阅读说明：本技术 用于核酸操纵的装置和方法 (Apparatus and method for nucleic acid manipulation ) 是由 J·雅各布森 D·力克 L-Y·A·昆 于 2018-05-22 设计创作，主要内容包括：提供了用于选择性排放和/或转移核酸分子的装置和方法。在一方面中,装置包括设置成与固体支持物上一个或多个特征部对齐的组件(例如,压电或声学组件),从而在使用时,所述组件(例如,压电或声学组件)产生机械力,以从固体支持物选择性排放和/或转移一份量或多份量的核酸。固体支持物可以包括多个离散的特征部,各特征部上具有按份量的核酸(例如,液滴)。可以包括电源以向组件(例如,压电或声学组件,如果存在的话)提供电流以产生机械力。该装置可以用于组装期间和/或之后的核酸单一化。(Devices and methods for selectively discharging and/or transferring nucleic acid molecules are provided. In one aspect, the device comprises a component (e.g., a piezoelectric or acoustic component) that is disposed in alignment with one or more features on the solid support, such that, in use, the component (e.g., the piezoelectric or acoustic component) generates a mechanical force to selectively discharge and/or transfer one or more quantities of nucleic acid from the solid support. The solid support can include a plurality of discrete features, each feature having a portion of nucleic acid (e.g., a droplet) thereon. A power source may be included to provide electrical current to a component (e.g., a piezoelectric or acoustic component, if present) to generate mechanical force. The device may be used for nucleic acid singulation during and/or after assembly.)

1. A device for selectively discharging nucleic acids, the device comprising:

a) a piezoelectric assembly disposed in alignment with one or more features on a solid support, such that, in use, the piezoelectric assembly generates a mechanical force to selectively discharge one or more quantities of nucleic acid from the solid support, wherein the solid support comprises a plurality of discrete features, each feature associated with a quantity of nucleic acid; and

b) a power source for providing an electrical current to the piezoelectric assembly to generate the mechanical force.

2. A device for selectively discharging nucleic acids, the device comprising:

a) a solid support comprising a plurality of discrete features, each feature associated with a portion of a nucleic acid;

b) a piezoelectric assembly configured to selectively discharge one or more quantities of nucleic acids from the solid support; and

c) a power source for providing an electrical current to the piezoelectric assembly to generate a mechanical force to discharge one or more quantities of nucleic acid.

3. The device of any one of claims 1 or 2, wherein each quantity of nucleic acid comprises one or more oligonucleotides.

4. The device of claim 3, wherein the one or more oligonucleotides are in a dry environment or a liquid environment.

5. The method of claim 3, wherein each quantity of nucleic acid is a droplet of solution.

6. The device of any one of claims 1 or 2, wherein each feature has a plurality of oligonucleotides immobilized thereon.

7. The device of claim 1 or 2, wherein the solid support is a multiwell plate or microarray comprising a plurality of beads.

8. The apparatus of claim 1 or 2, wherein the piezoelectric assembly comprises a matrix of piezoelectric elements, each piezoelectric element being arranged to correspond to a feature.

9. The device of claim 3, wherein the one or more oligonucleotides are released into the quantity of nucleic acid via chemical, enzymatic, and/or laser cleavage.

10. The device of claim 9, further comprising a laser for selectively releasing the one or more oligonucleotides into the quantity of nucleic acid by cleaving the photoactivatable linker.

11. The apparatus of claim 1 or 2, wherein the piezoelectric assembly comprises a single piezoelectric element.

12. The apparatus of claim 11, wherein the single piezoelectric element is a needle.

13. The device of claim 12, further comprising a transport assembly configured to move the needle to a desired feature.

14. A method of nucleic acid assembly, comprising:

a) providing a first solid support comprising a plurality of discrete features, each feature associated with a portion of an acid;

b) selectively discharging one or more quantities of a nucleic acid from a first feature to a second feature using a piezoelectric assembly, wherein the first feature comprises a first oligonucleotide having sequence complementarity with or overlapping a second oligonucleotide in the second feature; and

c) assembling the first and second oligonucleotides.

15. The method of claim 14, wherein the piezoelectric assembly comprises a matrix of piezoelectric elements, each piezoelectric element being disposed to correspond to a feature.

