Scavenger compounds for improved sequencing-by-synthesis

• US 10,036,011 B2
• Filed: 02/08/2017
• Issued: 07/31/2018
• Est. Priority Date: 02/11/2016
• Status: Active Grant

• Current Assignee: IsoPlexis Corporation (Bruker Corporation)
• Original Assignee: Qiagen Waltham, Inc. (Qiagen NV)
• Inventors: Andruzzi, Luisa
• Primary Examiner(s): Pande, Suchira
• Application Number:
  US15/427,832
• Publication Number:
  US 20170233725A1
• Time in Patent Office: 538 Days
• Field of Search: None
• CPC Class Codes: C12N 15/10 Processes for the isolation, preparation or purification of DNA or RNA chemical preparation of DNA or RNA C07H21/00; preparation of non-structural polynucleotides from microorganisms or with enzymes C12P19/34
• CPC Class Codes: C12N 15/1068 Template (nucleic acid) mediated chemical library synthesis, e.g. chemical and enzymatical DNA-templated organic molecule synthesis, libraries prepared by non ribosomal polypeptide synthesis [NRPS], DNA/RNA-polymerase mediated polypeptide synthesis
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2527/125 Specific component of sample, medium or buffer
• CPC Class Codes: C12Q 2535/113 Cycle sequencing

Abstract

The present invention discloses methods of applications of indole-3-propionic acid, L-carnitine and O-acetyl-L-carnitine in one or more different reactive steps of a sequencing-by-synthesis workflow. The reactive steps employing these compounds include, but are not limited to, cleaving, imaging, incorporating bases and washing. The use of these new compounds provides improved sequencing performance including, but not limited to, lower error rates, higher sequence outputs and/or longer read lengths.

Claims

1. A method of incorporating labeled nucleotides, comprising:

a) providing i) a plurality of nucleic acid primers and template molecules, ii) a polymerase, iii) a cleave reagent comprising a scavenger selected from the group consisting of indole-3-propionic acid and a carnitine-based compound, and iv) a plurality of nucleotide analogues wherein at least a portion of said nucleotide analogues is labeled with a label attached through a cleavable disulfide linker to the base;

b) hybridizing at least a portion of said primers to at least a portion of said template molecules so as to create hybridized primers;

c) incorporating a first labeled nucleotide analogue with said polymerase into at least a portion of said hybridized primers so as to create extended primers comprising an incorporated nucleotide analogue; and

d) cleaving the cleavable linker of said incorporated nucleotide analogues with said cleave reagent.

2. The method of claim 1, wherein said scavenger is indole-3-propionic acid.

3. The method of claim 1, wherein said scavenger is L-carnitine.

4. The method of claim 1, wherein said scavenger is O-acetyl-L-carnitine.

5. The method of claim 1, wherein said incorporated nucleotide analogues of step c) further comprise a removable chemical moiety capping the 3′

-OH group.

6. The method of claim 5, wherein the cleaving of step d) removes the removable chemical moiety capping the 3′

-OH group.

7. The method of claim 6, wherein the method further comprises:

d) incorporating a second nucleotide analogue with said polymerase into at least a portion of said extended primers.

8. The method of claim 7, wherein said incorporating of a second nucleotide analogue is performed in the presence of a scavenger selected from the group consisting of indole-3-propionic acid and a carnitine-based compound.

9. A method of incorporating nucleotides, comprising:

a) providing i) a plurality of nucleic acid primers and template molecules, and ii) an extend reagent, said extend reagent comprising polymerase, a plurality of nucleotide analogues, and a scavenger selected from the group consisting of indole-3-propionic acid and a carnitine-based compound;

b) hybridizing at least a portion of said primers to at least a portion of said template molecules so as to create hybridized primers; and

c) exposing said hybridized primers to said extend reagent under conditions such that a first nucleotide analogue is incorporated into at least a portion of said hybridized primers so as to create extended primers comprising an incorporated nucleotide analogue.

10. The method of claim 9, wherein said incorporated nucleotide analogue comprises a label attached through a cleavable disulfide linker to the base.

