HIGH THROUGHPUT GENE ASSEMBLY IN DROPLETS

• US 20150361422A1
• Filed: 11/21/2014
• Published: 12/17/2015
• Est. Priority Date: 06/16/2014
• Status: Abandoned Application

• Current Assignee: Agilent Technologies, Inc.
• Original Assignee: Agilent Technologies, Inc.
• Inventors: Sampas, Nicholas M., Myerson, Joel, Anderson, Paige, Curry, Bo, Sampson, Jeffrey R.
• Application Number:
  US14/550,713
• Publication Number:
  US 20150361422A1
• Time in Patent Office: Days
• US Class Current: 1/1
• 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 2219/00587 High throughput processes
• CPC Class Codes: B01J 2219/00596 Solid-phase processes
• CPC Class Codes: B01J 2219/00608 DNA chips
• CPC Class Codes: B01J 2219/00659 Two-dimensional arrays
• CPC Class Codes: B01J 2219/00711 Light-directed synthesis
• CPC Class Codes: B01J 2219/00722 Nucleotides
• 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: C12N 15/1075 by coupling phenotype to genotype, not provided for in other groups of this subclass
• CPC Class Codes: C12N 15/1093 General methods of preparing gene libraries, not provided for in other subgroups
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 2525/301 Hairpin oligonucleotides
• CPC Class Codes: C12Q 2565/501 being an array of oligonucleotides
• CPC Class Codes: C12Q 2565/537 characterised by the capture oligonucleotide acting as a primer

Abstract

Provided herein, among other things, is a method comprising: (a) obtaining a mixture of multiple sets of oligonucleotides, wherein the oligonucleotides within each set each comprise a terminal indexer sequence can be assembled to produce a synthon; and (b) hybridizing the oligonucleotide mixture to an array, thereby spatially-separating the different sets of oligonucleotides from one another. Other embodiments are also provided.

Claims

1. A method comprising:

(a) obtaining a mixture of multiple sets of oligonucleotides, wherein the oligonucleotides within each set each comprise a terminal indexer sequence and can be assembled to produce a synthon; and

(b) hybridizing the oligonucleotide mixture to an array, thereby spatially-separating the different sets of oligonucleotides from one another.

2. The method of claim 1, wherein the oligonucleotides are single-stranded oligonucleotides.

3. The method of claim 1, wherein the mixture comprises double-stranded oligonucleotides.

4. The method of claim 1, wherein the oligonucleotides are single stranded and comprise a 3′

hairpin.

5. The method of claim 4, comprising:

contacting the array with a solution comprising a polymerase and nucleotides, thereby extending the hairpin and producing, for each feature bound by the oligonucleotides, a set of double-stranded extension products.

6. The method of claim 1, wherein the oligonucleotides hybridize directly to oligonucleotides that are immobilized on the array.

7. The method of claim 1, wherein the oligonucleotides hybridize via an adaptor to oligonucleotides that are immobilized on the array.

8. The method of claim 7, wherein the method comprises:

contacting the array with a solution comprising a polymerase and nucleotides, thereby extending the adaptor and producing, for each feature bound by the oligonucleotides, a set of double-stranded extension products.

9. The method of claim 1, further comprising

(c) contacting the array with a solution, thereby producing, for each feature bound by the oligonucleotides, a discrete droplet comprising one or more features.

10. The method of claim 9, further comprising placing an immiscible liquid over the droplets, thereby producing, for each feature bound by the oligonucleotides, a discrete reaction chamber defined by a droplet.

11. The method of claim 10, further comprising incubating the array under conditions by which a synthon is assembled in each of the reaction chambers.

12. The method of claim 11, wherein the droplets comprise double-stranded oligonucleotides or double-stranded extension products, and the solution comprises a Type IIs restriction endonuclease, a DNA ligase and ATP, wherein the products of digestion of the double-stranded oligonucleotides or double-stranded extension products by the Type IIs restriction endonuclease are ligated to one another in a defined order by the DNA ligase in the discrete reaction chambers, thereby producing a synthon.

13. The method of claim 11, wherein the oligonucleotides are single-stranded oligonucleotides and the method comprises:

cleaving the terminal indexer sequence from the oligonucleotides to release assembly sequences from at least some of the oligonucleotides; and

assembling the synthon from the assembly sequences by polymerase chain assembly or by ligation.

14. The method of claim 13 wherein the cleaving the terminal indexer sequence from the oligonucleotide comprises cleaving a photocleavable linkage.

15. The method of claim 1, wherein the oligonucleotides are double-stranded oligonucleotides that comprise staggered photocleavable or chemically cleavable linkages, and wherein the method comprises cleaving said staggered cleavable linkages using light or a chemical treatment to produce fragments that are ligatable to one another in order.

16. The method of claim 11, further comprising separating the synthons from the array.

17. A composition comprising multiple sets of oligonucleotides, wherein the oligonucleotides within each set comprise:

(i) a terminal indexer sequence and (ii) an assembly sequence, wherein the terminal indexer sequence and the assembly sequence are separated by a photocleavable or chemically cleavable linker and the assembly sequences of each set of oligonucleotides can be assembled to produce a synthon.

18. The composition of claim 17, wherein the assembly sequences of each set comprise overlapping complementary sequences that can be ligated directly to one another after cleavage of the terminal indexer sequences.

19. The composition of claim 17, wherein the assembly sequences of each set comprise overlapping complementary sequences that can be assembled by polymerase chain assembly after cleavage of the terminal indexer sequences.

20. An apparatus comprising:

a planar support,

a plurality of spatially distinct droplets on a surface of the planar support, and

an immiscible liquid covering the droplets,

wherein the apparatus comprises a plurality of reaction chambers defined by the droplets, and each reaction chamber comprises a different synthon.