TEMPLATE SWITCH-BASED METHODS FOR PRODUCING A PRODUCT NUCLEIC ACID

• US 20160304946A1
• Filed: 06/27/2016
• Published: 10/20/2016
• Est. Priority Date: 10/24/2012
• Status: Active Grant

• Current Assignee: Takara Bio Usa, Inc. (Ningbo Junsheng Investment Group Co., Ltd.)
• Original Assignee: Clontech Laboratories Incorporated
• Inventors: Betts, Craig, Farmer, Andrew Alan
• Granted Patent: US 11,001,882 B2
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: C12N 15/1096 cDNA Synthesis; Subtracted cDNA library construction, e.g. RT, RT-PCR
• CPC Class Codes: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides
• CPC Class Codes: C12Q 1/6853 using modified primers or templates
• CPC Class Codes: C12Q 2521/107 RNA dependent DNA polymerase,(i.e. reverse transcriptase)
• CPC Class Codes: C12Q 2525/191 incorporating an adaptor
• CPC Class Codes: C12Y 207/07007 DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
• CPC Class Codes: C12Y 301/00 Hydrolases acting on ester bonds (3.1)

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

1-25. -25. (canceled)

26. A composition comprising a template deoxyribonucleic acid (DNA) and a template switch oligonucleotide each hybridized to adjacent regions of a nucleic acid strand.

27. The composition of claim 26, wherein the template switch oligonucleotide comprises a 3′

hybridization domain and a 5′

adapter sequence.

28. The composition of claim 27, wherein the 3′

hybridization domain comprises a homo-trinucleotide.

29. The composition of claim 26, wherein the composition is present in a reaction tube.

30. A kit comprising:

a 3′

overhang-generating enzyme;

a polymerase having terminal transferase activity; and

a template switch nucleic acid having a 3′

end complementary to a nucleotide stretch produced by the polymerase via non-tem plated nucleotide addition.

31. The kit according to claim 30, wherein the 3′

overhang-generating enzyme is a restriction endonuclease.

32. The kit according to claim 30, wherein the 3′

overhang-generating enzyme is a terminal transferase.

33. The kit according to claim 30, wherein the template switch nucleic acid comprises a defined sequence at a region 5′

of the 3′

overhang.

34. The kit according to claim 33, further comprising a primer having the defined sequence at the region 5′

of the 3′

overhang.

35. The kit according to claim 30, wherein the template switch nucleic acid comprises a cloning vector element.

36. The kit according to claim 35, wherein the cloning vector element is selected from the group consisting of:

a selectable marker, a reporter, a promoter, an origin of replication, a multiple cloning site, and combinations thereof.