Coupling adaptors to a target nucleic acid

• US 10,655,170 B2
• Filed: 07/05/2017
• Issued: 05/19/2020
• Est. Priority Date: 07/06/2016
• Status: Active Grant

• Current Assignee: Takara Bio Usa, Inc. (Ningbo Junsheng Investment Group Co., Ltd.)
• Original Assignee: Takara Bio Usa, Inc. (Ningbo Junsheng Investment Group Co., Ltd.)
• Inventors: Charizanis, Konstantinos, Gonzalez-Hernandez, Marta, McNulty, Amanda, Hecker, Karl, Kamberov, Emmanuel, Langmore, John
• Primary Examiner(s): Vivlemore, Tracy
• Assistant Examiner(s): Kaup, Sahana S
• Application Number:
  US15/642,263
• Publication Number:
  US 20180010178A1
• Time in Patent Office: 1,049 Days
• Field of Search: None
• CPC Class Codes: C12N 15/1093 General methods of preparing gene libraries, not provided for in other subgroups
• CPC Class Codes: C12N 15/66 General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6855 Ligating adaptors
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2521/501 Ligase
• CPC Class Codes: C12Q 2525/185 incorporating bases where the precise position of the bases in the nucleic acid string is important
• CPC Class Codes: C12Q 2525/191 incorporating an adaptor
• CPC Class Codes: C12Q 2535/122 Massive parallel sequencing
• CPC Class Codes: C12Q 2563/179 the label being a nucleic acid

Abstract

Methods of coupling adaptors to a target nucleic acid include coupling a first adaptor to a first end of the target nucleic acid to form a coupled first adaptor. A portion of a second adaptor is hybridized to a portion of the coupled first adaptor to form a hybridized second adaptor having a single-stranded 3′-end. The hybridized second adaptor is coupled to a second end of the target nucleic acid to form an adaptor-flanked product having at least a part of the first adaptor coupled to the first end of the target nucleic acid and at least a part of the second adaptor coupled to the second end of the target nucleic acid. These methods can minimize the formation of adaptor-dimers that may be problematic in subsequent complementary nucleic acid strand synthesis, amplification, and sequencing.

Claims

1. A method of coupling adaptors to a target nucleic acid, the method comprising:

coupling a first adaptor to a first end of the target nucleic acid to form a coupled first adaptor;

hybridizing a portion of a second adaptor to a portion of the coupled first adaptor to produce a hybridized structure having a hybridized second adaptor in which the hybridized second adaptor comprises a single-stranded 3′

-end; and

coupling the single-stranded 3′

-end of the hybridized second adaptor of the hybridized structure to a second end of the target nucleic acid to form an adaptor-flanked product, the adaptor-flanked product comprising at least a part of the first adaptor coupled to the first end of the target nucleic acid and at least a part of the second adaptor coupled to the second end of the target nucleic acid.

2. The method of claim 1, wherein the first adaptor comprises an adenylated 5′

-end and a blocked 3′

-end, and coupling the first adaptor to the first end of the target nucleic acid comprises ligating the adenylated 5′

-end of the first adaptor to a 3′

-end of the target nucleic acid comprising a hydroxyl group.

3. The method of claim 1, wherein the second adaptor is configured to hybridize to the first adaptor so that a 5′

-end of the first adaptor is double-stranded.

4. The method of claim 1, wherein the hybridized second adaptor comprises a blocked 5′

-end and the single-stranded 3′

-end of the hybridized second adaptor comprises a hydroxyl group.

5. The method of claim 4, wherein coupling the hybridized second adaptor to the second end of the target nucleic acid comprises ligating the single-stranded 3′

-end comprising the hydroxyl group of the second adaptor to a 5′

-end of the target nucleic acid including a phosphate group.

6. The method of claim 1, further comprising synthesizing a complementary nucleic acid strand to the target nucleic acid of the adaptor-flanked product.

7. The method of claim 6, wherein the target nucleic acid comprises RNA and synthesizing a complementary nucleic acid strand comprises reverse transcription of the RNA.

8. The method of claim 6, further comprising amplifying the complementary nucleic acid strand using at least one amplification primer, the at least one amplification primer configured to hybridize to the complementary nucleic acid strand.