16. The method of claim 14, wherein each quantity of nucleic acid comprises one or more oligonucleotides.

17. The method of claim 16, wherein the one or more oligonucleotides are in a dry environment or a liquid environment.

18. The method of claim 16, further comprising releasing the one or more oligonucleotides into the quantity of nucleic acid via chemical, enzymatic, and/or laser cleavage.

19. The method of claim 14, wherein the solid support is a multiwell plate or microarray comprising a plurality of beads.

20. The method of claim 14, wherein each feature has a plurality of oligonucleotides immobilized thereon.

21. The method of claim 14, wherein the first feature and the second feature are located on the same solid support.

22. The method of claim 14, wherein the first feature is located on the first solid support and the second feature is located on a second solid support.

23. A device for selectively discharging nucleic acids, the device comprising:

a) an assembly disposed in alignment with one or more features on a solid support, such that, in use, the assembly generates a mechanical force to selectively discharge one or more quantities of nucleic acid from the solid support, wherein the solid support comprises a plurality of discrete features, each feature associated with a quantity of nucleic acid; and

b) a power source for providing an electrical current to the piezoelectric assembly to generate the mechanical force.

24. A device for selectively discharging nucleic acids, the device comprising:

a) a solid support comprising a plurality of discrete features, each feature associated with a portion of nucleic acid;

b) an assembly configured to selectively discharge one or more quantities of nucleic acid from the solid support; and

c) a power source for providing an electrical current to the assembly to generate a mechanical force to discharge the one or more quantities of nucleic acid.

25. The apparatus of claim 23 or 24, wherein the component is an acoustic component or a piezoelectric component.

26. The device of any one of claims 23 or 24, wherein each quantity of nucleic acid comprises one or more oligonucleotides.

27. The device of claim 26, wherein the one or more oligonucleotides are in a dry environment or a liquid environment.

28. The method of claim 26, wherein each quantity of nucleic acid is a droplet of solution.

29. The device of any one of claims 23 or 24, wherein each feature has a plurality of oligonucleotides immobilized thereon.

30. The device of claim 23 or 24, wherein the solid support is a multiwell plate or microarray comprising a plurality of beads.

31. Apparatus according to claim 23 or 24, wherein the assembly comprises a matrix of elements, each element being arranged to correspond to a feature.

32. The device of claim 26, wherein the one or more oligonucleotides are released into the quantity of nucleic acid via chemical, enzymatic, and/or laser cleavage.

33. The apparatus of claim 32, further comprising a laser for selectively releasing the one or more oligonucleotides into the quantity of nucleic acid by cleaving the photoactivatable linker.

34. The device of claim 23 or 24, wherein the assembly comprises a unitary element.

35. The device of claim 34, wherein the unitary element is a needle.

36. The device of claim 35, further comprising a transport assembly configured to move the needle to a desired feature.

37. A method of nucleic acid assembly, comprising:

a) providing a first solid support comprising a plurality of discrete features, each feature associated with a serving amount of nucleic acid;

b) selectively discharging one or more quantities of nucleic acid from a first feature to a second feature using a component, wherein the first feature comprises a first oligonucleotide having sequence complementarity with or overlapping a second oligonucleotide in the second feature; and

c) assembling the first and second oligonucleotides.

38. The apparatus of claim 37, wherein the component is an acoustic component or a piezoelectric component.

39. The method of claim 37, wherein the assembly comprises a matrix of elements, each element being arranged to correspond to a feature.

40. The method of claim 37, wherein each quantity of nucleic acid comprises one or more oligonucleotides.

41. The method of claim 40, wherein the one or more oligonucleotides are in a dry environment or a liquid environment.

42. The method of claim 40, further comprising releasing the one or more oligonucleotides into the quantity of nucleic acid via chemical, enzymatic, and/or laser cleavage.

43. The method of claim 37, wherein the solid support is a multiwell plate or microarray comprising a plurality of beads.

44. The method of claim 37, wherein each feature has a plurality of oligonucleotides immobilized thereon.

45. The method of claim 37, wherein the first feature and the second feature are located on the same solid support.

46. The method of claim 37, wherein the first feature is located on the first solid support and the second feature is located on a second solid support.

