Programmable oligonucleotide synthesis

• US 8,367,335 B2
• Filed: 04/06/2012
• Issued: 02/05/2013
• Est. Priority Date: 08/23/2006
• Status: Active Grant

• Current Assignee: Telesis Bio, Inc.
• Original Assignee: Synthetic Genomics, Inc.
• Inventors: Staehler, Peer, Carapito, Raphael
• Primary Examiner(s): Babic, Christopher M.
• Application Number:
  US13/441,186
• Publication Number:
  US 20120196757A1
• Time in Patent Office: 305 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 molecule having a desired correct sequence from a mixture of nucleic acids, comprising:

(a) monoclonizing a mixture of nucleic acids on a solid reaction support;

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

(c) localizing a nucleic acid molecule with the desired correct sequence in the nucleic acids of step (b),

(d) isolating the nucleic acid molecule from step (c), and

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

14. The method of claim 1 wherein the further processing comprises assembling nucleic acid molecules of step (d) to a desired nucleic acid.

15. The method of claim 1 wherein the solid reaction support is selected from the group consisting of:

one or more beads, one or more particles, one or more chips, and a microfluidic reaction support.

16. The method of claim 15 wherein the solid reaction support is one or more beads or a chip.

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

(a) monoclonizing a mixture of nucleic acids on a solid reaction support;

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

(c) localizing a nucleic acid with the desired sequence in the nucleic acids of step (b),

(d) isolating the nucleic acid from step (c), and

(e) optionally further processing of isolated nucleic acid obtained in step (d) wherein the isolation takes place by at least one of steps (i) to (iv);

(i) isolating one or more beads on which said nucleic acid molecule is immobilized,

(ii) cleaving off the nucleic acid molecule from a support on which said nucleic acid molecule is immobilized,

(iii) selectively amplifying the nucleic acid molecule by spatially-resolved addition of PCR reagents,

(iv) eluting the nucleic acid molecule by laser capture method.

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

(a) monoclonizing a plurality of the different nucleic acids by emulsion-based PCR on beads

(b) immobilizing the beads in a reaction support

(c) parallel sequencing the monoclonized nucleic acids of step (a)

(d) localizing an individual nucleic acid molecule with the desired correct sequence in the nucleic acids of step (c)

(e) isolating the beads comprising the nucleic acid molecules localized in step (d)

(f) obtaining the clonal nucleic acid sequences by detachment of the clonal nucleic acid sequence from the bead or by copying

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

4. A method for quality control of assembled nucleic acid molecules, said method comprising:

(a) providing a mixture of assembled nucleic acid molecules suspected of containing sequence defects,

(b) amplifying the mixture of (a) to receive a clonal population thereof,

(c) immobilizing the clonal population of (b) on a support,

(d) parallel sequencing the clonal population immobilized according to (c),

(e) localizing at least one clonal nucleic acid molecule that meets a predefined criterion

(f) obtaining the at least one clonal nucleic acid molecule of (e).

6. The method of claim 4, wherein the assembled sequences provided in step (a) are built each from one or more oligonucleotides.

7. The method of claim 4, wherein the assembled sequences represent genes, gene clusters, chromosomes, genomes or fragments thereof.

8. The method of claim 4, wherein amplification in step (b) comprises an emulsion-based PCR.

9. The method of claim 4, wherein immobilizing the clonal population according to step (c) comprises immobilization on beads.

10. The method of claim 4, wherein the predetermined criterion in step (e) is the desired correct sequence.

11. The method of claim 4, wherein the obtaining the clonal nucleic acid molecule of (f) comprises at least one of:

(i) detachment of the clonal nucleic acid molecule from the support,

(ii) copying of the clonal nucleic acid molecule,

(iii) laser capture, or/and

(iv) amplification by spatially resolved PCR.

12. A method of assembly of nucleic acid molecules comprising:

(i) providing single-stranded nucleic acid molecules to be assembled into a target nucleic acid molecule,

(ii) assembling by hybridization complementary regions of the single-strands nucleic acids of (i),

(iii) controlling the quality of the mixture of assembled nucleic acid molecules obtained in step (ii) by the method of claim 4.

13. The method of claim 12, wherein step (ii) results in a gene, gene cluster, chromosome, genome or a fragment thereof.

5. A method for isolation of a sequence-verified nucleic acid clone, comprising:

(a) providing a mixture of nucleic acid molecules suspected of containing sequence defects,

(b) amplifying the mixture of (a) to receive a clonal population thereof,

(c) immobilizing the clonal population of (b) on a support,

(d) parallel sequencing the clonal population immobilized according to (c)

(e) localizing at least one clonal nucleic acid molecule that meets a predefined criterion, and

(f) obtaining the at least one clonal nucleic acid molecule of (e).

17. The method of claim 5 wherein the solid reaction support is selected from the group consisting of:

one or more beads, one or more particles, one or more chips, and a microfluidic reaction support.

18. The method of claim 17 wherein the solid reaction support is one or more beads or a chip.