SYNTHESIS OF SEQUENCE-VERIFIED NUCLEIC ACIDS

• US 20100216648A1
• Filed: 02/19/2010
• Published: 08/26/2010
• Est. Priority Date: 02/20/2009
• Status: Abandoned Application

• Current Assignee: Synthetic Genomics, Inc.
• Original Assignee: Febit Holding GmbH (FEBIT)
• Inventors: CARAPITO, Raphael, BEIER, Markus, LEONARD, Jack T., JAEGER, Joachim, MATZAS, Mark, STAEHLER, Cord F., STAEHLER, Peer F.
• Application Number:
  US12/708,783
• Publication Number:
  US 20100216648A1
• Time in Patent Office: Days
• US Class Current: 506/6
• CPC Class Codes: C12N 15/1093 General methods of preparing gene libraries, not provided for in other subgroups
• CPC Class Codes: C12Q 1/6869 Methods for sequencing

Abstract

The invention relates to methods and devices for preparing synthetic nucleic acids.

Claims

1. A method to retrieve sequence verified nucleic acids from a plate, carrier or/and substrate, comprising the steps:

(1) provision of a mixture of nucleic acids,

(2) turning the nucleic acid mixture into a clonal library,

(3) sequencing the clonal library,

(4) retrieval of the sequence-verified nucleic acid molecules,

(5) optionally use of the retrieved molecules in continuative steps/procedures, or/and

(6) optionally repeating steps (2)-(5) at least one time.

2. The method of claim 1, comprising

(a) provision of a mixture of nucleic acids,

(b) individualizing the nucleic acids of the mixture of (1),

(c) sequencing the individualized nucleic acids of (2),

(d) isolating the sequenced nucleic acid molecules of (3),

(e) optionally use of the retrieved molecules in continuative steps/procedures, or/and

(f) optionally repeating steps (2)-(5) at least one time.

3. The method of claim 1, wherein step (2) comprises separation so that individual nucleic acids are present at a defined location.

4. The method of claim 1, wherein the individualized nucleic acids of (2) are presented on a solid carrier.

5. The method of claim 4, wherein the carrier is selected from glass slides, gels, polymers, capillaries, microfluidic carriers, membranes, porous carriers, plastics, silicon, ordered or chaotic pores, sponge structures, cubes, 2D and 3D, emulsions, dendrimers, beads, particles, resins, metals, nano-particles and nano-structures or combinations thereof.

6. The method of claim 5, wherein the carrier comprises beads.

7. The method of claim 5, wherein the carrier is provided as a or within a microtiter plate, a nanotiter plate, or/and a picotiter plate.

8. The method of claim 1, wherein step (2) comprises an amplification reaction of the individualized nucleic acids.

9. The method of claim 8, wherein amplification comprises emulsion PCR, bridge amplification, MegaPlex PCR, creation of polonies, or/and rolling circle amplification, wherein emulsion PCR is preferred.

10. The method of claim 1, wherein step (3) comprises single molecule sequencing without amplification reaction in step (2).

11. The method of claim 1, wherein step (3) comprises sequencing-by-synthesis or sequencing-by-ligation techniques.

12. The method of claim 1, wherein isolation in step (4) comprises physical retrieval, physical recovery or/and extraction.

13. The method of claim 1, wherein isolation in step (4) comprises enzymatic amplification of the nucleic acids presented on the carrier.

14. The method of claim 13, comprising copying-off the nucleic acid presented on the carrier by a polymerase.

15. The method of claim 12, comprising isolating the carrier.

16. The method of claim 1, wherein step (5) comprises production of sequence-verified nucleic acid fragments.

17. The method of claim 16, comprising creation of respositories comprising the sequence-verified nucleic acid fragments.

18. The method of claim 16, comprising the production of a bead-array.

19. The method of claim 16, comprising the production of a micro-array.

20. The method of claim 16, comprising the production of a sequence-verified nucleic acid library.

21. The method of claim 16, comprising the fabrication of a synthetic gene.

22. The method of claim 16, comprising re-formatting a library or mix of nucleic acid sequences into separated vessels, fractions, pots or/and wells.

23. The method of claim 1, wherein repeating steps (2) to (5) at least one time comprises performing steps (2) to (5) in a second cycle with a sub-population selected from the nucleic acid molecules obtained in step (5) of a first cycle.

24. The method of claim 23, wherein the sub-population of nucleic acids obtained in a first cycle of steps (2)-(5) comprises nucleic acids each having a predetermined sequence.

