Scar-less multi-part DNA assembly design automation

• US 9,361,427 B2
• Filed: 02/01/2012
• Issued: 06/07/2016
• Est. Priority Date: 02/01/2011
• Status: Active Grant

• Current Assignee: Regents of the University of California (University of California)
• Original Assignee: Regents of the University of California (University of California)
• Inventors: Hillson, Nathan J.
• Primary Examiner(s): NEGIN, RUSSELL SCOTT
• Application Number:
  US13/364,285
• Publication Number:
  US 20120259607A1
• Time in Patent Office: 1,588 Days
• Field of Search: None
• US Class Current: 1/1
• CPC Class Codes: G16B 15/00 ICT specially adapted for analysing two-dimensional or three-dimensional molecular structures, e.g. structural or functional relations or structure alignment
• CPC Class Codes: G16B 20/00 ICT specially adapted for functional genomics or proteomics, e.g. genotype-phenotype associations
• CPC Class Codes: G16B 20/20 Allele or variant detection, e.g. single nucleotide polymorphism [SNP] detection
• CPC Class Codes: G16B 25/00 ICT specially adapted for hybridisation; ICT specially adapted for gene or protein expression
• CPC Class Codes: G16B 25/20 Polymerase chain reaction [PCR]; Primer or probe design; Probe optimisation
• CPC Class Codes: G16B 30/00 ICT specially adapted for sequence analysis involving nucleotides or amino acids
• CPC Class Codes: G16B 30/20 Sequence assembly

Abstract

The present invention provides a method of a method of designing an implementation of a DNA assembly. In an exemplary embodiment, the method includes (1) receiving a list of DNA sequence fragments to be assembled together and an order in which to assemble the DNA sequence fragments, (2) designing DNA oligonucleotides (oligos) for each of the DNA sequence fragments, and (3) creating a plan for adding flanking homology sequences to each of the DNA oligos. In an exemplary embodiment, the method includes (1) receiving a list of DNA sequence fragments to be assembled together and an order in which to assemble the DNA sequence fragments, (2) designing DNA oligonucleotides (oligos) for each of the DNA sequence fragments, and (3) creating a plan for adding optimized overhang sequences to each of the DNA oligos.

Claims

1. A method of designing the parts and protocol for assembling a DNA construct via a synthetic method utilizing flanking overlap sequences, said method comprising:

receiving a list of DNA sequence fragments comprising the parts to be assembled together and an order in which to assemble said DNA sequence fragments to form said construct;

determining with a bioCAD computer system flanking overlap sequences for said fragments, wherein said determining comprises consideration of a plurality of flanking overlap sequences, where said plurality comprises flanking overlap sequences having different lengths and nucleotide sequence to determine the melting temperatures (Tm) of each of said different flanking overlap sequences with the corresponding flanking overlap sequence on an assembly piece that will be a neighboring assembly piece in the DNA sequence fragments to be assembled, and selecting from said plurality particular flanking overlap sequences for each assembly piece, where said flanking overlap sequences each have a nucleotide sequence, melting temperature, and number of base pairs so that said assembly pieces will assemble to each other in the sequence order required to form said construct;

designing with said bioCAD computer system oligonucleotide primers for the PCR amplification of said fragments with flanking overlap sequences where determined;

creating a plan and protocol for the preparation of said fragments with flanking overlap sequences and/or the assembly of said fragments to form said construct; and

performing PCR and/or SOE utilizing said primers to generate the DNA sequence fragments comprising said parts with flanking overlap sequences.

2. The method of claim 1 wherein the receiving further comprises searching for at least one DNA sequence within a collection of physically existing DNA sequences in a registry of biological parts and/or a local collection of DNA sequences.

3. The method of claim 1 wherein said determining further comprises ensuring that the flanking overlap sequences of the DNA sequence fragments do not have stable single-stranded DNA secondary structure at a temperature at which said assembly pieces assemble to each other to form said construct.

4. The method of claim 1 wherein the receiving comprises receiving the list of DNA sequence fragments wherein at least one of the DNA sequence fragments has been physically instantiated.

5. The method of claim 1 wherein the receiving comprises receiving the list of DNA sequence fragments wherein at least one of the DNA sequence fragments has not been physically instantiated.

6. The method of claim 1 wherein the receiving comprises receiving at least one instruction to use at least one of the DNA sequence fragments at more than one place in the order.

7. The method of claim 1 wherein the creating comprises planning the flanking overlap sequences with respect to assembling a combinatorial library of constructs without scars.

8. The method of claim 7, further comprising assembling said DNA sequence fragments into a combinatorial construct library using an assembly method selected from the group consisting of SLIC, Gibson, CPEC, and SLiCE.

