Minimization of blooming in high-density arrays by using reactive wash reagents

• US 6,184,347 B1
• Filed: 11/19/1998
• Issued: 02/06/2001
• Est. Priority Date: 11/19/1998
• Status: Expired due to Fees

• Current Assignee: Agilent Technologies, Inc.
• Original Assignee: Agilent Technologies, Inc.
• Inventors: Kanemoto, Roy H., Perbost, Michel G. M.
• Primary Examiner(s): Marschel, Ardin H.
• Application Number:
  US09/195,869
• Time in Patent Office: 810 Days
• Field of Search: 530/333, 530/334, 536/25.3, 536/25.31, 422/50, 422/68.1
• US Class Current: 530/333
• CPC Class Codes: B01J 2219/00497 Features relating to the solid phase supports
• CPC Class Codes: B01J 2219/00527 Sheets
• CPC Class Codes: B01J 2219/00536 in the shape of disks
• 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 2219/00617 by chemical means
• CPC Class Codes: B01J 2219/00626 Covalent
• CPC Class Codes: B01J 2219/00659 Two-dimensional arrays
• CPC Class Codes: B01J 2219/00722 Nucleotides
• CPC Class Codes: C07B 2200/11 Compounds covalently bound to a solid support
• 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: C40B 40/06 Libraries containing nucleotides or polynucleotides, or derivatives thereof

Abstract

A wash reagent employed for the bulk washing of the surface of a high-density array to remove unreacted reactants from cells of the array while, at the same time, reacting with the unreacted monomer in order to prevent reaction of the reacted monomer with functional groups on the surface of the HDA outside of the region of the surface to which the reactive monomer is applied. The wash reagent is chosen for a particular solid-state synthesis so that the unreacted reactants and catalyzing agents are soluble in the wash reagent, so that the wash reagent does not react with, or catalyze, reactions of the substrate or the biopolymers bound to the substrate, and so that the wash reagent reacts with unreacted reactive monomer in order to prevent subsequent reactions of the unreacted reactive monomer.

Claims

1. A method used, during the solid-state synthesis of surface-bound polymers on a solid substrate, for removing a reaction solution including unreacted reactive reagents from the surface of the solid substrate and from any nascent polymers bound to the solid substrate, the method comprising:

selecting a reactive wash solution that is not reactive toward, and does not catalyze reactions with, the solid substrate or any nascent polymers bound to the solid substrate but that reacts with, and deactivates, the reactive reagents and that is miscible with the reaction solution; and

applying the reactive wash solution to the surface of the solid substrate in order to react with, and deactivate, any unreacted reactive reagents and to remove the reaction solution from the surface of the solid substrate and from any nascent polymers bound to the substrate.

2. The method of claim 1 wherein the reaction solution includes reactive monomers and a catalyzing reagent that catalyzes the coupling of reactive monomers to the nascent polymers.

3. The method of claim 2 wherein the reactive monomers are deoxynucleoside phosphoramidites and the polymers are oligonucleotides.

4. The method of claim 3 wherein the solid substrate is a high-density array comprising cells in which different oligonucleotides are synthesized, the method further including:

applying the reactive wash solution separately to each cell of the high-density array in order to react with, and deactivate, any unreacted deoxynucleoside phosphoramidites s; and

rinsing the surface of the solid substrate with the reactive wash solution to remove the deactivated unreacted deoxynucleoside phosphoramidites and catalyzing reagent from the surface of the solid substrate and from any nascent polymers bound to the substrate.

5. The method of claim 3 wherein the solid substrate is a high-density array comprising cells in which different oligonucleotides are synthesized, the method further including:

applying the reactive wash solution separately to each cell of the high-density array in order to react with, and deactivate, any unreacted deoxynucleoside phosphoramidites;

allowing the applied reactive wash solution and reaction solution to evaporate; and

rinsing the surface of the solid substrate with the reactive wash solution to remove the deactivated deoxynucleoside phosphoramidites and catalyzing agent from the surface of the solid substrate and from any nascent polymers bound to the substrate.

6. The method of claim 3 wherein the reactive wash solution includes a chemical compound containing a hydroxyl functional group.

7. The method of claim 6 wherein the reactive wash solution is methanol.

8. The method of claim 1 wherein the reaction solution includes a reactive dye.

9. The method of claim 1 wherein the reaction solution includes a reactive radio-labeled marker.

10. A method for coupling a reactive monomer molecule to a nascent polymer bound to surface of a solid substrate, the method comprising:

applying monomer molecules and any reagents required to catalyze the coupling of the reactive monomer molecule with the nascent polymer to the surface of the solid substrate; and

applying a reactive wash solution to the surface of the solid substrate to react with, and deactivate, any remaining reactive monomers on the surface of the solid substrate and to dissolve and remove the remaining deactivated monomers and any reagents required to catalyze the coupling of the reactive monomer molecule to the polymer from the surface of the solid substrate and from any synthesized polymers bound to the substrate.

11. The method of claim 10 wherein the reactive monomers are deoxynucleoside phosphoramidites and the polymers are oligonucleotides.

12. The method of claim 11 wherein the solid substrate is a high-density array comprising cells in which different oligonucleotides are synthesized, the method further including:

applying the reactive wash solution separately to each cell of the high-density array in order to react with, and deactivate, any unreacted deoxynucleoside phosphoramidite s; and

rinsing the surface of the solid substrate with the reactive wash solution to remove the deactivated unreacted deoxynucleoside phosphoramidite s and catalyzing reagent from the surface of the solid substrate and from any nascent polymers bound to the substrate.

13. The method of claim 12 wherein the solid substrate is a high-density array comprising cells in which different oligonucleotides are synthesized, the method further including:

applying the reactive wash solution separately to each cell of the high-density array in order to react with, and deactivate, any unreacted deoxynucleoside phosphoramidite s;

allowing the applied reactive wash solution and solution to evaporate; and

rinsing the surface of the solid substrate with the reactive wash solution to remove the deactivated deoxynucleoside phosphoramidite s and catalyzing agent from the surface of the solid substrate and from any nascent polymers bound to the substrate.

14. The method of claim 11 wherein the reactive wash solution includes a chemical compound containing a hydroxyl functional group.

15. The method of claim 14 wherein the reactive wash solution is methanol.