阅读说明：本技术 使用珠寡核苷酸的mda (MDA using bead oligonucleotides ) 是由 R·雷伯弗斯基 J·阿格雷丝迪 G·卡琳-纽曼 于 2018-06-19 设计创作，主要内容包括：提供了改善的多重置换扩增(MDA)试剂和方法。(Improved Multiple Displacement Amplification (MDA) reagents and methods are provided.)

1. A method of performing a multiple displacement amplification, the method comprising,

providing a plurality of oligonucleotides, each oligonucleotide comprising a 3 ' random sequence of at least four consecutive nucleotides, a barcode sequence, and optionally an intervening sequence, annealing the oligonucleotides to a complementary nucleic acid that is complementary to the barcode sequence, the intervening sequence, or both the barcode sequence and the intervening sequence, wherein the complementary nucleic acid is not complementary to the 3 ' random sequence, leaving the 3 ' random sequence as a single strand;

contacting a plurality of oligonucleotides with a sample DNA under conditions that anneal complementary nucleic acids to the oligonucleotides and allow annealing of 3' random sequences to the sample DNA; and

extending the 3 'random sequence in a template-dependent manner with a strand displacing polymerase to produce an extended oligonucleotide comprising a 3' sequence complementary to the DNA.

2. The method of claim 1, wherein the plurality comprises at least 25 different oligonucleotides having different random sequences.

3. The method of claim 1, wherein the oligonucleotide further comprises a 5' tag sequence.

4. The method of claim 3, wherein the tag sequence is 2-40 nucleotides in length.

5. The method of claim 1, wherein the oligonucleotide lacks an intervening sequence and the complementary nucleic acid is complementary to a barcode sequence or a contiguous portion of at least 6 nucleotides thereof.

6. The method of claim 1, wherein the barcode sequence is discontinuous and an intervening sequence is between two or more portions of the barcode sequence.

7. The method of claim 1 or 6, wherein the complementary nucleic acid does not comprise a sequence complementary to a barcode sequence.

8. The method of claim 1, wherein the oligonucleotide is covalently linked to separate copies of a complementary nucleic acid such that the oligonucleotide forms a polynucleotide hairpin.

9. The method of claim 1, wherein the complementary nucleic acid is not covalently linked to an oligonucleotide.

10. The method of claim 9, wherein the 5' tag sequence is covalently attached to a solid support bead.

11. The method of any one of claims 1-10, wherein the method is performed in a partition.

12. The method of claim 11, wherein said partitions comprise on average 1-3 solid support beads.

13. The method of claim 12 or 13, wherein the partition is a droplet in an emulsion.

14. The method of any one of claims 11-13, further comprising combining the contents of the partitions into a bulk reaction mixture after the extending.

15. The method of any one of claims 1-14, wherein the complementary nucleic acid comprises one or more nucleotides that are incompatible with a strand displacing polymerase.

16. The method of claim 15, wherein one or more nucleotides are one or more uracils.

17. The method of claim 15, wherein the one or more nucleotides are biotinylated.

18. The method of claim 17, wherein the biotinylated nucleotide is bound to streptavidin.

19. The method of any one of claims 1-15, wherein the strand displacing polymerase is a Φ 29 polymerase.

20. The method of any one of claims 1-19, wherein the random sequence is 4-10 nucleotides in length.

21. The method of any one of claims 1-19, wherein the barcode sequence is 8-50 nucleotides in length.

22. The method of any one of claims 1-19, wherein the intervening sequence is 6-40 nucleotides in length.

23. The method of any one of claims 1-22, wherein the method comprises providing sample DNA encapsulated in hydrogel beads, positioning the hydrogel beads in a droplet comprising at least one of the oligonucleotides, and releasing the sample DNA from the hydrogel, thereby contacting the oligonucleotides with the sample DNA.

24. The method of claim 23, wherein the sample DNA in the hydrogel consists of DNA from one or more cells.

25. The method of claim 23 or 24, comprising:

encapsulating one or more cells in hydrogel beads,

lysing the one or more cells and optionally contacting the lysed cells with one or more proteases; and is

The cell lysate that diffused out of the hydrogel beads was separated from the hydrogel beads.