47. A method of nucleic acid assembly, comprising:

a) providing a first solid support comprising a plurality of discrete features, each feature associated with a serving amount of nucleic acid;

b) selectively transferring one or more quantities of nucleic acid from a first feature to a second feature, wherein the first feature comprises a first oligonucleotide having sequence complementarity with or overlapping a second oligonucleotide in the second feature; and

c) assembling the first and second oligonucleotides.

48. The method of claim 47, wherein each quantity of nucleic acid comprises one or more oligonucleotides.

49. The method of claim 48, wherein the one or more oligonucleotides are in a dry environment or a liquid environment.

50. The method of claim 48, further comprising releasing the one or more oligonucleotides into the quantity of nucleic acid via chemical, enzymatic, and/or laser cleavage.

51. The method of claim 47, wherein the solid support is a multiwell plate or microarray comprising a plurality of beads.

52. The method of claim 47, wherein each feature has a plurality of oligonucleotides immobilized thereon.

53. The method of claim 47, wherein the first feature and the second feature are located on the same solid support.

54. The method of claim 47, wherein the first feature is located on the first solid support and the second feature is located on a second solid support.

Technical Field

The devices and methods disclosed herein relate to nucleic acid manipulation, particularly during multiplex nucleic acid assembly.

Background

Recombinant and synthetic nucleic acids have many applications in research, industry, agriculture, and medicine. Recombinant and synthetic nucleic acids can be used to express and obtain a wide variety of polypeptides, including enzymes, antibodies, growth factors, receptors, and other polypeptides useful for a variety of medical, industrial, or agricultural purposes. Recombinant and synthetic nucleic acids can also be used to produce genetically modified organisms, including modified bacteria, yeast, mammalian, plant, and other organisms. Genetically modified organisms can be used in research (e.g., animal models of disease, tools to understand biological processes, etc.), industry (e.g., as host organisms for protein expression, bioreactors for producing industrial products, tools for environmental remediation, isolating or modifying natural compounds with industrial applications, etc.), agriculture (e.g., modified crops with increased yield or increased resistance to disease or environmental stress, etc.), and in other applications. Recombinant and synthetic nucleic acids can also be used as therapeutic compositions (e.g., for modifying gene expression, for gene therapy, etc.) or as diagnostic tools (e.g., probes for disorders, etc.).

In fact, nucleic acid synthesis is an important area of synthetic biology. "synthetic biology" is "the design and large-scale construction of new biological components and systems, and the redesign of existing natural biological systems for personalization purposes, combining engineering and computer-aided design approaches with biological research" as described in the U.S. department of energy (doe) in its report filed 2013 in 7 months to congress. DNA synthesis and assembly have been identified in DOE reports as a fundamental challenge to the continuing development of synthetic biology. In particular, one of the main limitations of "synthetic biology experiments is the synthesis and assembly of large DNA constructs, which are still expensive, slow and error prone". Engineering new biological manufacturing systems would require new methods for rapid, inexpensive, and accurate synthesis and assembly of genetic designs.

Various techniques have been developed for modifying existing nucleic acids (e.g., naturally occurring nucleic acids) to produce recombinant nucleic acids. For example, many different combinations of nucleic acid amplification, mutagenesis, nuclease digestion, ligation, cloning, and other techniques can be used to produce many different recombinant nucleic acids. Chemically synthesized polynucleotides are often used as primers or adapters for nucleic acid amplification, mutagenesis, and cloning.

Techniques for de novo nucleic acid synthesis on solid supports are also under development. For example, single stranded oligonucleotides of predetermined nucleic acid sequences can be synthesized in situ on a common support, where each predetermined nucleic acid sequence is synthesized on separate or discrete features (spots) on the support.

There are also techniques available for de novo nucleic acid assembly, in which nucleic acids are prepared (e.g., chemically synthesized on a support) and assembled to produce longer target nucleic acids of interest. For example, different multiplex assembly techniques have been developed for assembling oligonucleotides into larger synthetic nucleic acids that can be used in research, industry, agriculture, and/or medicine.

However, despite current advances, one limitation of currently available support-based synthesis and assembly techniques is the ability to identify and select one or more targets of interest. Therefore, high precision, high selectivity nucleic acid singulation (singularization) and assembly techniques are needed.

