Solid support for solid phase amplification and sequencing and method for preparing the same nucleic acid

• US 6,274,351 B1
• Filed: 07/11/1997
• Issued: 08/14/2001
• Est. Priority Date: 10/28/1994
• Status: Expired due to Fees

• Current Assignee: Serono Genetics Institute S.A. (Merck & Co., Inc.)
• Original Assignee: Genset SA
• Inventors: Peponnet, Christine
• Primary Examiner(s): Jones, W. Gary
• Assistant Examiner(s): Tung, Joyce
• Application Number:
  US08/836,031
• Time in Patent Office: 1,495 Days
• Field of Search: 435/91.2, 435/91.1, 435/402, 536/24.2, 536/22.1, 536/23.1
• US Class Current: 435/91.1
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 1/6846 Common amplification features
• CPC Class Codes: C12Q 2525/197 incorporating a spacer/coupling moiety
• CPC Class Codes: C12Q 2565/537 characterised by the capture oligonucleotide acting as a primer

Abstract

A support for solid phase amplification or sequencing of nucleic acids has a functionalized solid support, a linker arm having functional groups covalently bound to the solid support through at least one binding site of the functional groups, and an oligonucleotide primer bound at its 5′ end to the linker, the primer, thereby, being immobilized on the solid support.

Claims

1. A support for the solid phase amplification or sequencing of nucleic acids comprising:

a polyfunctionalized solid support;

a linker having a multiplicity of functional groups capable of binding to said solid support; and

an oligonucleotide primer bound at a 5′

end to said linker;

wherein said primer is immobilized on said solid support via said linker by means of at least one covalent bond between said functional groups and said solid support.

5. The support of claim 1, wherein said functional groups are chosen from amine, hydroxyl, carboxyl, aldehyde, thiol, and phosphate groups.

6. The support of claim 5, wherein said functional groups are chosen from amine, hydroxyl, and phosphate.

10. The support of claim 1, wherein said primer is selected from the group consisting of:

A-10T, A-20T, A-30-T, A-5T-Ph, A-10T-Ph, A-5A-Ph, A-10A-Ph, A-5C,

A-5C-Ph, A-10C-Ph, A-triethylene glycol)₃[3]Ph and A-5T-5NH₂[2],

wherein

A- is a oligonucleotide primer, Tafter the integer is thymine, A after the integer is adenine,

C after the integer is cytosine, Ph is a terminal 5′

phosphate group, and the integer represents the number of T, A, C, Ph, or NH₂ groups present on said linker.

14. The support of claim 1, wherein said polyfunctionalized support is comprised of organic or inorganic polymer material.

15. The support of claim 14, wherein said polyfunctionalized solid support is comprised of a heat-resistant plastic material functionalized by corona treatment or gamma-irradiation.

16. The support of claim 15, wherein said heat-resistant plastic material consists of heat-resistant modified polystyrene such as a styrene/acrylonitrile copolymer or polycarbonate.

18. The support of claim 1, wherein said functionalized solid support is heat resistant.

2. The support of claim, wherein said linker i is a polymer molecule.

3. The support of claim 2, wherein said polymer molecule contains monomer units, each monomer unit bearing at least one of said functional groups.

4. The support of claim 3, wherein said polymer molecule contains at least 5 and 10 monomer units.

7. The support of claim 2, wherein the polymer molecule is a homopolynucleotide or a polynucleotide analog.

8. The support of claim 7, wherein said polymer molecule is a homopolynucleotide containing between 10 and 50 nucleotides.

9. The support of claim 8, wherein the polymer molecule is a poly-T, a poly-A, or a poly-C.

11. The support of claim 7, wherein said polymer molecule is a polynucleotide analog of formula:

wherein R is an aliphatic residue bearing at least one functional group and n is an integer from 2 to 50.

12. The support of claim 11, wherein said functional group is an amine group or a hydroxyl group.

13. The support of claim 2, wherein said polymer molecule has a terminal phosphate group at an end which is not linked to said primer.

19. The support of claim 3, wherein said polymer molecule contains at least 10 monomer units.

17. A method for preparing a support for solid phase amplification or sequencing of nucleic acids, comprising:

providing a linker covalently bond to a 5′

end of an oligonucleotide primer, said linker having functional groups which provide a multiplicity of potential binding sites; and

contacting an amount of said linker with a polyfunctionalized solid support under reaction conditions whereby various covalent bonds are formed between at least one of said potential binding sites of each linker and said solid support.