26. The method of any one of claims 23-25, comprising denaturing the sample DNA encapsulated by the hydrogel beads.

27. The method of claim 26, further comprising hybridizing the denatured DNA with random oligonucleotides to maintain the denatured DNA.

28. The method of any one of claims 23 to 28, comprising encapsulating the hydrogel beads in separate aqueous compartments, bringing the hydrogel beads into solution within the compartments, and then extending in the aqueous compartments.

29. The method of claim 28, wherein the partitions are droplets.

30. A method of producing a partially double-stranded oligonucleotide, the method comprising,

providing a solid support bead covalently attached to the 5' end of an oligonucleotide comprising, in the following order: a 3' random sequence of at least four consecutive nucleotides, a consensus universal sequence, and a barcode sequence;

annealing the oligonucleotide primers to the consensus universal sequence; and

the annealed oligonucleotide primers are extended with a polymerase in a template-dependent manner to generate a second strand nucleic acid complementary to the consensus universal sequence and the barcode sequence, thereby generating a partially double-stranded oligonucleotide, one of which is covalently attached to a solid support bead and has a single-stranded 3' random sequence.

31. The method of claim 30, wherein the oligonucleotide further comprises an intervening sequence.

32. The method of claim 31, wherein the barcode sequence is discontinuous and an intervening sequence is between two or more portions of the barcode sequence.

33. The method of claim 30, wherein the oligonucleotide further comprises a 5' tag sequence.

34. The method of claim 30, wherein the extending is performed in the presence of dUTP such that uracil is incorporated into the second strand nucleic acid.

35. The method of claim 34, wherein dUTP is biotinylated such that uracil incorporated into the second strand nucleic acid is biotinylated.

36. The method of claim 35, further comprising contacting the second strand nucleic acid with streptavidin.

37. The method of any one of claims 30-36, wherein the random sequence is 4-10 nucleotides in length.

38. The method of any one of claims 30-36, wherein the barcode sequence is 8-50 nucleotides in length.

39. The method of any one of claims 30-36, wherein the intervening sequence is 6-40 nucleotides in length.

40. A plurality of oligonucleotides having different sequences, each oligonucleotide covalently linked to a separate solid support bead, each oligonucleotide comprising a 3 'random sequence of at least four consecutive nucleotides, a barcode sequence and optionally an intervening sequence, wherein the oligonucleotides differ by having different 3' random sequences, each oligonucleotide annealing to a complementary nucleic acid that is complementary to the barcode sequence, the intervening sequence or both, wherein the complementary nucleic acid is not complementary to the 3 'random sequence, leaving the 3' random sequence single-stranded.

41. The method of claim 1, wherein the plurality comprises at least 25 different oligonucleotides having different random sequences.

42. The plurality of oligonucleotides of claim 40, wherein the oligonucleotides further comprise a 5' tag sequence.

43. The plurality of oligonucleotides of claim 42, wherein the tag sequence is 2-40 nucleotides in length.

44. The plurality of oligonucleotides of claim 40, wherein the oligonucleotides lack an intervening sequence and the complementary nucleic acid is complementary to a barcode sequence or a contiguous portion of at least 6 nucleotides thereof.

45. The plurality of oligonucleotides of claim 40, wherein the barcode sequence is unique for each solid support bead.

46. The plurality of oligonucleotides of claim 40, wherein the barcode sequence is discontinuous and the intervening sequence is between two or more portions of the barcode sequence.

47. The plurality of oligonucleotides of any one of claims 40-46, wherein the complementary nucleic acid does not comprise a sequence complementary to a barcode sequence.

48. The plurality of oligonucleotides of claim 40, wherein the oligonucleotides are covalently linked to separate copies of a complementary nucleic acid such that the oligonucleotides form a polynucleotide hairpin.

49. The plurality of oligonucleotides of claim 40, wherein the complementary nucleic acid is not covalently linked to the oligonucleotide.

50. The plurality of oligonucleotides of any one of claims 40-49, wherein the complementary nucleic acid comprises one or more nucleotides that are incompatible with a strand displacing polymerase.

51. The plurality of oligonucleotides of claim 50, wherein the one or more nucleotides is uracil.

52. The plurality of oligonucleotides of claim 50 or 51, wherein the one or more nucleotides are biotinylated and bound to streptavidin.

