Method and system for the generation of large double stranded DNA fragments

• US 20070196834A1
• Filed: 08/17/2006
• Published: 08/23/2007
• Est. Priority Date: 09/09/2005
• Status: Abandoned Application

• Current Assignee: Wisconsin Alumni Research Foundation (University of Wisconsin System)
• Original Assignee: Wisconsin Alumni Research Foundation (University of Wisconsin System)
• Inventors: Belshaw, Peter, Cerrina, Francesco, Li, Mo-Huang, Richmond, Kathryn, Sussman, Michael, Kaysen, James, Chu, Larry
• Application Number:
  US11/506,150
• Publication Number:
  US 20070196834A1
• Time in Patent Office: Days
• US Class Current: 435/5
• CPC Class Codes: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides
• CPC Class Codes: C12Q 1/6813 Hybridisation assays

Abstract

Synthesis of long chain molecules such as DNA is carried out rapidly and efficiently to produce relatively large quantities of the desired product. The synthesis of an entire gene or multiple genes formed of many hundreds or thousands of base pairs can be accomplished rapidly and, if desired, in a fully automated process requiring minimal operator intervention, and in a matter of hours, a day or a few days rather than many days or weeks. Production of a desired gene or set of genes having a specified base pair sequence is initiated by analyzing the specified target sequence and determining an optimal set of subsequences of base pairs that can be assembled to form the desired final target sequence. The set of oligonucleotides are then synthesized utilizing automated oligonucleotide synthesis techniques. The synthesized oligonucleotides are subsequently selectively released from the substrate and used in a sequential assembly process.

Claims

1. A method for the generation of a long double stranded DNA target sequence comprising:

(a) synthesizing a set of oligonucleotides that contain sections of the target sequence, each oligonucleotide attached to a support by a cleavable linker;

(b) cleaving the linker to release selected oligonucleotides in a desired sequence, bringing the released oligonucleotides together, and joining selected oligonucleotides to form a set of subsequences which are parts of the desired target sequence; and

(c) assembling the subsequences to form the desired target sequence.

2. The method of claim 1 further including, before the step of synthesizing, identifying the set of subsequences that can be assembled to form the target sequence, and further identifying the set of oligonucleotides that can be assembled together to form each subsequence.

3. The method of claim 1 further including carrying out error correction on the oligonucleotides and on the subsequences.

4. The method of claim 3 wherein the error correction is carried out by DNA coincidence filtering.

5. The method of claim 4 wherein the DNA coincidence filtering is carried out by passing double stranded oligonucleotides and subsequences through a filter containing MutS protein to bind DNA duplexes containing mismatched bases while allowing error free duplexes to pass through.

6. The method of claim 1 wherein the synthesized oligonucleotides are held to the support by photocleavable linkers and wherein releasing selected oligonucleotides comprises illuminating one or more areas of the support containing the selected oligonucleotides to photocleave the linkers holding the oligonucleotides to the support.

7. The method of claim 1 wherein the synthesized oligonucleotides are held to the support by chemically labile linkers and wherein releasing selected oligonucleotides comprises applying a reagent that cleaves the linker to one or more areas of the support containing the selected oligonucleotides to cleave the linkers holding the oligonucleotides to the support.

8. The method of claim 1 wherein the oligonucleotides are synthesized with primer sequences at their ends, the method further including the step of conducting polymerase chain reaction amplification of the oligonucleotides after release of the oligonucleotides from the support and before assembling the oligonucleotides to form the subsequences.

9. The method of claim 1 further including carrying out polymerase chain reaction amplification of the subsequences before assembly of the subsequences into the target sequence.

10. The method of claim 1 wherein synthesizing the set of oligonucleotides is carried out in a maskless array synthesizer having a reaction chamber in which DNA synthesis reactions are performed on the support with an active surface in which arrays of different oligonucleotides are formed, a flow cell enclosing the active surface of the support and having ports for supplying reagents into the flow cell which can be flowed over the active surface of the support, a DNA synthesizer reagent supply connected to supply reagents to the flow cell, and an image former for providing a high precision, array light image projected onto the substrate active support.

