IP Library › Granted Patent US 12,454,721
Granted Patent B2
US 12,454,721 · App. 17/225,511 · Granted Oct 28, 2025

Template switch-based methods for producing a product nucleic acid

Inventors: Craig Betts (Mountain View, CA); Andrew Alan Farmer (Los Altos, CA); Nathalie Bolduc (Mountain View, CA)
Assignee: Takara Bio USA, Inc.
C12Q1/6853C12N15/1096C12P19/34C12Y207/07007C12Y301/00C12Q2521/107C12Q2525/191
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Quick Facts
Patent No.
US 12,454,721
App. No.
17/225,511
Granted
Oct 28, 2025
Kind
B2
Abstract

Provided are methods of producing a product nucleic acid. The methods include combining a template deoxyribonucleic acid (DNA), a polymerase, a template switch oligonucleotide, and dNTPs into a reaction mixture. The components are combined into the reaction mixture under conditions sufficient to produce a product nucleic acid that includes the template DNA and the template switch oligonucleotide each hybridized to adjacent regions of a single product nucleic acid that includes a region polymerized from the dNTPs by the polymerase. Aspects of the invention further include compositions and kits.

Claims (24)

1 . A population of double stranded nucleic acids, the population of double stranded nucleic acids comprising:

double stranded nucleic acids that comprise a target template DNA adjacent to a template switch oligonucleotide, wherein the target template DNA and the template switch oligonucleotide are hybridized to a single product nucleic acid, wherein the single product nucleic acid is a single stranded cDNA, wherein the single stranded cDNA comprises a sequence complementary to the template switch oligonucleotide adjacent to a sequence complementary to the target template DNA, and wherein the sequence complementary to the template switch oligonucleotide is at the 3′ end of the single stranded cDNA.

2 . The population of double stranded nucleic acids according to claim 1 , wherein the target DNA template is present in a nucleic acid sample isolated from the group consisting of: genomic DNA or fragments thereof, complementary DNA (cDNA), and recombinant DNA.

3 . The population of double stranded nucleic acids according to claim 2 , wherein the target DNA template is present in a nucleic acid sample isolated from genomic DNA or fragments thereof.

4 . The population of double stranded nucleic acids according to claim 1 , wherein the template switch oligonucleotide comprises a 3′ hybridization domain and a 5′ adapter sequence.

5 . The population of double stranded nucleic acids according to claim 4 , wherein the 3′ hybridization domain comprises a homo-trinucleotide.

6 . The population of double stranded nucleic acids according to claim 4 , wherein the 3′ hybridization domain comprises a hetero-trinucleotide.

7 . The population of double stranded nucleic acids according to claim 6 , wherein the hetero-trinucleotide comprises a nucleotide comprising cytosine and a nucleotide comprising guanine.

8 . The population of double stranded nucleic acids according to claim 1 , wherein the single product nucleic acid comprises a sequencing platform adapter domain complementary to the sequence of the template switch oligonucleotide.

9 . The population of double stranded nucleic acids according to claim 1 , wherein the single product nucleic acid is extended from a first primer that hybridizes to the target DNA template.

10 . The population of double stranded nucleic acids according to claim 9 , wherein the first primer comprises a first domain that hybridizes to the target DNA template and a second domain that does not hybridize to the target DNA template.

11 . The population of double stranded nucleic acids according to claim 10 , wherein the first domain has a defined sequence.

12 . The population of double stranded nucleic acids according to claim 10 , wherein the first domain has an arbitrary sequence.

13 . The population of double stranded nucleic acids according to claim 10 , wherein the second domain comprises a sequence that is identical to a sequence present in the template switch oligonucleotide.

14 . The population of double stranded nucleic acids according to claim 13 , wherein the single product nucleic acid comprises the same adapter sequence at its 5′ and 3′ ends.

15 . The population of double stranded nucleic acids according to claim 10 , wherein the second domain comprises a nucleotide sequence that is not present in the template switch oligonucleotide.

16 . The population of double stranded nucleic acids according to claim 15 , wherein the single product nucleic acid comprises different adapter sequences at its 5′ and 3′ ends.

17 . A plurality of constructs, the plurality of constructs comprising:

constructs that comprise a target genomic DNA template adjacent to a template switch oligonucleotide, wherein the target genomic DNA template and the template switch oligonucleotide are hybridized to a first strand cDNA, wherein the first strand cDNA comprises in the 5′ to 3′ order: a 5′ adapter sequence, a nucleic acid fragment complementary to a target genomic DNA template, and an adapter sequence complementary to a template switch oligonucleotide.

18 . The plurality of constructs according to claim 17 , wherein the 5′ adapter sequence comprises a sequence from a first primer.

19 . The plurality of constructs according to claim 17 , wherein the adapter sequence derived from a template switch oligonucleotide and the 5′ adapter sequence comprise the same nucleotide sequence.

20 . The plurality of constructs according to claim 17 , wherein the adapter sequence derived from a template switch oligonucleotide and the 5′ adapter sequence comprise different nucleotide sequences.

21 . The plurality of constructs according to claim 20 , wherein the adapter sequence derived from a template switch oligonucleotide is a sequencing platform adapter domain and the 5′ adapter sequence is a sequencing platform adapter domain.

22 . The plurality of constructs according to claim 17 , wherein the library is present in a reaction tube.

Assignments (3)
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Apr 9, 2021
From: BETTS, CRAIG; FARMER, ANDREW ALAN
To: CLONTECH LABORATORIES, INC.
Reel/Frame 055875/0515 →
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Apr 9, 2021
From: BOLDUC, NATHALIE
To: TAKARA BIO USA, INC.
Reel/Frame 055875/0598 →
CHANGE OF NAME Recorded Apr 9, 2021
From: CLONTECH LABORATORIES, INC.
To: TAKARA BIO USA, INC.
Reel/Frame 055875/0611 →
Continuity (4)
Continuation 15194414 · Jun 27, 2016
Division 14058038 · Oct 18, 2013
Provisional Application 61717973 · Oct 24, 2012
Related Publication 20210222236A1 · Jul 22, 2021
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