53. The plurality of oligonucleotides of any one of claims 40-52, wherein the random sequence is 4-10 nucleotides in length.

54. The plurality of oligonucleotides of any one of claims 40-52, wherein the barcode sequence is 8-50 nucleotides in length.

55. The plurality of oligonucleotides of any one of claims 40-52, wherein the intervening sequence is 6-40 nucleotides in length.

56. The plurality of oligonucleotides of any one of claims 40-52, wherein the random sequence is 4-10 nucleotides in length.

Background

Multiple Displacement Amplification (MDA) is a non-PCR based DNA amplification technique that involves the use of random oligonucleotides that are primed at random positions on a DNA sample. In many cases, the random oligonucleotide is a random hexamer primer that anneals to DNA. The primer is then extended with a polymerase, e.g., a strand displacing polymerase such as Φ 29DNA polymerase, at a constant temperature. The resulting extension products can then be sequenced and aligned to generate DNA sequences. Examples of single cell whole genome MDA are described, for example, by Spits et al, Nature Protocols 1, 1965-.

Summary of The Invention

In some aspects, a method of performing multiple displacement amplification is provided. In some embodiments, the method comprises,

providing a plurality of oligonucleotides, each oligonucleotide comprising a 3 ' random sequence of at least four consecutive nucleotides, a barcode sequence, and optionally an intervening sequence, annealing the oligonucleotides to a complementary nucleic acid that is complementary to the barcode sequence, the intervening sequence, or both the barcode sequence and the intervening sequence, wherein the complementary nucleic acid is not complementary to the 3 ' random sequence, leaving the 3 ' random sequence as a single strand;

contacting a plurality of oligonucleotides with a sample DNA under conditions that anneal complementary nucleic acids to the oligonucleotides and allow annealing of 3' random sequences to the sample DNA; and

the 3 'random sequence is extended in a template-dependent manner with a strand displacing polymerase to produce an extended oligonucleotide comprising a 3' sequence complementary to the DNA.

In some embodiments, the plurality comprises at least 25 different oligonucleotides having different random sequences.

In some embodiments, the oligonucleotide further comprises a 5' tag sequence. In some embodiments, the tag sequence is 2-40 nucleotides in length.

In some embodiments, the oligonucleotide lacks an intervening sequence, and the complementary nucleic acid is complementary to the barcode sequence or a contiguous portion of at least 6 nucleotides thereof. In some embodiments, the barcode sequence is discontinuous and the intervening sequence is between two or more portions of the barcode sequence.

In some embodiments, the complementary nucleic acid does not comprise a sequence complementary to a barcode sequence.

In some embodiments, the oligonucleotide is covalently linked to separate copies of a complementary nucleic acid such that the oligonucleotide forms a polynucleotide hairpin.

In some embodiments, the complementary nucleic acid is not covalently linked to the oligonucleotide. In some embodiments, the 5' tag sequence is covalently attached to a solid support bead.

In some embodiments, the method is performed in partition(s). In some embodiments, the partitions comprise on average 1-3 solid support beads. In some embodiments, the partition is a droplet within the emulsion.

In some embodiments, the method further comprises combining the contents of the partitions into a bulk (bulk) reaction mixture after the extending.

In some embodiments, the complementary nucleic acid comprises one or more nucleotides that are incompatible with a strand displacing polymerase. In some embodiments, the one or more nucleotides are one or more uracils. In some embodiments, the one or more nucleotides are biotinylated. In some embodiments, the biotinylated nucleotide is bound to streptavidin.

In some embodiments, the strand displacing polymerase is a Φ 29 polymerase.

In some embodiments, the random sequence is 4-10 nucleotides in length.

In some embodiments, the barcode sequence is 8-50 nucleotides in length.

In some embodiments, the intervening sequence is 6-40 nucleotides in length.

In some embodiments, the method comprises providing sample DNA encapsulated in hydrogel beads, positioning the hydrogel beads in a droplet comprising at least one of the oligonucleotides, and releasing the sample DNA from the hydrogel, thereby contacting the oligonucleotides with the sample DNA. In some embodiments, the sample DNA in the hydrogel consists of DNA from one or more cells. In some embodiments, the method comprises: encapsulating the one or more cells in hydrogel beads, lysing the one or more cells, and optionally contacting the lysed cells with one or more proteases; and separating the cell lysate that diffuses out of the hydrogel beads from the hydrogel beads.

