Microarray synthesis and assembly of gene-length polynucleotides

• US 7,563,600 B2
• Filed: 09/12/2002
• Issued: 07/21/2009
• Est. Priority Date: 09/12/2002
• Status: Expired due to Term

• Current Assignee: Gen9, Inc. (Ginkgo Bioworks Holdings, Inc.)
• Original Assignee: CombiMatrix Corporation
• Inventors: Oleinikov, Andrew V.
• Primary Examiner(s): Kim; Young J
• Application Number:
  US10/243,367
• Publication Number:
  US 20060035218A1
• Time in Patent Office: 2,504 Days
• Field of Search: None
• US Class Current: 435/91.1
• CPC Class Codes: B01J 19/0046 Sequential or parallel reactions, e.g. for the synthesis of polypeptides or polynucleotides; Apparatus and devices for combinatorial chemistry or for making molecular arrays synthesis methods per se C40B50/00
• CPC Class Codes: B01J 2219/00378 Piezoelectric or ink jet dispensers
• CPC Class Codes: B01J 2219/00385 Printing
• CPC Class Codes: B01J 2219/00432 Photolithographic masks
• CPC Class Codes: B01J 2219/00454 by chemical cleavage from the solid support
• CPC Class Codes: B01J 2219/005 Beads
• CPC Class Codes: B01J 2219/00527 Sheets
• CPC Class Codes: B01J 2219/00585 Parallel processes
• CPC Class Codes: B01J 2219/00596 Solid-phase processes
• CPC Class Codes: B01J 2219/00605 the compounds being directly bound or immobilised to solid supports
• CPC Class Codes: B01J 2219/00608 DNA chips
• CPC Class Codes: B01J 2219/00626 Covalent
• CPC Class Codes: B01J 2219/00639 the compounds being trapped in or bound to a porous medium
• CPC Class Codes: B01J 2219/00641 the porous medium being continuous, e.g. porous oxide substrates
• 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 2219/00677 Ex-situ synthesis followed by deposition on the substrate
• CPC Class Codes: B01J 2219/00689 using computers
• CPC Class Codes: B01J 2219/00713 Electrochemical 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: C12N 15/1068 : Template (nucleic acid) mediated chemical library synthesis, e.g. chemical and enzymatical DNA-templated organic molecule synthesis, libraries prepared by non ribosomal polypeptide synthesis [NRPS], DNA/RNA-polymerase mediated polypeptide synthesis
• CPC Class Codes: C12Q 1/6837 : using probe arrays or probe chips C12Q1/6874 takes precedence
• 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 80/00 : Linkers or spacers specially adapted for combinatorial chemistry or libraries, e.g. traceless linkers or safety-catch linkers

Abstract

There is disclosed a process for in vitro synthesis and assembly of long, gene-length polynucleotides based upon assembly of multiple shorter oligonucleotides synthesized in situ on a microarray platform. Specifically, there is disclosed a process for in situ synthesis of oligonucleotide fragments on a solid phase microarray platform and subsequent, “on device” assembly of larger polynucleotides composed of a plurality of shorter oligonucleotide fragments.

Claims

1. A composition for the manufacture of a polynucleotide sequence comprising:

a first oligonucleotide comprising at its 5′

end a sequence region of about 10 to about 50 bases that is the same as a sequence region at the 3′

end of another oligonucleotide in the composition;

a second oligonucleotide comprising at its 3′

end a sequence region of about 10 to about 50 bases that is the same as a sequence region at the 5′

end of another oligonucleotide in the composition; and

a plurality of oligonucleotides wherein each oligonucleotide of the plurality comprises a first sequence region of about 10 to about 50 bases at its 5′

end that is the same as a sequence region at the 3′

end of another oligonucleotide in the composition and a second distinct sequence region of about 10 to about 50 bases at its 3′

end that is the same as a sequence region at the 5′

end of a different oligonucleotide in the composition;

wherein the oligonucleotides of the composition are attached to a solid or porous surface and wherein the oligonucleotides of the composition together comprise the polynucleotide sequence.

2. The composition of claim 1, further comprising (i) a primer that binds specifically to a sequence region at the 3′

end of the first oligonucleotide, and (ii) a polymerase.

3. The composition of claim 1, wherein the solid or porous surface is in the form of a microarray device.

4. The composition of claim 3, wherein the microarray device is a semiconductor device having a plurality of electrodes for synthesizing oligonucleotides in situ using electrochemical means to couple and decouple nucleotide bases.