Methods of nucleic acid sample preparation

• US 10,704,082 B2
• Filed: 09/15/2017
• Issued: 07/07/2020
• Est. Priority Date: 09/15/2016
• Status: Active Grant

• Current Assignee: ArcherDX, LLC (Danaher Corporation)
• Original Assignee: ArcherDX, LLC (Danaher Corporation)
• Inventors: Stahl, Joshua, Myers, Jason, Culver, Brady, Kudlow, Brian
• Primary Examiner(s): Bertagna, Angela M.
• Application Number:
  US15/706,642
• Publication Number:
  US 20180127806A1
• Time in Patent Office: 1,026 Days
• Field of Search: None
• CPC Class Codes: C12N 15/1013 by using magnetic beads
• CPC Class Codes: C12N 15/1096 cDNA Synthesis; Subtracted cDNA library construction, e.g. RT, RT-PCR
• 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 1/6853 using modified primers or templates
• CPC Class Codes: C12Q 1/6855 Ligating adaptors
• CPC Class Codes: C12Q 2521/101 DNA polymerase
• CPC Class Codes: C12Q 2521/131 Terminal transferase
• CPC Class Codes: C12Q 2521/501 Ligase
• CPC Class Codes: C12Q 2525/117 incorporating modified base
• CPC Class Codes: C12Q 2525/173 incorporating a polynucleotide run, e.g. polyAs, polyTs
• CPC Class Codes: C12Q 2525/191 incorporating an adaptor
• CPC Class Codes: C12Q 2531/113 PCR
• CPC Class Codes: C12Q 2535/122 Massive parallel sequencing
• CPC Class Codes: C12Q 2537/159 Reduction of complexity, e.g. amplification of subsets, removing duplicated genomic regions
• CPC Class Codes: C12Q 2549/119 using nested primers
• CPC Class Codes: C12Q 2563/131 the label being a member of a cognate binding pair, i.e. extends to antibodies, haptens, avidin
• CPC Class Codes: C12Q 2563/143 Magnetism, e.g. magnetic label

Abstract

Aspects of the technology disclosed herein relate to methods of preparing and analyzing nucleic acids. In some embodiments, methods for preparing nucleic acids for sequence analysis (e.g., using next-generation sequencing) are provided herein.

Claims

1. A method of preparing nucleic acids for analysis, the method comprising:

(a) adding one or more nucleotides to a 3′

end of a double-stranded nucleic acid comprising a target nucleotide sequence, wherein at least one of the one or more nucleotides is a capture moiety modified nucleotide;

(b) ligating an adapter nucleic acid to the double-stranded nucleic acid to which the capture moiety modified nucleotide has been added to produce a ligation product, wherein a sequence of one or more nucleotides at a 3′

end of the adapter nucleic acid is complementary with the one or more nucleotides added to the 3′

end of the double-stranded nucleic acid in step (a);

(c) capturing the ligation product by contacting the ligation product with a binding partner of the capture moiety modified nucleotide; and

(d) amplifying the ligation product by polymerase chain reaction using a first target-specific primer that specifically anneals to the target nucleotide sequence and a first adapter primer that specifically anneals to a complementary sequence of the adapter nucleic acid.

2. The method of claim 1, wherein step (b) comprises combining the adapter nucleic acid, the double-stranded nucleic acid, and a ligase under conditions in which the ligase ligates the adapter nucleic acid to the double-stranded nucleic acid, wherein the adapter nucleic acid that is combined with the double-stranded nucleic acid comprises a duplex portion and an overhang sequence, wherein the overhang sequence comprises the sequence of one or more nucleotides at the 3′

end of the adapter nucleic acid that is complementary with the one or more nucleotides added to the 3′

end of the double stranded nucleic acid in step (a).

3. The method of claim 1, wherein step (b) comprises combining the adapter nucleic acid, the double-stranded nucleic acid, and a ligase under conditions in which the ligase ligates the adapter nucleic acid to the double-stranded nucleic acid, wherein the adapter nucleic acid that is combined with the double-stranded nucleic acid is single-stranded.

4. The method of claim 1 further comprising:

(e) amplifying an amplification product of step (d) by polymerase chain reaction using a second adapter primer and a second target-specific primer.

5. The method of claim 4, wherein the second target-specific primer is nested relative to the first target-specific primer.

6. The method of claim 4, wherein the second target-specific primer comprises a 5′

tail that does not anneal to the target nucleotide sequence.

7. The method of claim 6, further comprising adding an additional primer comprising a 3′

portion that is identical to the 5′

tail of the second target-specific primer.

