Method for synthesizing a specific, surface-bound polymer uniformly over an element of a molecular array

• US 6,936,472 B2
• Filed: 10/05/2001
• Issued: 08/30/2005
• Est. Priority Date: 04/28/1999
• Status: Expired due to Fees

• Current Assignee: Agilent Technologies, Inc.
• Original Assignee: Agilent Technologies, Inc.
• Inventors: Earhart, Jonathan P., Perbost, Michel G. M.
• Primary Examiner(s): Horlick, Kenneth R.
• Assistant Examiner(s): Lu, Frank
• Application Number:
  US09/972,256
• Publication Number:
  US 20020034830A1
• Time in Patent Office: 1,425 Days
• Field of Search: 435/6, 435/91.1, 435/183, 436/94, 536/23.1, 536/24.3, 536/24.33, 536/25.3, 536/25.34
• US Class Current: 436/94
• CPC Class Codes: B01J 2219/00378 Piezoelectric or ink jet dispensers
• CPC Class Codes: B01J 2219/00385 Printing
• CPC Class Codes: B01J 2219/00529 DNA chips
• 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 2219/00608 DNA chips
• CPC Class Codes: B01J 2219/00617 by chemical means
• CPC Class Codes: B01J 2219/00626 Covalent
• CPC Class Codes: B01J 2219/00637 by coating it with another layer
• CPC Class Codes: B01J 2219/00675 In-situ synthesis on the substrate
• 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: 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: 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
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

Abstract

A method for specifically and uniformly synthesizing desired polymers within molecular array elements. Droplets containing a reactive monomer are successively applied to the elements of a molecular array in order to synthesize a substrate-bound polymer. Application of an initial droplet, having a first volume, defines the position and size of a molecular array element. Subsequent droplets are applied, to add successive reactive monomers to growing nascent polymers within the molecular array element, with covering volumes so that, even when application of the subsequent droplets is misregistered, the entire surfaces of the elements of the molecular array are exposed to the subsequently applied droplets. Following application of initial droplets, the surface of the molecular array is exposed to a solution containing a very efficient capping agent in order to chemically cap any unreacted nascent growing polymers and any unreacted substrate molecules.

Claims

1. An improved method for repeatedly applying solutions of reactive nucleotide monomers to a particular feature on a chemically-prepared surface of a solid substrate of a molecular array in order to synthesize a substrate-bound oligonucleotide polymer within the feature having a predetermined monomer sequence, the method comprising:

applying an initial volume of an initial solution of a reactive nucleotide monomer to the feature to react with reactive substrate-bound groups of the feature to produce a nascent substrate-bound oligonucleotide polymer within a region of the surface of the solid substrate in contact with the initial volume of solution; and

repeatedly;

applying a solution containing an activating agent to the feature in order to activate nascent oligonucleotide polymers within the feature for reaction with a subsequently applied reactive nucleotide monomer; and

applying an additional covering volume of a solution of a reactive nucleotide monomer to the feature in order to chemically bind the reactive nucleotide monomer to the nascent polymers within the feature.

2. The method of claim 1, additionally comprising capping unreacted reactive substrate-bound groups of the chemically-prepared surface of the solid substrate by applying a solution of a reactive capping agent to the surface of the solid substrate.

3. The method of claim 2 wherein the capping is performed following application of the initial volume and before applying the activating agent and the additional covering volume.

4. The method of claim 1 further including:

rinsing the surface of the feature following application of a solution of reactive nucleotide monomer; and

rinsing the surface of the feature following application of a solution containing an activating agent to the surface of the solid substrate.

5. The method of claim 1 wherein the reactive monomers are 5′

-protected deoxynucleoside phosphoramidites.

6. The method of claim 5 wherein a deoxynucleoside phosphoramidite is 5′

protected by covalent bonding of a dimethoxytrityl group to the 5′

-hyrdoxyl oxygen of the deoxynucleoside phosphoramidite.

