DNA sequencing by parallel oligonucleotide extensions

• US 6,306,597 B1
• Filed: 03/29/1999
• Issued: 10/23/2001
• Est. Priority Date: 04/17/1995
• Status: Expired due to Term

• Current Assignee: Illumina Incorporated, Solexa Incorporated (Illumina Incorporated), Stephen C. Macevicz, Applera Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: Lynx Therapeutics, Inc. (Illumina Incorporated)
• Inventors: Macevicz, Stephen C.
• Primary Examiner(s): Whisenant, Ethan
• Assistant Examiner(s): LU, FRANK WEI MIN
• Application Number:
  US09/280,270
• Time in Patent Office: 939 Days
• Field of Search: 435/6, 435/91.1, 435/91.2, 435/183, 536/23.1, 536/24.3, 536/24.33, 536/25.3, 436/94
• US Class Current: 435/6.12
• CPC Class Codes: C07H 21/02 with ribosyl as saccharide radical
• CPC Class Codes: C07H 21/04 with deoxyribosyl as saccharide radical
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2521/501 Ligase
• CPC Class Codes: C12Q 2525/186 incorporating a non-extendable or blocking moiety
• CPC Class Codes: C12Q 2533/107 Probe or oligonucleotide ligation
• CPC Class Codes: Y02P 20/582 Recycling of unreacted starting or intermediate materials
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

Abstract

The present invention includes, in one aspect, a kit comprising one or more sets of oligonucleotide probes whose perfectly matched duplexes with complementary sequences have similar or substantially the same free energy of duplex formation.

Claims

1. A method for identifying a sequence of nucleotides in a polynucleotide, the method comprising the steps of:

(a) extending an initializing oligonucleotide along the polynucleotide by ligating an oligonucleotide probe thereto to form an extended duplex;

(b) identifying one or more nucleotides of the polynucleotide; and

(c) repeating steps (a) and (b) until the sequence of nucleotides is determined.

2. A kit for DNA sequence analysis, the kit comprising one or more sets of oligonucleotide probes, wherein

(i) each probe set contains at least 50 different-sequence, single-stranded oligonucleotide probes,

(ii) the oligonucleotide probes of a set have a selected length of up to 12 nucleotides, and

(iii) for at least one said probe set, the different-sequence, single stranded oligonucleotide probes in that set have annealing temperatures whose maximum and minimum values differ from each other by no more than 1°

C.

3. The kit of claim 2, wherein each probe set contains from 50 to 10,000 different different-sequence, single-stranded oligonucleotide probes.

4. The kit of claim 2 wherein said annealing temperatures have a value from 22°

C. to 70°

C.

5. The kit of claim 4 wherein the oligonucleotide probes have lengths of 8, 9, or 12 nucleotides.

6. The kit of claim 2 which further includes an initializing oligonucleotide having a length of from 20 to 30 nucleotides, for binding to a target nucleic acid.

7. The kit of claim 3 further including one or more initializing oligonucleotides and one or more solid phase supports having templates attached thereto, the templates each comprising a target polynucleotide having a binding region capable of forming a perfectly matched duplex with one or more of the initializing oligonucleotides.

8. The kit of claim 3 wherein each probe set contains 50 to 500 different-sequence, single-stranded oligonucleotide probes.