8. The method of claim 1, wherein the capture moiety is a biotin moiety, optionally wherein the biotin moiety is covalently linked to the nucleotide via a linker of 5 to 20 atoms in length.

9. The method of claim 1, wherein the capture moiety modified nucleotide comprises a nucleobase selected from the group consisting of adenine, guanine, thymine, uracil, and cytosine, or derivative(s) thereof.

10. The method of claim 9, wherein the capture moiety modified nucleotide comprises an adenine nucleobase or derivative thereof, optionally wherein the capture moiety is covalently linked to the adenine nucleobase or derivative thereof at position 5, 6, 7 or 8, optionally wherein position 7 of the adenine nucleobase is a carbon atom.

11. The method of claim 1, wherein the binding partner is streptavidin, optionally wherein the streptavidin is attached to a paramagnetic bead.

12. The method of claim 1, wherein, in step (a), one nucleotide is added to the 3′

end of the double-stranded nucleic acid comprising the target nucleotide sequence.

13. The method of claim 1 further comprising a washing step after step (a) and before step (b).

14. The method of claim 1 further comprising, prior to step (a), 5′

phosphorylating the double-stranded nucleic acid.

15. The method of claim 1 further comprising, prior to step (a):

i) preparing cDNA by conducting a randomly-primed first strand synthesis reaction using an RNA preparation as a template and a second strand synthesis reaction using a product of the randomly-primed first strand synthesis reaction as a template; and

ii) end repairing the cDNA to produce a blunt-ended, double-stranded nucleic acid.

16. The method of claim 1, wherein, in step (b), the double-stranded nucleic acid is ligated to the adapter nucleic acid in the presence of a crowding agent, optionally wherein the crowding agent is polyethylene glycol in an amount representing 5% to 50% of the ligation mixture.

17. The method of claim 1, wherein the double-stranded nucleic acid is blunt-ended.

18. A method of preparing nucleic acids for analysis, the method comprising:

(a) preparing a cDNA by conducting a randomly-primed first strand synthesis reaction using a nucleic acid preparation comprising an RNA as a template and a second strand synthesis reaction using a product of the randomly-primed first strand synthesis reaction as a template, wherein the RNA comprises a target nucleotide sequence;

(b) end repairing the cDNA to produce a blunt-ended, double-stranded nucleic acid comprising the target nucleotide sequence;

(c) washing the blunt-ended, double-stranded nucleic acid;

(d) adding one or more nucleotides to the 3′

end of the nucleic acid washed in step (c), wherein at least one of the one or more nucleotides is a capture moiety modified nucleotide;

(e) washing the nucleic acid produced in step (d);

(f) ligating an adapter nucleic acid that comprises a ligatable duplex portion and an overhang sequence to the nucleic acid washed in step (e) to produce a ligation product, wherein the overhang sequence is complementary with the one or more nucleotides added in step (d);

(g) amplifying the ligation product by polymerase chain reaction using a first target-specific primer that specifically anneals to the target nucleotide sequence and a first adapter primer that specifically anneals to a complementary sequence of the adapter nucleic acid;

(h) amplifying an amplification product of step (g) by polymerase chain reaction using a second adapter primer and a second target-specific primer, wherein the second target-specific primer is nested relative to the first target-specific primer; and

(i) washing the amplification product of step (h).

19. The method of claim 18, wherein the method further comprises, following step (f) and before step (g), capturing the ligation product using an immobilized binding partner of the capture moiety modified nucleotide.

20. The method of claim 19, further comprising purifying the captured ligation product.

21. The method of claim 19, wherein the binding partner is streptavidin, optionally wherein the streptavidin is attached to a paramagnetic bead.

22. The method of claim 18, wherein the capture moiety modified nucleotide comprises a nucleobase selected from the group consisting of adenine, guanine, thymine, uracil, and cytosine, or a derivative thereof.

23. The method of claim 22, wherein the capture moiety modified nucleotide comprises an adenine nucleobase or derivative thereof, optionally wherein the capture moiety is covalently linked to the adenine nucleobase or derivative thereof at position 5, 6, 7 or 8, optionally wherein position 7 of the adenine nucleobase is a carbon atom.

24. The method of claim 18, wherein the capture moiety is a biotin moiety.

25. The method of claim 24, wherein the biotin moiety is covalently linked to the nucleotide via a linker of 5 to 20 atoms in length.

26. The method of claim 18, wherein the washing steps are performed using a solid-phase reversible immobilization technique.

27. The method of claim 18, wherein the second adapter primer is nested relative to the first adapter primer.