Methods for adding adapters to nucleic acids and compositions for practicing the same

Patent metadata

Field	Value
Publication number	US-9719136-B2
Application number	US-201414566445-A
Country	US
Kind code	B2
Filing date	Dec 10, 2014
Priority date	Dec 17, 2013
Publication date	Aug 1, 2017
Grant date	Aug 1, 2017

Abstract

Provided are methods of adding adapters to nucleic acids. The methods include combining in a reaction mixture a template nucleic acid, a template switch oligonucleotide, a polymerase, and dNTPs. The reaction mixture components are combined under conditions sufficient to produce a product nucleic acid that includes the template nucleic acid and the template switch oligonucleotide each hybridized to adjacent regions of a single product nucleic acid including a region polymerized from the dNTPs by the polymerase. The methods further include attaching sequencing platform adapter constructs to ends of the product nucleic acid or a derivative thereof. Aspects of the invention further include compositions and kits.

First claim

What is claimed is:

1. A method comprising:
(a) combining:
a template nucleic acid;
a template switch oligonucleotide comprising a 3′ hybridization domain and one or more ribonucleotides 5′ of the 3′ hybridization domain;
a polymerase; and
dNTPs;

in a reaction mixture under conditions sufficient to produce a first strand cDNA complementary to the template nucleic acid and the template switch oligonucleotide;
b) degrading the template switch oligonucleotide; and
c) amplifying the first strand cDNA.

2. The method according to claim 1 , wherein the template switch oligonucleotide comprises two or more ribonucleotides 5′ of the 3′ hybridization domain.

3. The method according to claim 1 , wherein the template switch oligonucleotide is degraded using a ribonuclease.

4. The method according to claim 1 , wherein the amplifying is performed with a primer comprising a barcode.

5. The method according to claim 1 , wherein said template nucleic acid is from a single cell.

6. The method according to claim 1 , wherein the 3′ hybridization domain comprises a homo-trinucleotide.

7. The method according to claim 1 , wherein the 3′ hybridization domain comprises a hetero-trinucleotide.

8. The method according to claim 1 , wherein the template switch oligonucleotide comprises a sequencing platform nucleic acid domain selected from the group consisting of: a domain that specifically binds to a surface-attached sequencing platform oligonucleotide, a sequencing primer binding domain, a barcode domain, a barcode sequencing primer binding domain, a molecular identification domain, a complement of any such domains, and combinations thereof.

9. The method according to claim 1 , wherein the template nucleic acid is sheared or fragmented template nucleic acid.

10. The method according to claim 9 , wherein the method further comprises shearing or fragmenting the template nucleic acid prior to the combining.

11. The method according to claim 1 , wherein the method comprises heating the reaction mixture.

12. The method according to claim 1 , wherein the reaction mixture includes a first strand synthesis primer comprising at least one exo-sample nucleotide.

13. The method according to claim 1 , wherein the degrading comprises degrading dissociated template switch oligonucleotide.

14. A method comprising:
(a) combining:
a template nucleic acid;
a template switch oligonucleotide comprising one or more exo-sample nucleotides selected from the group consisting of: bromodeoxyuridine, 7-methylguanine, 5,6-dihydroxydeoxythymidine, and 3-methyldeoxadenosine, or any combination thereof and a 3′ hybridization domain;
a polymerase; and
dNTPs;

in a reaction mixture under conditions sufficient to produce a first strand cDNA complementary to the template nucleic acid and the template switch oligonucleotide;
b) degrading a portion of the template switch oligonucleotide that is 5′ of the 3′ hybridization domain; and
c) amplifying the first strand cDNA.

15. The method according to claim 14 , wherein the template switch oligonucleotide is degraded using one or more enzymes.

16. The method according to claim 15 , wherein the one or more enzymes are selected from the group consisting of: a uracil-N-glycosylase, a lyase, a DNA glycosylase-lyase and combinations thereof.

17. The method according to claim 16 , wherein the DNA glycosylase-lyase is Endonuclease VIII.

18. The method according to claim 14 , wherein the template switch oligonucleotide further comprises a ribonucleotide 5′ of the 3′ hybridization domain.

19. The method according to claim 14 , wherein the template switch oligonucleotide comprises a sequencing platform nucleic acid domain selected from the group consisting of: a domain that specifically binds to a surface-attached sequencing platform oligonucleotide, a sequencing primer binding domain, a barcode domain, a barcode sequencing primer binding domain, a molecular identification domain, a complement of any such domains, and combinations thereof.

Assignees

• Clontech Lab Inc
• Takara Bio Usa Inc

Inventors

• Betts Craig
• Farmer Andrew Alan

Classifications

• C12N15/1096 Primary

  cDNA Synthesis; Subtracted cDNA library construction, e.g. RT, RT-PCR · CPC title

• C12Q1/6869 Primary

  Methods for sequencing · CPC title

• C12Q2525/191

  incorporating an adaptor · CPC title

• C12Q1/6876

  Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes · CPC title