Methods for solid-phase nucleic acid amplification and sequencing

• US 6,277,604 B1
• Filed: 10/29/1999
• Issued: 08/21/2001
• 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 (Merck & Co., Inc.)
• Inventors: Peponnet, Christine
• Primary Examiner(s): Jones, W. Gary
• Assistant Examiner(s): Tung, Joyce
• Application Number:
  US09/430,035
• Time in Patent Office: 662 Days
• Field of Search: 435/91.2, 435/91.1, 435/6, 935/77, 536/24.3
• 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

Methods for amplifying and sequencing nucleic acids are disclosed. In addition, supports for the solid phase amplification or sequencing of nucleic acids are disclosed. The supports comprise a solid support, a linker arm having functional groups capable of binding to the solid support, and an oligonucleotide bound to the linker arm.

Claims

1. A method for the solid-phase amplification of nucleic acids, which comprises contacting a support 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 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;

with a sample comprising at least one nucleic acid complementary to the primer bound to the solid support in the presence of a polymerase, and under reaction conditions which effect elongation of said primer.

2. The method according to claim 1, further comprising sequencing at least one nucleic acid sequence.

8. The method according to claim 1, further comprising reacting an amplified nucleic acid with suitable reagents under appropriate conditions for the sequencing of at least one nucleotide.

11. The method according to claim 1, wherein said solid support is heat resistant.

12. The method according to claim 1, wherein the linker is a polymer molecule.

13. The method according to claim 12, wherein the linker contains at least 5 monomer units.

3. A method for the solid-phase amplification of nucleic acids, which comprises contacting a support comprising a polyfunctionalized solid support;

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

a first oligonucleotide primer bound 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;

with, in solution comprising at least one, nucleic acid complementary to the primer bound to the solid support in the presence of a polymerase, and a second amplification primer, under reaction conditions which effect elongation of said primer bound to the solid support and said second amplification primer, and amplification of said nucleic acid.

4. The method according to claim 3, wherein the primer bound to the solid support is also present in solution, but in a lower amount than said second amplification primer.

5. The method according to claim 3, wherein said second amplification primer is present in solution in an amount of about 5 to 10 pmol.

6. The method according to claim 4, wherein the amount of the primer bound to the solid support in solution is 8 to 16 times less than that of said second amplification primer.

7. The method according to claim 3, wherein said amplification occurs in a liquid phase, during which said amplification is exponential, until said second amplification primer is depleted, followed by amplification in a solid phase, during which amplification is linear.

9. The method according to claim 3, further comprising reacting an amplified nucleic acid with suitable reagents under appropriate conditions for the sequencing of at least one nucleotide.

10. A method for the solid-phase sequencing of nucleic acids, which comprises contacting a support comprising:

a poly solid support;

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

an oligonucleotide primer bound to said linker wherein said primer is immobilized on said solid support by means of at least one covalent bond between said primer and said solid support;

with a sample comprising at least one nucleic acid complementary the primer bound to said support in the presence of suitable reagents, including a polymerase, and under appropriate reaction conditions for sequencing at least one nucleic acid sequence.