9. The method of claim 1 wherein the creating comprises creating the plan to add flanking DNA fragments containing the flanking overlap sequences via splicing by overlap extension (SOE) to each of said oligonucleotide primers after the primers have been incorporated at the ends of a product of a polymerase chain reaction.

10. The method of claim 1 further comprising determining an assembly strategy for each of the DNA sequence fragments that minimizes cost of fragment preparation.

11. The method of claim 10 wherein the designing comprises designing said oligonucleotide primers to provide the DNA sequence fragments at a minimum cost.

12. The method of claim 10 wherein the creating comprises creating a protocol that minimizes cost of construction and assembly of said construct.

13. The method of claim 1 further comprising checking the protocol against oligonucleotide mis-priming and against assembly piece incompatibility events.

14. The method of claim 13 wherein the checking further comprises providing a hierarchical assembly protocol, wherein the hierarchical assembly protocol is designed to mitigate the assembly piece incompatibility events.

15. The method of claim 13 further comprising outputting an experimental protocol of the DNA assembly based on the checked plan.

16. The method of claim 15 wherein the outputting comprises outputting the experimental protocol, wherein the protocol dictates the spatial arrangement of polymerase chain reactions (PCRs) to implement the DNA assembly on a multi-well thermo-cycler block with an annealing temperature gradient.

17. The method of claim 1 further comprising:

checking the plan; and

issuing a warning if at least one significant DNA sequence repeat exists in the plan.

18. The method of claim 1 further comprising outputting an experimental protocol for the DNA assembly based on the plan.

19. The method of claim 18 wherein the protocol dictates how polymerase chain reactions (PCRs) to implement the DNA assembly are to be spatially arranged on a multi-well thermo-cycler block with an annealing temperature gradient.

20. The method of claim 1 further comprising outputting a DNA sequence file of the DNA assembly based on the plan.

21. The method of claim 20 wherein the outputting further comprises providing annotations of features within the DNA assembly.

22. The method of claim 1, wherein said parts and protocol are for an assembly method selected from the group consisting of SLIC, Gibson, CPEC, and SLiCE.

23. The method of claim 1, further comprising assembling said DNA sequence fragments into said construct using an assembly method selected from the group consisting of SLIC, Gibson, CPEC, and SLiCE.

24. The method of claim 1, wherein said determining comprises selecting flanking sequences having a melting temperature and number of base pairs so that said assembly pieces will assemble to each other to form said construct where said construct will be assembled from more than two DNA sequence fragments.

25. A method of designing the parts and protocol for assembling a DNA construct via a synthetic method utilizing flanking overlap sequences, said method comprising:

receiving a list of DNA sequence fragments comprising the parts to be assembled together and an order in which to assemble said DNA sequence fragments;

determining with a bioCAD computer system flanking overlap sequences for said fragments, wherein said determining comprises consideration of a plurality of flanking overlap sequences, where said plurality comprises flanking overlap sequences having different lengths and nucleotide sequence to determine the melting temperatures (Tm) of each of said different flanking overlap sequences with the corresponding flanking overlap sequence on an assembly piece that will be a neighboring assembly piece in the DNA sequence fragments to be assembled, and selecting from said plurality particular flanking overlap sequences for each assembly piece, where said flanking overlap sequences each have a nucleotide sequence, melting temperature, and number of base pairs so that said assembly pieces will assemble to each other in the sequence order required to form said construct;

utilizing said computer system to design direct synthesis pieces and oligonucleotide primers for the preparation of the DNA sequence fragments with said flanking overlap sequences;

creating a plan and/or protocol for the preparation of said fragments with flanking overlap sequences and/or the assembly of said fragments; and

executing PCR utilizing said primers and providing direct synthesis to generate the DNA sequence fragments comprising said parts with the flanking overlap sequences.

26. The method of claim 25 wherein the receiving further comprises searching for at least one DNA sequence within a collection of physically existing DNA sequences in a registry of biological parts and/or a local collection of DNA sequences.

27. The method of claim 25 wherein said determining further comprises ensuring that the ends of the DNA sequence fragments do not have stable single-stranded DNA secondary structure at a temperature at which said assembly pieces assemble to each other to form said construct.

28. The method of claim 25 wherein the receiving comprises receiving the list of DNA sequence fragments wherein at least one of the DNA sequence fragments has been physically instantiated.

29. The method of claim 25 wherein the receiving comprises receiving the list of DNA sequence fragments wherein at least one of the DNA sequence fragments has not been physically instantiated.

30. The method of claim 25 wherein the receiving comprises receiving at least one instruction to use at least one of the DNA sequence fragments at more than one place in the order.

31. The method of claim 25 wherein the creating comprises planning the flanking overlap sequences with respect to assembling a combinatorial library of DNA constructs without scars.

