Method for immobilizing nucleic acid molecules

• US 5,610,287 A
• Filed: 11/16/1994
• Issued: 03/11/1997
• Est. Priority Date: 12/06/1993
• Status: Expired due to Fees

• Current Assignee: Beckman Coulter Incorporated (Danaher Corporation)
• Original Assignee: Molecular Tool, Inc. (Laboratory Corporation Of America Holdings)
• Inventors: Nikiforov, Theo, Knapp, Michael R.
• Primary Examiner(s): Naff, David M.
• Application Number:
  US08/341,148
• Time in Patent Office: 846 Days
• Field of Search: 435/180, 435/6, 435/172.3, 536/24.3, 536/24.31, 536/24.32, 536/25.3, 536/24.33
• US Class Current: 536/24.3
• CPC Class Codes: C12N 15/1006 by means of a solid support carrier, e.g. particles, polymers
• CPC Class Codes: C12Q 1/6834 Enzymatic or biochemical coupling of nucleic acids to a solid phase

Abstract

Synthetic nucleic acid molecules are non-covalently immobilized in the presence of a salt or cationic detergent on a hydrophilic polystyrene solid support containing an --OH, --C═O or --COOH hydrophilic group or on a glass solid support. The support is contacted with a solution having a pH of about 6 to about 8 containing the synthetic nucleic acid and the cationic detergent or salt. Preferably, the cationic detergent is 1-ethyl-3-(3'"'"'-dimethylaminopropyl)-1,3-carbodiimide hyrochloride at a concentration of about 30 mM to about 100 mM or octyldimethylamine hydrochloride at a concentration of about 50 mM to about 150 mM. The salt is preferably NaCl at a concentration of about 50 mM to about 250 mM. When the detergent is 1-ethyl-3-(3'"'"'-dimethylaminopropyl)-1,3-carbodiimide hyrochloride, the glass support or the hydrophilic polystyrene support is used. When NaCl or octyldimethylamine hydrochloride is used, the support is the hydrophilic polystyrene. After immobilization, the support containing the immobilized nucleic acid may be washed with an aqueous solution containing a non-ionic detergent. The immobilized nucleic acid may be used in nucleic acid hybridization assays, nucleic acid sequencing and in analysis of genomic polymorphisms.

Claims

1. A method for non-covalently immobilizing a synthetic nucleic acid molecule on a solid support which is a hydrophilic polystyrene solid support containing a hydrophilic group selected from the group consisting of --OH, --C═

O, and --COOH, or a glass solid support, said method comprising the steps;

(a) contacting said support with a solution having a pH of from about 6 to about 8, and containing said nucleic acid and (1) a cationic detergent selected from the group consisting of 1-ethyl-3-(3'"'"'-dimethylaminopropyl)-1,3-carbodiimide hydrochloride provided at a concentration of from about 30 mM to about 100 mM, and octyldimethylamine hydrochloride provided at a concentration of from about 50 mM to about 150 mM or (2) NaCl provided at a concentration of from about 50 mM to about 250 mM, to thereby non-covalently immobilize said nucleic acid to said support, wherein;

(i) when said cationic detergent is 1-ethyl-3-(3'"'"'-dimethylaminopropyl)carbodiimide-1,3-hydrochloride, said support is selected from the group consisting of glass or said hydrophilic polystyrene;

(ii) when said cationic detergent is octyldimethylamine hydrochloride, said support is said hydrophilic polystyrene; and

(iii) when said solution contains said NaCl, said support is said hydrophilic polystyrene; and

(b) subsequently washing said solid support with an aqueous solution.

2. The method of claim 1, wherein said solid support is said hydrophilic polystyrene support.

3. The method of claim 1, wherein said solid support is said glass support.

4. The method of claim 1, wherein said solid support is in the form of a bead or membrane.

5. The method of claim 1, wherein said cationic detergent is 1-ethyl-3-(3'"'"'-dimethylaminopropyl)-1,3-carbodiimide hydrochloride.

6. The method of claim 1, wherein said cationic detergent is octyldimethylamine hydrochloride.

7. The method of claim 1, wherein in step (b), said aqueous solution contains a non-ionic detergent.

8. The method of claim 7, wherein said non-ionic detergent is polyoxyethylene (20) sorbitan.

9. The method of claim 8, wherein said polyoxyethylene (20) sirbitan is provided in a solution that additionally contains buffered saline.

10. The method of claim 1, wherein said synthetic nucleic acid molecule is an oligonucleotide having a minimum length of at least 12 nucleotide residues and a maximum length of about 100 residues.

11. The method of claim 10, wherein said oligonucleotide is chemically modified.

12. The method of claim 2, wherein said hydrophilic polystyrene support is in the form of a 96-well microtiter plate.

13. The method of claim 2, wherein said hydrophilic polystyrene support is in the form of a 96-pin array designed to fit into a 96-well microtiter plate.

14. The method of claim 3, wherein said glass support is in the form of a microscope slide.

15. The method of claim 1, wherein said immobilized synthetic nucleic acid molecule is a polynucleotide and wherein said method additionally comprises the steps of:

(A'"'"') capturing from solution at least one strand of a specific polynucleotide analyte by hybridization to said immobilized polynucleotide; and

(B'"'"') detecting the presence of the captured analyte.

16. The method of claim 1, wherein said immobilized synthetic nucleic acid molecule is a polynucleotide and wherein said method additionally comprises the steps of:

(A") amplifying a specific region of a specific genome using a polymerase chain reaction to produce an amplified specific region of said genome, said region having a sequence complementary to said immobilized polynucleotide; and

(B") capturing from solution at least one strand of said amplified specific region of said genome by hybridization to said immobilized polynucleotide; and

(C") detecting the presence of said specific region of said genome.

17. The method of claim 1, wherein said immobilized synthetic nucleic acid molecule is a polynucleotide primer and wherein said method additionally comprises the steps of:

(A'"'"'") incubating a sample of nucleic acid of a target organism, containing a single nucleotide polymorphism in the presence of said immobilized polynucleotide primer and a polymerase and at least one dideoxynucleotide derivative, under conditions sufficient to permit a polymerase mediated, template-dependent extension of said primer, said extension causing the incorporation of a single dideoxynucleotide derivative that is complementary to a polymorphic nucleotide of said single nucleotide polymorphism of said target organism nucleic acid;

(B'"'"'") permitting said template-dependent extension of said primer molecule, and said incorporation of said single dideoxynucleotide; and

(C'"'"'") determining the identity of the dideoxynucleotide derivative incorporated that is complementary to said polymorphic nucleotide.

18. The method of claim 1, wherein said solid support is in the form of a filter.

19. The method of claim 1, wherein said solid support is in the form of an affinity column.