11. A method for the generation of nucleotides having a desired sequence comprising:

(a) synthesizing a set of double stranded nucleotides that are intended to contain the desired sequence;

(b) carrying out coincidence filtering error correction on the nucleotides by passing double stranded nucleotides through a filter that binds DNA duplexes containing mismatched bases while allowing error free duplexes to pass through.

12. The method of claim 11 wherein the filter contains MutS protein to bind DNA duplexes containing mismatched bases.

13. The method of claim 11 wherein the synthesized nucleotides are held to a support by photocleavable linkers and wherein releasing selected nucleotides comprises illuminating one or more areas of the support containing the selected nucleotides to photocleave the linkers holding the nucleotides to the support.

14. The method of claim 11 wherein the synthesized nucleotides are held to the support by chemically labile linkers and wherein releasing selected nucleotides comprises applying a reagent that cleaves the linker to one or more areas of the support containing the selected nucleotides to cleave the linkers holding the nucleotides to the support.

15. The method of claim 11 wherein the nucleotides are synthesized with primer sequences at their ends, the method further including the step of conducting polymerase chain reaction amplification of the nucleotides.

16. The method of claim 11 wherein synthesizing a set of nucleotides is carried out in a maskless array synthesizer having a reaction chamber in which DNA synthesis reactions are performed on a support with an active surface in which arrays of different nucleotides are formed, a flow cell enclosing the active surface of the support and having ports for supplying reagents into the flow cell which can be flowed over the active surface of the support, a DNA synthesizer reagent supply connected to supply reagents to the flow cell, and an image former for providing a high precision, array light image projected onto the support active surface.

17. A method for the generation of a long double stranded DNA target sequence comprising:

(a) synthesizing a set of oligonucleotides that contain sections of the target sequence, each oligonucleotide attached to a support by a cleavable linker, wherein synthesizing is carried out in a maskless array synthesizer having a reaction chamber in which DNA synthesis reactions are performed on the support with an active surface in which arrays of different oligonucleotides are formed, a flow cell enclosing the active surface of the support and having ports for supplying reagents into the flow cell which can be flowed over the active surface of the support, a DNA synthesizer reagent supply connected to supply reagents to the flow cell, and an image former for providing a high precision, array light image projected onto the support active surface;

(b) cleaving the linker to release selected oligonucleotides in a desired sequence, bringing the released oligonucleotides together, and joining selected oligonucleotides to form a set of subsequences which are parts of the desired target sequence; and

(c) assembling the subsequences to form the desired target sequence.

18. The method of claim 17 further including, before the step of synthesizing, identifying the set of subsequences that can be assembled to form the target sequence and identifying the set of oligonucleotides that can be assembled to form each subsequence.

19. The method of claim 17 further including carrying out error correction on the oligonucleotides and on the subsequences.

20. The method of claim 19 wherein the error correction is carried out by DNA coincidence filtering.

21. The method of claim 20 wherein the DNA coincidence filtering is carried out by passing double stranded oligonucleotides and subsequences through a filter containing MutS protein to bind DNA duplexes containing mismatched bases while allowing error free duplexes to pass through.

22. The method of claim 17 wherein the synthesized oligonucleotides are held to the support by photocleavable linkers and wherein releasing selected oligonucleotides comprises illuminating one or more areas of the support containing the selected nucleotides to photocleave the linkers holding the oligonucleotides to the support.

23. The method of claim 17 wherein the synthesized oligonucleotides are held to the support by chemically labile linkers and wherein releasing selected oligonuceotides comprises applying a reagent that cleaves the linker to one or more areas of the support containing the selected oligonucleotides to cleave the linkers holding the oligonucleotides to the support.

24. The method of claim 17 wherein the oligonucleotides are synthesized with a primer sequences at their ends, the method further including the step of conducting polymerase chain reaction amplification of the oligonucleotides after release of the oligonucleotides from the support and before assembling the oligonucleotides to form the subsequences.

25. The method of claim 17 further including carrying out polymerase chain reaction amplification of the subsequences before assembly of the subsequences into the target sequence.