32. The method of claim 31, further comprising assembling said DNA sequence fragments into a combinatorial construct library using an assembly method selected from the group consisting of SLIC, Gibson, CPEC, and SLiCE.

33. The method of claim 25 wherein the creating comprises creating the plan to add flanking DNA fragments containing the flanking overlap sequences via splicing by overlap extension (SOE) to each of said oligonucleotide primers after the primers have been incorporated at the ends of a product of a polymerase chain reaction.

34. The method of claim 25 further comprising determining an assembly strategy for each of the DNA sequence fragments that minimizes cost of fragment preparation.

35. The method of claim 34 wherein the designing comprises designing the direct synthesis pieces and said oligonucleotide primers to provide the DNA sequence fragments at a minimum cost.

36. The method of claim 34 wherein the creating comprises creating a protocol that minimizes cost of construction and assembly of said construct.

37. The method of claim 25 further comprising checking the protocol against oligonucleotide mis-priming and against assembly piece incompatibility events.

38. The method of claim 37 wherein the checking further comprises providing a hierarchical assembly protocol, wherein the hierarchical assembly protocol is designed to mitigate the assembly piece incompatibility events.

39. The method of claim 37 further comprising outputting an experimental protocol and a DNA sequence file of the DNA assembly based on the checked plan.

40. The method of claim 39 wherein the outputting comprises outputting the experimental protocol, wherein the protocol dictates the spatial arrangement of polymerase chain reactions (PCRs) to implement the DNA assembly on a multi-well thermo-cycler block with an annealing temperature gradient.

41. The method of claim 25 further comprising:

checking the plan; and

issuing a warning if at least one significant DNA sequence repeat exists in the plan.

42. The method of claim 25 further comprising outputting an experimental protocol for the DNA assembly based on the plan.

43. The method of claim 42 wherein the outputting comprises outputting the experimental protocol, wherein the protocol dictates how polymerase chain reactions (PCRs) to implement the DNA assembly are to be spatially arranged on a multi-well thermo-cycler block with an annealing temperature gradient.

44. The method of claim 25 further comprising outputting a DNA sequence file of the DNA assembly based on the plan.

45. The method of claim 44 wherein the outputting further comprises providing annotations of features within the DNA assembly.

46. The method of claim 25, wherein said parts and protocol are for an assembly method selected from the group consisting of SLIC, Gibson, CPEC, and SLiCE.

47. The method of claim 25, further comprising assembling said DNA sequence fragments into said construct using an assembly method selected from the group consisting of SLIC, Gibson, CPEC, and SLiCE.

48. The method of claim 25, wherein said determining comprises selecting flanking sequences having a melting temperature and number of base pairs so that said assembly pieces will assemble to each other to form said construct where said construct will be assembled from more than two DNA sequence fragments.

49. A method of designing the parts and protocol for assembling a DNA construct via a synthetic method utilizing flanking overlap sequences, said method comprising:

receiving a list of DNA sequence fragments comprising the parts to be assembled together and an order in which to assemble said DNA sequence fragments;

determining with a bioCAD computer system flanking overlap sequences for said fragments, wherein said determining comprises consideration of a plurality of flanking overlap sequences, where said plurality comprises flanking overlap sequences having different lengths and nucleotide sequence to determine the melting temperatures (Tm) of each of said different flanking overlap sequences with the corresponding flanking overlap sequence on an assembly piece that will be a neighboring assembly piece in the DNA sequence fragments to be assembled, and selecting from said plurality particular flanking overlap sequences for each assembly piece, where said flanking overlap sequences each have a nucleotide sequence, melting temperature, and number of base pairs so that said assembly pieces will assemble to each other in the sequence order required to form said construct;

utilizing said computer system to design direct synthesis pieces for the DNA sequence fragments;

creating a plan and/or protocol for the preparation of the direct synthesis pieces;

providing the direct synthesis pieces; and

performing PCR and/or SOE utilizing said primers to generate the DNA sequence fragments comprising said parts with flanking overlap sequences;

or executing PCR utilizing said primers and providing direct synthesis to generate the DNA sequence fragments comprising said parts with flanking overlap sequences.

50. The method of claim 49 wherein the receiving further comprises searching for at least one DNA sequence within a collection of physically existing DNA sequences in a registry of biological parts and/or a local collection of DNA sequences.

51. The method of claim 49 wherein said determining further comprises ensuring that the ends of the DNA sequence fragments do not have stable single-stranded DNA secondary structure at a temperature at which said assembly pieces assemble to each other to form said construct.

52. The method of claim 49 wherein the receiving comprises receiving the list of DNA sequence fragments wherein at least one of the DNA sequence fragments has been physically instantiated.

53. The method of claim 49 wherein the receiving comprises receiving the list of DNA sequence fragments wherein at least one of the DNA sequence fragments has not been physically instantiated.