Disclosure of Invention

In one aspect, there is provided a device for selectively discharging and/or transferring nucleic acids, the device comprising a piezoelectric assembly disposed in alignment with one or more features on a solid support, such that, in use, the piezoelectric assembly generates a mechanical force to selectively discharge and/or transfer one or more quantities of nucleic acids from the solid support. The solid support includes a plurality of discrete features, each feature having or associated with a serving of nucleic acid. The power supply provides an electrical current to the piezoelectric assembly to generate a mechanical force.

In one aspect, there is provided a device for selectively discharging nucleic acids, the device comprising: a) a piezoelectric assembly disposed in alignment with one or more features on the solid support, such that, in use, the piezoelectric assembly generates a mechanical force to selectively discharge one or more aliquots of nucleic acid from the solid support, wherein the solid support comprises a plurality of discrete features, each feature having an aliquot of nucleic acid thereon or associated with an aliquot of nucleic acid; and b) a power source for providing an electrical current to the piezoelectric assembly to generate a mechanical force.

In another aspect, there is provided a device for selectively discharging nucleic acids, the device comprising: (a) a solid support comprising a plurality of discrete features, each feature having or being associated with a serving of nucleic acid; (b) a piezoelectric assembly configured to selectively discharge one or more quantities of nucleic acid from the solid support; and (c) a power source for providing an electrical current to the piezoelectric assembly to generate a mechanical force to discharge the one or more quantities of nucleic acid.

In various embodiments of various devices of the present disclosure, the volume of nucleic acid selectively discharged by the device can comprise one or more oligonucleotides. In various embodiments, the volume of nucleic acid selectively discharged by the device may comprise one or more oligonucleotides. The one or more oligonucleotides can be in a dry environment (e.g., associated with a solid bead) or a liquid environment (e.g., in an aqueous solution). The one or more oligonucleotides may be initially immobilized (covalently or non-covalently) to the one or more features and may be released into the quantity of nucleic acid via chemical, enzymatic and/or laser cleavage. In one embodiment, a laser may be used to selectively release one or more oligonucleotides into the portion of nucleic acid by cleaving the photoactivatable linker.

In some embodiments, the solid support of the device can have a plurality of oligonucleotides immobilized thereon. For example, each oligonucleotide having a different sequence can be located on a discrete, addressable feature. In some embodiments, each feature can comprise a plurality of oligonucleotides immobilized thereon. In some embodiments, the solid support can be a multiwell plate or microarray comprising a plurality of beads.

In some embodiments, the piezoelectric assembly includes a matrix of piezoelectric elements, where each piezoelectric element may be disposed to correspond to a feature.

In some embodiments, the piezoelectric assembly comprises a single piezoelectric assembly. In some embodiments, the single piezoelectric element may be a needle. The device may further include a transport assembly configured to move the needle to the desired feature.

In another aspect, a method for nucleic acid assembly is provided, the method comprising: (a) providing a first solid support comprising a plurality of discrete features, each feature having or being associated with a serving of nucleic acid; (b) selectively discharging (and/or moving) one or more quantities of nucleic acid from a first feature to a second feature using a piezoelectric assembly; wherein the first feature comprises a first oligonucleotide having a sequence that is complementary to or overlaps with a second oligonucleotide in the second feature; and (c) assembling the first and second oligonucleotides.

In some embodiments, the piezoelectric assembly includes a matrix of piezoelectric elements, where each piezoelectric element may be disposed to correspond to a feature. In some embodiments, the piezoelectric assembly includes a matrix of piezoelectric elements, wherein each piezoelectric element is disposed to correspond to a feature. In some embodiments, each quantity of nucleic acid comprises one or more oligonucleotides. In some embodiments, each quantity of nucleic acid may comprise one or more oligonucleotides. The one or more oligonucleotides can be in a dry environment (e.g., associated with a solid bead) or a liquid environment (e.g., in an aqueous solution). In some embodiments, each feature can comprise a plurality of oligonucleotides immobilized thereon. The one or more oligonucleotides may be released into the quantity of nucleic acid via chemical, enzymatic and/or laser cleavage. In some embodiments, the first feature and the second feature may be located on the same solid support. In certain embodiments, the first feature may be located on a first solid support and the second feature may be located on a second solid support.

