Programmable oligonucleotide synthesis

• US 8,173,368 B2
• Filed: 08/23/2007
• Issued: 05/08/2012
• Est. Priority Date: 08/23/2006
• Status: Active Grant

• Current Assignee: Telesis Bio, Inc.
• Original Assignee: Febit Holding GmbH (FEBIT)
• Inventors: Staehler, Peer, Carapito, Raphael
• Primary Examiner(s): Babic, Christopher M
• Application Number:
  US12/438,425
• Publication Number:
  US 20100015668A1
• Time in Patent Office: 1,720 Days
• Field of Search: None
• US Class Current: 435/6.1
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]

Abstract

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

Claims

1. A method for isolating an individual nucleic acid having a desired sequence from a mixture of nucleic acids, comprising

(a) providing a mixture of different nucleic acids,

(b) monoclonizing a plurality of the different nucleic acids to form an array of monoclonized individual nucleic acids,

(c) parallel sequencing the monoclonized nucleic acids of step (b) by sequencing-by-synthesis or/and sequencing-by-ligation,

(d) identifying an individual nucleic acid with the desired sequence from the nucleic acids of step (c),

(e) isolating the nucleic acid from step (d) by separating said individual nucleic acid with the desired sequence from said array of different nucleic acids, and

(f) optionally further processing of the isolated nucleic acid obtained in step (d).

2. The method of claim 1, wherein monoclonizing in step (b) comprises emulsion-based PCR, use of individual molecular arrays or/and creation of polonies.

3. The method of claim 1, wherein steps (d) and (e) are repeated to isolate additional individual nucleic acids with desired sequences from said array of different nucleic acids.

4. The method of claim 3, wherein the additional isolated nucleic acids and the isolated nucleic acid are employed in one or more assembly reactions.

5. The method of claim 1, wherein isolation takes place by at least one of steps (i) to (iv):

(i) isolating one or more beads comprising the nucleic acid,

(ii) cleaving off the nucleic acids from a support on which said nucleic acids are immobilized,

(iii) selectively amplifying by spatially-resolved addition of PCR reagents, and

(iv) elution by a laser capture method.

6. The method of claim 1, wherein the nucleic acid mixture employed in step (a) is obtained by assembling oligonucleotides.

7. The method of claim 1, wherein the nucleic acid mixture employed in step (a) is obtained by parallel synthesis.

8. The method of claim 3, wherein a target sequence is built up from the additional isolated nucleic acids and the isolated nucleic acid.

9. The method of claim 3, wherein the additional isolated nucleic acids and the isolated nucleic acid are inserted in a target organism, which constructs an assembled genome from them in vivo.

10. The method of claim 9, wherein the target organism is Deinococcus radiodurans.

11. A method of parallel sequencing at least one nucleic acid in a mixture comprising assembled nucleic acids, wherein said mixture is suspected of containing defective nucleotides, comprising:

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

(b) sequencing of at least one clonal population from step (a) by sequencing-by-synthesis or/and sequencing-by-ligation,

(c) identifying at least one assembled nucleic acid containing defective nucleotides and/or at least one assembled nucleic acid which is correct from the nucleic acids sequenced in step (b), and

(d) isolating at least one nucleic acid, which is correct by separating it from said nucleic acid containing defective nucleotides.

12. The method of claim 11, wherein amplification in step (a) comprises emulsion-based PCR, use of individual molecular arrays or/and creation of polonies.

13. The method of claim 11, wherein isolation takes place by at least one of steps (i) to (iv):

(i) isolating one or more beads comprising the nucleic acid,

(ii) cleaving off the nucleic acids from a support on which said nucleic acids are immobilized,

(iii) selectively amplifying by spatially-resolved addition of PCR reagents, and

(iv) elution by a laser capture method.

14. The method of claim 11, wherein a plurality of nucleic acids is isolated and a target sequence is built up from the isolated nucleic acids.

15. The method of claim 11, wherein the isolated nucleic acids are inserted in a target organism, which constructs an assembled genome from them in vivo.

16. The method of claim 15, wherein the target organism is Deinococcus radiodurans.

17. A method for isolating an individual nucleic acid having a desired sequence from a mixture of nucleic acids, comprising:

(a) providing a mixture of different nucleic acids;

(b) monoclonizing a plurality of the different nucleic acids by emulsion-based PCR on beads to form an array of monoclonized individual bead-bound nucleic acids,

(c) parallel sequencing the monoclonized nucleic acids of step (b) by sequencing-by-synthesis,

(d) identifying an individual nucleic acid with the desired sequence from the nucleic acids of step (c), and

(e) isolating the beads comprising the nucleic acid identified in step (d) by separating said beads comprising the nucleic acid with the desired sequence from said mixture of nucleic acids.

18. A method of parallel sequencing at least one nucleic acid in a mixture comprising assembled nucleic acids, wherein said mixture is suspected to contain nucleic acids containing defective nucleotides, comprising the steps:

(a) monoclonizing the mixture of nucleic acids by emulsion-based PCR on beads,

(b) forming an array of the beads in or on a reaction support,

(c) parallel sequencing the monoclonized nucleic acids of step (a) by sequencing-by-synthesis,

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

(e) isolating the beads comprising the nucleic acid identified in step (d) by physically separating beads having the correct nucleic acid sequence from said reaction support.

19. A method of assembling a combined nucleic acid having a desired correct sequence comprising:

(a) providing a mixture of different nucleic acids,

(b) monoclonizing a plurality of the different nucleic acids to form an array of monoclonized individual nucleic acids,

(c) parallel sequencing the monoclonized individual nucleic acids of step (b),

(d) isolating from said array a plurality of individual nucleic acids each having a correct, desired, and different sequence as building blocks, and

(e) binding or covalently linking at least two of said building blocks that are different to assemble said combined nucleic acid.