54. The method of claim 49 wherein the receiving comprises receiving at least one instruction to use at least one of the DNA sequence fragments at more than one place in the order.

55. The method of claim 49 wherein the creating comprises planning the flanking overlap sequences to assemble a combinatorial library of DNA constructs.

56. The method of claim 55, further comprising assembling said DNA sequence fragments into a combinatorial construct library using an assembly method selected from the group consisting of SLIC, Gibson, CPEC, and SLiCE.

57. The method of claim 49 wherein the creating comprises creating the plan to add flanking DNA fragments containing the flanking overlap sequences via said direct synthesis.

58. The method of claim 49 further comprising determining an assembly strategy for each of the DNA sequence fragments that minimizes cost of fragment preparation.

59. The method of claim 58 wherein the designing comprises designing the direct synthesis pieces to provide the DNA sequence fragments at a minimum cost.

60. The method of claim 58 wherein the creating comprises creating a protocol that minimizes cost of construction and assembly of said construct.

61. The method of claim 49 further comprising checking the plan against assembly piece incompatibility events.

62. The method of claim 61 wherein the checking further comprises providing a hierarchical assembly protocol, wherein the hierarchical assembly protocol is designed to mitigate the assembly piece incompatibility events.

63. The method of claim 61 further comprising outputting an experimental protocol and a DNA sequence file of the DNA assembly based on the checked plan.

64. The method of claim 49 further comprising:

checking the plan; and

issuing a warning if at least one significant DNA sequence repeat exists in the plan.

65. The method of claim 49 further comprising outputting an experimental protocol for the DNA assembly based on the plan.

66. The method of claim 49 further comprising outputting a DNA sequence file of the DNA assembly based on the plan.

67. The method of claim 66 wherein the outputting further comprises providing annotations of features within the DNA assembly.

68. The method of claim 49, wherein said determining comprises selecting flanking sequences having a melting temperature and number of base pairs so that said assembly pieces will assemble to each other to form said construct where said construct will be assembled from more than two DNA sequence fragments.

69. A method of designing the parts and protocol for assembling a DNA construct via a synthetic method utilizing flanking overlap sequences, said method comprising:

receiving a list of DNA sequence fragments comprising the parts to be assembled together and an order in which to assemble said DNA sequence fragments;

determining with a bioCAD computer system flanking overlap sequences for said fragments, wherein said determining comprises consideration of a plurality of flanking overlap sequences, where said plurality comprises flanking overlap sequences having different lengths and nucleotide sequence to determine the melting temperatures (Tm) of each of said different flanking overlap sequences with the corresponding flanking overlap sequence on an assembly piece that will be a neighboring assembly piece in the DNA sequence fragments to be assembled, and selecting from said plurality particular flanking overlap sequences for each assembly piece, where said flanking overlap sequences each have a nucleotide sequence, melting temperature, and number of base pairs so that said assembly pieces will assemble to each other in the sequence order required to form said construct;

designing with said bioCAD computer system said oligonucleotide primers for the PCR amplification of said fragments with flanking overlap sequences where determined, and/or utilizing a said computer system to design direct synthesis pieces for the DNA sequence fragments with flanking overlap sequences where determined;

creating a plan and protocol for the preparation of said fragments with flanking overlap sequences and/or the assembly of said fragments;

outputting said plan and protocol to a user; and

performing PCR and/or SOE utilizing said primers to generate the DNA sequence fragments comprising said parts with flanking overlap sequences;

or executing PCR utilizing said primers and providing direct synthesis to generate the DNA sequence fragments comprising said parts with flanking overlap sequences.

70. The method of claim 69 wherein the receiving further comprises searching for at least one DNA sequence within a collection of physically existing DNA sequences in a registry of biological parts and/or a local collection of DNA sequences.

71. The method of claim 69 wherein the receiving comprises receiving the list of DNA sequence fragments wherein at least one of the DNA sequence fragments has been physically instantiated.

72. The method of claim 71, wherein said method further comprises providing the direct synthesis pieces.

73. The method of claim 69, wherein the creating comprises planning the flanking overlap sequences with respect to assembling a combinatorial library of constructs without scars.

74. The method of claim 69, wherein said parts and protocol are for an assembly method selected from the group consisting of SLIC, Gibson, CPEC, and SLiCE.

75. The method of claim 69, further comprising assembling said DNA sequence fragments into said construct using an assembly method selected from the group consisting of SLIC, Gibson, CPEC, and SLiCE.

76. The method of claim 69, wherein said determining comprises selecting flanking sequences having a melting temperature and number of base pairs so that said assembly pieces will assemble to each other to form said construct where said construct will be assembled from more than two DNA sequence fragments.