In other aspects, there is provided a device for selectively discharging nucleic acids, the device comprising: a) an assembly disposed in alignment with one or more features on a solid support, such that, in use, the assembly generates a mechanical force to selectively discharge one or more quantities of nucleic acid from the solid support, wherein the solid support comprises a plurality of discrete features, each feature associated with a quantity of nucleic acid; and b) a power source for providing an electrical current to the assembly to generate a mechanical force.

In other aspects, there is provided a device for selectively discharging nucleic acids, the device comprising: (a) a solid support comprising a plurality of discrete features, each feature associated with a quantity of nucleic acid; (b) a module configured to selectively discharge one or more quantities of nucleic acid from the solid support; and (c) a power source for providing an electrical current to the assembly to generate a mechanical force to discharge the one or more quantities of nucleic acid. In some embodiments, the assembly is configured to interact with one or more features and effect transfer of one or more quantities of nucleic acid by mechanical displacement. In some embodiments, the component is an acoustic component or a piezoelectric component.

In some embodiments, each quantity of nucleic acid comprises one or more oligonucleotides. In certain embodiments, one or more oligonucleotides are in a dry environment or a liquid environment. In some embodiments, each quantity of nucleic acid is a droplet of solution.

In some embodiments, each feature has a plurality of oligonucleotides immobilized thereon. In certain embodiments, the solid support is a multiwell plate or microarray comprising a plurality of beads. In some embodiments, the assembly includes a matrix of press elements, wherein each element is arranged to correspond to a feature.

In some embodiments, the one or more oligonucleotides are released into the volume of nucleic acid via chemical, enzymatic, and/or laser cleavage.

In some embodiments, the device comprises a laser for selectively releasing one or more oligonucleotides into the quantity of nucleic acid by cleaving the photoactivatable linker. In some embodiments, the assembly comprises a single element. In some embodiments, the unitary element may be a needle.

In some embodiments, the device includes a transport assembly configured to move the needle to the desired feature.

In one aspect, a method of nucleic acid assembly is provided, the method comprising: (a) providing a first solid support comprising a plurality of discrete features, each feature associated with a portion of nucleic acid; (b) selectively discharging one or more quantities of nucleic acid from a first feature to a second feature using a component, wherein the first feature comprises a first oligonucleotide having a sequence that is complementary to or overlaps a second oligonucleotide in the second feature; and (c) assembling the first and second oligonucleotides. In some embodiments, the assembly is configured to interact with one or more features and effect transfer of one or more quantities of nucleic acid by mechanical displacement. In some embodiments, the component is an acoustic component or a piezoelectric component. In some embodiments, the assembly includes a matrix of press elements, wherein each element is arranged to correspond to a feature.

In certain embodiments, each quantity of nucleic acid comprises one or more oligonucleotides. In some embodiments, one or more oligonucleotides are in a dry environment or a liquid environment.

In a specific embodiment, the method comprises releasing one or more oligonucleotides into the volume of nucleic acid via chemical, enzymatic and/or laser cleavage. In some embodiments, the solid support is a multiwell plate or microarray comprising a plurality of beads. In certain embodiments, each feature has a plurality of oligonucleotides immobilized thereon.

In some embodiments, the first feature and the second feature are located on the same solid support. In certain embodiments, the first feature is located on a first solid support and the second feature is located on a second solid support.

In another aspect, a method of nucleic acid assembly comprises: a) providing a first solid support comprising a plurality of discrete features, each feature associated with a portion of nucleic acid; b) selectively transferring one or more quantities of nucleic acid from a first feature to a second feature, wherein the first feature comprises a first oligonucleotide having a sequence that is complementary to or overlaps a second oligonucleotide in the second feature; and c) assembling the first and second oligonucleotides.

In some embodiments, each quantity of nucleic acid comprises one or more oligonucleotides. In some embodiments, one or more oligonucleotides are in a dry environment or a liquid environment. In certain embodiments, the method further comprises releasing the one or more oligonucleotides to the portion of nucleic acid via chemical, enzymatic, and/or laser cleavage.

In some embodiments, the solid support is a multiwell plate or microarray comprising a plurality of beads. In some embodiments, each feature has a plurality of oligonucleotides immobilized thereon. In certain embodiments, the first feature and the second feature are located on the same solid support. In particular embodiments, the first feature is located on a first solid support and the second feature is located on a second solid support.