tethered nucleotides

Patent metadata

Field	Value
Publication number	US-11739316-B2
Application number	US-202016908567-A
Country	US
Kind code	B2
Filing date	Jun 22, 2020
Priority date	Jun 21, 2019
Publication date	Aug 29, 2023
Grant date	Aug 29, 2023

Abstract

The present disclosure describes oligonucleotide-tethered nucleotides, methods of making them, and methods of using them. The oligonucleotide-tethered nucleotides comprise, in some embodiments, a nucleotide linked to an oligonucleotide of from about 3 to about 100 nucleotides in length. These oligonucleotide-tethered nucleotides can be used to label a plurality of different types of nucleic acids in a plurality of different situations with a known oligonucleotide, which can serve as a barcode in some embodiments. The resulting oligonucleotide-labeled nucleic acids oligonucleotides can be used in a variety of nucleic acid sequencing methods.

First claim

What is claimed is:

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.

2. The method of claim 1 , wherein the sample is treated with an oligonucleotide-tethered binding agent (OTBA) prior to fixation.

3. The method of claim 2 , wherein the tethered oligonucleotide of the OTBA comprises a cell marker binding agent index, and wherein binding agent of the OTBA comprises an aptamer or an antibody or a functional fragment thereof.

4. The method of claim 1 , wherein a sample comprising one or more nucleic acids comprises more than one cell or cell nuclei, wherein the cells or cell nuclei may comprise one or more cell markers, wherein the sample is split into two or more first portions before step a, and wherein each first portion comprises a subpopulation of cells or cell nuclei of the original sample.

5. The method of claim 4 , wherein the first primer comprises a first universal handle sequence and a first barcode, said first barcode being common among the first primers in each first portion, but different from the first barcodes present in first primers in other first portions; and wherein the oligonucleotide of the oligonucleotide-tethered dideoxynucleotide comprises a second universal handle sequence.

6. The method of claim 5 , further comprising, before step c:
a. combining the first portions after formation of the first nucleic extension products and
b. splitting the combined first portions into two or more second portions, wherein the second portions comprise the splint oligonucleotide; wherein the splint oligonucleotide comprises:
i. an oligonucleotide sequence that anneals to the second universal handle on the tethered oligonucleotide;
ii. a template sequence for a second barcode, wherein the second barcodes of each second portion are common, but are different from the second barcodes of other second portions, and
iii. a template sequence for a third universal handle;

wherein the second extension products comprise the second barcode and third universal handle.

7. The method of claim 6 further comprising:
a. combining the second portions;
b. splitting the combined second portions into two or more third portions;
c. contacting each third portion with amplification primers, wherein the amplification primers are capable of hybridizing to and extending from the first universal handle and the third universal handle, and wherein the amplification primers optionally comprise third and/or fourth barcodes respectively, and first and/or second adapter sequences, respectively, to generate amplification products, wherein the combination of the first, second, and third barcode sequences (or complements thereof) of the amplification products are unique to the amplification products originating from a single cell or nucleus.

8. The method of claim 1 , wherein the primer, the tethered oligonucleotide, or both comprises a random sequence, a target-specific sequence or both.

9. The method of claim 1 , wherein one or more of the primer, the tethered oligonucleotide, or the splint oligonucleotide comprises a universal handle, a universal sequence, a unique molecular identifier, an adapter sequence, a promoter sequence, a barcode sequence, an index sequence, or any combination thereof.

10. The method of claim 1 , wherein the polymerase is a DNA polymerase or an RNA polymerase.

11. The method of claim 1 , wherein the nucleic acid is DNA or RNA.

Assignees

• Thermo Fisher Scientific Baltics Uab
• Life Technologies Corp

Inventors

• Lubys Arvydas
• Cikotiene Inga
• Kapustina Zana
• Berezniakovas Arturas
• Medziune Justina
• Zeimyte Simona
• Andersen Mark
• Allen Michael
• Chen Sihong

Classifications

• C12N15/1065 Primary

  Preparation or screening of tagged libraries, e.g. tagged microorganisms by STM-mutagenesis, tagged polynucleotides, gene tags · CPC title

• C12N15/115

  Aptamers, i.e. nucleic acids binding a target molecule specifically and with high affinity without hybridising therewith {; Nucleic acids binding to non-nucleic acids, e.g. aptamers} · CPC title

• C12Q1/6806

  Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence) · CPC title

• C12N2310/3517

  Marker; Tag · CPC title

• C07H21/00 Primary

  Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids · CPC title