9. The method of claim 8, wherein amplifying the complementary nucleic acid strand using at least one amplification primer comprises performing a polymerase chain reaction using a first amplification primer comprising a 3′

-end configured to hybridize to a 3′

region of the complementary nucleic acid strand and a second amplification primer comprising a 3′

-end able to hybridize to a sequence from a 5′

region of the complementary nucleic acid strand.

10. The method of claim 9, wherein at least one of the first amplification primer and the second amplification primer comprises a barcode.

11. The method of claim 1, wherein the hybridized second adaptor comprises a bridging oligonucleotide and a ligation oligonucleotide, the bridging oligonucleotide being the part of the second adaptor that hybridizes to the portion of the coupled first adaptor to form the hybridized second adaptor and comprising another portion that hybridizes with a portion of the ligation oligonucleotide, the ligation oligonucleotide comprising the single-stranded 3′

-end.

12. The method of claim 11, wherein the bridging oligonucleotide comprises a blocked 5′

-end, the ligation oligonucleotide comprises a blocked 5′

-end, and the single-stranded 3′

-end of the ligation nucleotide comprises a hydroxyl group.

13. The method of claim 11, wherein the single-stranded 3′

-end of the ligation oligonucleotide comprises a hydroxyl group and coupling the hybridized second adaptor to the second end of the target nucleic acid comprises ligating the single-stranded 3′

-end of the ligation oligonucleotide comprising the hydroxyl group to a 5′

-end of the target nucleic acid comprising a phosphate group.

14. The method of claim 1, wherein the hybridized second adaptor comprises at least one cleavable site between the portion of the second adaptor that hybridizes to the portion of the coupled first adaptor and another portion of the second adaptor that comprises the single-stranded 3′

-end.

15. The method of claim 14, wherein the hybridized second adaptor comprises DNA, the at least one cleavable site comprises uracil, the hybridized second adaptor comprises a blocked 5′

-end, and the single-stranded 3′

-end of the hybridized second adaptor comprises a hydroxyl group.

16. The method of claim 11, wherein the single-stranded 3′

-end of the hybridized second adaptor comprises a hydroxyl group and coupling the hybridized second adaptor to the second end of the target nucleic acid comprises ligating the single-stranded 3′

-end of the hybridized second adaptor comprising the hydroxyl group to a 5′

-end of the target nucleic acid comprising a phosphate group.

17. The method of claim 14, further comprising cleaving the at least one cleavable site after coupling the hybridized second adaptor to the second end of the target nucleic acid, the cleaving resulting in the portion of the second adaptor that hybridizes to the portion of the coupled first adaptor to comprise an extendible 3′

-end and the another portion of the second adaptor that comprises the single stranded 3′

-end coupled to the second end of the target nucleic acid.

18. The method of claim 14, further comprising extending the extendible 3′

-end and synthesizing a complementary nucleic acid strand.

19. The method of claim 1, wherein the hybridized second adaptor comprises a 5′

to 5′

reverse linker.

20. The method of claim 19, wherein the 5′

to 5′

reverse linker is between the portion of the second adaptor that hybridizes to the portion of the coupled first adaptor and another portion of the second adaptor that comprises the single-stranded 3′

-end.

21. The method of claim 19, wherein the single-stranded 3′

-end of the hybridized second adaptor comprises a first hydroxyl group and coupling the hybridized second adaptor to the second end of the target nucleic acid comprises ligating the single-stranded 3′

-end of the hybridized second adaptor comprising the first hydroxyl group to a 5′

-end of the target nucleic acid comprising a phosphate group.

22. The method of claim 19, wherein the second adaptor comprises another 3′

end.

23. The method of claim 22, wherein the another 3′

end comprises a second hydroxyl group.

24. The method of claim 22, further comprising extending the another 3′

end and synthesizing a complementary nucleic acid strand.

25. The method of claim 1, wherein the second adaptor comprises at least four non-random nucleotides at the 5′

end.

26. The method of claim 1, wherein the first adaptor is coupled to the 3′

-end of the target nucleic acid.