25. The method of claim 1, wherein parallel sequence verification is performed.

26. The method of claim 1, wherein in step (1), a mixture of at least 100, 1,000, 10,000, 100,000, 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹ or 10¹⁹ different nucleic acids and up to 1,000, 10,000, 100,000, 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹ or up to 10²⁰ different nucleic acids is provided.

27. The method of claim 1, wherein the method provides a set of nucleic acids with 2, 10, 100, 1,000, 10,000, 100,000, 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹ or up to 10²⁰ defined sequenced members.

28. The method of claim 1, comprising identifying locations or beads with clonal DNA of known sequence.

29. The method of claim 28, comprising identifying locations or beads by means of reference points in a microscopic picture of the carrier.

30. A method for re-formatting a library or mix of nucleic acid sequences with n members in up to n separate vessels, fractions, pots or/and wells, particularly involving the steps as defined in claim 1.

31. The method of claim 30, wherein n is an integer of at least 100, 1,000, 10,000, 100,000, 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹ or 10¹⁹ and up to 1,000, 10,000, 100,000, 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹ or 10²⁰.

32. A process to in vitro clone, sequence or/and make available for separate handling or recombination a set of nucleic acids with 2, 10, 100, 1,000, 10,000, 100,000, 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹ or up to 10²⁰ defined sequenced members, particularly involving the steps as defined in claim 1.

33. A method to physically separate, in vitro amplify to form a clone, and sequence a set of nucleic acids by means of using a parallel sequencing process as described herein, particularly involving the steps as defined in claim 1.

34. An apparatus with alignment capability to identify locations or beads with clonal DNA of known sequence.

35. The apparatus of claim 34, comprising means for alignment by reference points in a microscopic picture of the carrier.

36. An apparatus for retrieval of clonal sequence-verified nucleic acid fragments, including

(a) unit/means to link the sequence-information to the physical position or location of the nucleic acid fragment,

(b) unit/means to release location-specifically the sequence-verified nucleic acid fragments from the carrier (e.g. based on the sequence and location information provided), or/and

(c) unit/means to retrieve the released sequence-verified nucleic acid fragments from the carrier.

37. The apparatus of claim 36, wherein (a) comprises means for identifying the physical position or location of the nucleic acid fragment by reference points in a microscopic picture of the carrier.

38. An apparatus for producing sequence-verified nucleic acid fragments, including

(a) unit/means to generate a clonal representation of a nucleic acid mixture,

(b) unit/means to sequence the clonal representation of the said nucleic acid mixture, and generating the sequence-information and the physical location information for each of the nucleic acid fragments,

(c) unit/means to link the sequence-information to the physical position or location of the nucleic acid fragments,

(d) unit/means to release the sequence-verified nucleic acid fragments from the carrier, in particular based on the sequence and location information provided,

(e) unit/means to retrieve the released sequence-verified nucleic acid fragments from the carrier, or/and

(f) optional:

unit/means to reformat (and optionally store away) the retrieved sequence-verified nucleic acid fragments.

39. The apparatus of claim 38, wherein the clonal representation of a nucleic acid mixture comprises monoclonized nucleic acids obtained from the mixture.

40. The apparatus of claim 38, wherein (c) comprises means for identifying the physical position or location of the nucleic acid fragments by reference points in a microscopic picture of the carrier.

41. A method for isolation of a nucleic acid with the desired correct sequence from a mixture of nucleic acids, comprising the steps

(a) monoclonizing the mixture of nucleic acids,

(b) sequencing the monoclonized nucleic acids of step (a),

(c) identifying a nucleic acid with the desired sequence from the nucleic acids of step (b), and

(d) isolating the nucleic acid from step (c).

42. A method of sequencing at least one nucleic acid in a mixture comprising assembled nucleic acids, which possibly contain defective nucleotides, comprising the steps:

(a) amplification of a mixture comprising assembled nucleic acids, which are in each case built up from 2 or more nucleic acid fragments, to clonal populations,

(b) sequencing of at least one clonal population from step (a), and

(c) optionally isolation of at least one nucleic acid, which contains defects, or/and at least one nucleic acid, which is correct.

43. The method of claim 42, wherein sequencing is parallel sequencing of a multiplicity of sequences.

44. The method of claim 42, wherein said at least one nucleic acid, which contains defects, or/and said at least one nucleic acid, which is correct are retrieved and used in continuative steps/procedures, or/and steps (a)-(b) are repeated at least one time.