7. The method of claim 6 wherein the phosphoramidite is selected from among the group of phosphoramidites consisting of:

diethoxy-N,N-diisopropyl phosphoramidite;

diallyl-N,N-diisopropyl phosphoramidite;

a phosphoramidite having the formula (R¹—

O—

)(R²—

O)P—

N(CH(CH₃)₂)₂ where R¹ and R² may be similar or different alkyl side chains, side chains having a carboxylic group, and side chains with a double bond; and

dicyanoethyl-N,N-diisopropyl phosphoaramidite.

8. The method of claim 6 wherein the solution of a reactive nucleotide monomer additionally includes an activator which is selected from among the group of activators consisting of:

0.45 M tetrazole;

0.25 M 5-ethylthio-1H-tetrazole; and

0.25 M 4,5-dicyanoimidazole.

9. The method of claim 5 wherein the solution containing an activating agent comprises 3% acid in a solvent, wherein the acid is selected from among trichloroacetic acid and dichloroacetic acid, and wherein the solvent is selected from among dichloromethane and toluene.

10. The method of claim 5 further including:

following the reaction of each applied 5′

-protected deoxynucleoside phosphoramidite reactive monomer, the 3′

-phosphite group of the applied 5′

-protected deoxynucleoside phosphoramidite linking the applied 5′

-protected deoxynucleoside to a target hydroxyl oxygen atom is oxidized by application of an oxidizing solution to a phosphate group; and

the oxidizing solution is rinsed from the surface of the substrate following oxidation of the phosphite group.

11. The method of claim 1 used in an apparatus for automated production of molecular arrays.

12. An improved method for repeatedly applying solutions of reactive nucleotide monomers to a particular feature on a solid substrate of a molecular array in order to synthesize a substrate-bound oligonucleotide polymer within the feature having a predetermined monomer sequence, the method comprising:

applying an initial volume of an initial solution of a reactive nucleotide monomer to the feature to react with reactive substrate-bound groups of the chemically-prepared surface within the feature to produce a nascent substrate-bound oligonucleotide polymer within the feature in a region of the surface of the solid substrate in contact with the initial volume of solution; and

repeatedly

applying a solution containing an activating agent to the feature in order to activate nascent oligonucleotide polymers for reaction with a subsequently applied reactive nucleotide monomer; and

applying an additional covering volume of a solution of a reactive nucleotide monomer to the feature in order to chemically bind the reactive monomer to the nascent polymers within the feature.

13. The method of claim 12 additionally comprising capping unreacted reactive substrate-bound groups of the chemically-prepared surface of the solid substrate by applying a solution of a reactive capping agent to the surface of the solid substrate that reacts with greater than 90% efficiency with unreacted reactive substrate-bound groups of the chemically-prepared surface of the solid substrate.

14. The method of claim 13 wherein the capping is performed following application of the initial volume and before applying the activating agent and the additional covering volume.

15. The method of claim 12 wherein the additional covering volume of a solution of a reactive nucleotide monomer applied to the feature ensures that the additional volume of the solution will spread out and completely cover the surface containing nascent oligonucleotide polymers produced by application of the initial volume of the initial solution.

16. The method of claim 15 wherein the reactive monomers are 5′

-protected deoxynucleoside phosphoramidites.

17. The method of claim 14 wherein the capping agent initially used to cap unreacted reactive substrate-bound groups of the chemically-prepared surface of the solid substrate solution comprises a mixture of a first solution of approximately 0.1 M phosphoramidite in anhydrous acetonitrile and a second solution comprising an activator in acetonitrile.

18. The method of claim 17 wherein the phosphoramidite is selected from among the group of phosphoramidites consisting of:

diethoxy-N,N-diisopropyl phosphoramidite;

diallyl-N,N-diisopropyl phosphoramidite;

a phosphoramidite having the formula (R¹—

O—

)(R²—

O)P—

N(CH(CH₃)₂)₂ where R¹ and R² may be similar or different alkyl side chains, side chains having a carboxylic group, and side chains with a double bond; and

dicyanoethyl-N,N-diisopropyl phosphoaramidite; and

wherein the activator is selected from among the group of activators consisting of;

0.45 M tetrazole;

0.25 M 5-ethylthio-1H-tetrazole; and

0.25 M 4,5-dicyanoimidazole.

19. The method of claim 12 used in an apparatus for automated production of molecular arrays.