Method for the synthesis of DNA sequences

• US 7,183,406 B2
• Filed: 05/20/2002
• Issued: 02/27/2007
• Est. Priority Date: 05/18/2001
• Status: Expired due to Fees

• Current Assignee: Wisconsin Alumni Research Foundation (University of Wisconsin System)
• Original Assignee: Wisconsin Alumni Research Foundation (University of Wisconsin System)
• Inventors: Flickinger, Shane T., Cerrina, Francesco, Sussman, Michael J., Belshaw, Peter J
• Primary Examiner(s): Borin; Michael
• Assistant Examiner(s): Lin; Jerry
• Application Number:
  US10/152,046
• Publication Number:
  US 20030068633A1
• Time in Patent Office: 1,744 Days
• Field of Search: 435/287.2, 435/6, 702/20, 702/19, 536/24.3, 536/25.3
• US Class Current: 536/25.3
• CPC Class Codes: B01J 2219/00274 Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
• CPC Class Codes: B01J 2219/00439 using micromirror arrays
• CPC Class Codes: B01J 2219/00441 using lasers
• CPC Class Codes: B01J 2219/00454 by chemical cleavage from the solid support
• CPC Class Codes: B01J 2219/00497 Features relating to the solid phase supports
• CPC Class Codes: B01J 2219/00527 Sheets
• CPC Class Codes: B01J 2219/00585 Parallel processes
• CPC Class Codes: B01J 2219/0059 Sequential processes
• CPC Class Codes: B01J 2219/00596 Solid-phase processes
• CPC Class Codes: B01J 2219/00659 Two-dimensional arrays
• CPC Class Codes: B01J 2219/00675 In-situ synthesis on the substrate
• CPC Class Codes: B01J 2219/00689 using computers
• CPC Class Codes: B01J 2219/007 Simulation or vitual synthesis
• CPC Class Codes: B01J 2219/00722 Nucleotides
• CPC Class Codes: B82Y 30/00 Nanotechnology for materials or surface science, e.g. nanocomposites
• CPC Class Codes: C07B 2200/11 Compounds covalently bound to a solid support
• CPC Class Codes: C07F 9/2408 of hydroxyalkyl compounds
• CPC Class Codes: C07H 19/06 Pyrimidine radicals
• CPC Class Codes: C07H 19/10 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• CPC Class Codes: C07H 19/16 Purine radicals
• CPC Class Codes: C07H 19/20 : with the saccharide radical esterified by phosphoric or polyphosphoric acids
• 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: C12N 15/10 : Processes for the isolation, preparation or purification of DNA or RNA chemical preparation of DNA or RNA C07H21/00; preparation of non-structural polynucleotides from microorganisms or with enzymes C12P19/34
• CPC Class Codes: C12N 15/1031 : mutagenesis by gene assembly, e.g. assembly by oligonucleotide extension PCR
• CPC Class Codes: C40B 40/06 : Libraries containing nucleotides or polynucleotides, or derivatives thereof
• CPC Class Codes: C40B 50/14 : Solid phase synthesis, i.e. wherein one or more library building blocks are bound to a solid support during library creation; Particular methods of cleavage from the solid support
• CPC Class Codes: C40B 60/14 : for creating libraries

Abstract

A method is disclosed for the direct synthesis of double stranded DNA molecules of a variety of sizes and with any desired sequence. The DNA molecule to be synthesis is logically broken up into smaller overlapping DNA segments. A maskless microarray synthesizer is used to make a DNA microarray on a substrate in which each element or feature of the array is populated by DNA of a one of the overlapping DNA segments. The DNA segments are released from the substrate and held under conditions favoring hybridization of DNA, under which conditions the segments will spontaneously hybridize together to form the desired DNA construct. This method makes possible the remote assembly of DNA sequence, through a process analogous to facsimile transmission of documents, since the information on DNA to be made can be transmitted remotely to an instrument which can then synthesize any needed DNA sequence from the information.

Claims

1. A method for the fabrication of a DNA construct of a target sequence comprising the steps of

(a) defining a plurality of overlapping single stranded DNA sequence segments which in the aggregate would make up the target DNA sequence, wherein the single stranded segments overlap in the regions where the segments are complementary to each other;

(b) making a DNA microarray on a substrate, the microarray made up of features having single stranded DNA molecules, the single stranded DNA molecules selected to be overlapping sequence segments of the target sequence, the single stranded DNA molecules being made attached by linkers to the substrate, the linkers being stable under the conditions of DNA synthesis for the microarray, the linkers also being capable of being rendered photo-labile by chemical treatment;

(c) detaching the single stranded DNA molecules from the substrate by first applying a chemical modification to the linkers to render the linkers light labile and then by selectively applying light to cleave the labile linkers in areas of single stranded DNA molecules thus detaching the single stranded DNA molecules from the substrate; and

(d) allowing the single stranded DNA molecules detached in step (c) to assemble into a double stranded target DNA construct by providing conditions favoring hybridization of the single stranded DNA molecules to each other using the 3′

overhang contained on either strand.

2. The method as claimed in claim 1 wherein the step of defining the overlapping sequence segments is a logical not a physical step performed by computer.

3. The method as claimed in claim 1 wherein the step of making a DNA microarray is performed using a maskless array synthesizer instrument.

4. The method as claimed in claim 1 wherein the step of allowing the segments to assemble includes cooling the DNA segments under conditions favoring hybridization of DNA segments.

5. The method as claimed in claim 1 wherein the step of assembling the DNA segments also includes the use of a DNA ligase, a DNA polymerase, or a DNA kinase.

7. The method of claim 1 wherein some of the DNA molecules that are detached are physically separated from others of the detached DNA molecules so that hybridization of desired DNA sub-assemblies is permitted while incorrect assemblies are inhibited.

6. A method for the fabrication of a target DNA construct of comprising the steps of

(a) defining a plurality of overlapping single stranded DNA sequence segments which in the aggregate make up the target DNA construct, wherein the single stranded segments overlap in the regions where the segments are complementary to each other;

(b) making the single stranded DNA molecules in an array on a substrate, the single stranded DNA molecules linked to the substrate by base labile linkers;

(c) releasing the single stranded DNA molecules from the substrate by alkaline treatment of the array to cause the base labile linkers to release the single stranded DNA molecules; and

(d) allowing the single stranded DNA molecules to assemble into a double stranded target DNA construct by providing conditions favoring hybridization of the single stranded DNA molecules to each other using the 3′

overhang contained on either strand.

8. The method of claim 6 wherein the step of allowing the DNA molecules to assemble also includes the addition of a DNA ligase, a DNA polymerase or a DNA kinase.

9. A method for the fabrication of a DNA construct of a target sequence comprising the steps of

(a) defining a plurality of overlapping single stranded DNA sequence segments which in the aggregate would make up the target DNA sequence, wherein the single stranded segments overlap in the regions where the segments are complementary to each other;

(b) making a DNA microarray on a substrate in which single stranded DNA molecules constructed in the microarray are the overlapping sequence segments, the DNA molecules each being attached to the substrate by a linker which is stable while the microarray is being made, the linker being capable of being rendered photo-labile by chemical treatment;

(c) releasing the single stranded DNA molecules from the substrate by chemical treatment to modify the linkers to become light labile and then using light to cleave the linkers to release the single stranded DNA molecules;

(d) allowing the single stranded DNA molecules released in step (c) to assemble into a double stranded target DNA construct by providing conditions favoring hybridization of the single stranded DNA molecules to each other using the 3′

overhang contained on either strand; and

(e) ligating said assembled DNA molecules into a DNA molecule of the target sequence.