11. The method of claim 10, wherein said label is fluorescent.

12. The method of claim 9, wherein said extend reagent further comprises cystamine.

13. The method of claim 10, further comprising:

d) cleaving the cleavable linker of said incorporated nucleotide analogue with a cleave reagent comprising a scavenger selected from the group consisting of indole-3-propionic acid and a carnitine-based compound.

14. The method of claim 9, wherein said scavenger is indole-3-propionic acid.

15. The method of claim 9, wherein said scavenger is L-carnitine.

16. The method of claim 9, wherein said scavenger is O-acetyl-L-carnitine.

17. The method of claim 13, wherein said incorporated nucleotide analogues prior to step d) further comprise a removable chemical moiety capping the 3′

-OH group.

18. The method of claim 17, wherein the cleaving of step d) removes the removable chemical moiety capping the 3′

-OH group.

19. A method of incorporating nucleotides, comprising:

a) providing i) a plurality of nucleic acid primers and template molecules, ii) an extend reagent comprising polymerase and a plurality of nucleotide analogues, and iii) a wash reagent comprising a scavenger selected from the group consisting of indole-3-propionic acid and a carnitine-based compound;

b) hybridizing at least a portion of said primers to at least a portion of said template molecules so as to create hybridized primers;

c) exposing said hybridized primers to said extend reagent under conditions such that a first nucleotide analogue is incorporated into at least a portion of said hybridized primers so as to create extended primers comprising an incorporated nucleotide analogue;

d) washing said extended primers with said wash reagent.

20. The method of claim 19, wherein said incorporated nucleotide analogue comprises a label attached through a cleavable disulfide linker to the base.

21. The method of claim 20, wherein said label is fluorescent.

22. The method of claim 20, wherein said extend reagent further comprises cystamine.

23. The method of claim 20, further comprising:

e) detecting said label of a first labeled nucleotide analogue.

24. The method of claim 23, wherein said detecting of step e) is performed in the presence of a scavenger selected from the group consisting of indole-3-propionic acid and a carnitine-based compound.

25. The method of claim 19, wherein said scavenger is indole-3-propionic acid.

26. The method of claim 19, wherein said scavenger is L-carnitine.

27. The method of claim 19, wherein said scavenger is O-acetyl-L-carnitine.

28. A method of incorporating labeled nucleotides, comprising:

a) providing i) a plurality of nucleic acid primers and template molecules, ii) an extend reagent comprising polymerase and a plurality of nucleotide analogues wherein at least a portion of said nucleotide analogues is labeled, and iii) an image reagent comprising a scavenger selected from the group consisting of indole-3-propionic acid and a carnitine-based compound;

b) hybridizing at least a portion of said primers to at least a portion of said template molecules so as to create hybridized primers;

c) exposing said hybridized primers to said extend reagent under conditions such that a first labeled nucleotide analogue is incorporated into at least a portion of said hybridized primers so as to create extended primers comprising an incorporated nucleotide analogue; and

d) detecting said label of said first labeled nucleotide analogue with said image reagent.

29. The method of claim 28, wherein said label is attached through a cleavable disulfide linker to the base.

30. The method of claim 29, wherein said label is fluorescent.

31. The method of claim 28, wherein said extend reagent further comprises cystamine.

32. The method of claim 29, further comprising:

e) cleaving the cleavable linker of said incorporated nucleotide analogue with a cleave reagent comprising a scavenger selected from the group consisting of indole-3-propionic acid and a carnitine-based compound.

33. The method of claim 28, wherein said scavenger is indole-3-propionic acid.

34. The method of claim 28, wherein said scavenger is L-carnitine.

35. The method of claim 28, wherein said scavenger is O-acetyl-L-carnitine.

36. The method of claim 32, wherein said incorporated labeled nucleotide analogue of step d) further comprises a removable chemical moiety capping the 3′

-OH group.

37. The method of claim 36, wherein the cleaving of step e) removes the removable chemical moiety capping the 3′

-OH group.