In some embodiments, the method comprises denaturing the sample DNA encapsulated by the hydrogel beads. In some embodiments, the method further comprises hybridizing the denatured DNA with random oligonucleotides to maintain the denatured DNA.

In some embodiments, the method comprises encapsulating hydrogel beads in separate aqueous partitions, bringing the hydrogel beads into solution within the partitions, and then extending in the aqueous partitions. In some embodiments, the partitions are droplets.

In some aspects, methods of producing partially double-stranded oligonucleotides are provided. In some embodiments, the method comprises,

providing a solid support bead covalently attached to the 5' end of an oligonucleotide comprising, in the following order: a 3' random sequence of at least four consecutive nucleotides, a consensus universal sequence, and a barcode sequence;

annealing the oligonucleotide primers to the consensus universal sequence; and

the annealed oligonucleotide primers are extended with a polymerase in a template-dependent manner to generate a second strand nucleic acid complementary to the consensus universal sequence and the barcode sequence, thereby generating a partially double-stranded oligonucleotide, one of which is covalently attached to a solid support bead and has a single-stranded 3' random sequence.

In some embodiments, the oligonucleotide further comprises an intervening sequence. In some embodiments, the barcode sequence is discontinuous and the intervening sequence is between two or more portions of the barcode sequence.

In some embodiments, the oligonucleotide further comprises a 5' tag sequence.

In some embodiments, the extension is performed in the presence of dUTP such that uracil is incorporated into the second strand nucleic acid. In some embodiments, dUTP is biotinylated such that uracil incorporated into the second strand nucleic acid is biotinylated. In some embodiments, the method further comprises contacting the second strand nucleic acid with streptavidin.

In some embodiments, the random sequence is 4-10 nucleotides in length. In some embodiments, the barcode sequence is 8-50 nucleotides in length. In some embodiments, the intervening sequence is 6-40 nucleotides in length.

In some aspects, a plurality of oligonucleotides having different sequences is provided. In some embodiments, each oligonucleotide is covalently linked to a separate solid support bead, each oligonucleotide comprising a 3 'random sequence of at least four consecutive nucleotides, a barcode sequence, and optionally an intervening sequence, wherein the oligonucleotides differ by having different 3' random sequences, each oligonucleotide anneals to a complementary nucleic acid that is complementary to the barcode sequence, the intervening sequence, or both, wherein the complementary nucleic acid is not complementary to the 3 'random sequence, leaving the 3' random sequence single-stranded.

In some embodiments, the plurality comprises at least 25 different oligonucleotides having different random sequences.

In some embodiments, the oligonucleotide further comprises a 5' tag sequence.

In some embodiments, the tag sequence is 2-40 nucleotides in length.

In some embodiments, the oligonucleotide lacks an intervening sequence, and the complementary nucleic acid is complementary to the barcode sequence or a contiguous portion of at least 6 nucleotides thereof.

In some embodiments, the barcode sequence is unique to each solid support bead.

In some embodiments, the barcode sequence is discontinuous and the intervening sequence is between two or more portions of the barcode sequence.

In some embodiments, wherein the complementary nucleic acid does not comprise a sequence complementary to a barcode sequence.

In some embodiments, the oligonucleotide is covalently linked to separate copies of a complementary nucleic acid such that the oligonucleotide forms a polynucleotide hairpin.

In some embodiments, the complementary nucleic acid is not covalently linked to the oligonucleotide.

In some embodiments, the complementary nucleic acid comprises one or more nucleotides that are incompatible with a strand displacing polymerase.

In some embodiments, the one or more nucleotides are uracils. In some embodiments, the one or more nucleotides are biotinylated and bound to streptavidin.

In some embodiments, the random sequence is 4-10 nucleotides in length. In some embodiments, the barcode sequence is 8-50 nucleotides in length. In some embodiments, the intervening sequence is 6-40 nucleotides in length. In some embodiments, wherein the random sequence is 4-10 nucleotides in length.