DNA sequencing by parallel oligonucleotide extensions

• US 5,750,341 A
• Filed: 04/17/1995
• Issued: 05/12/1998
• 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): Jones, W. Gray
• Assistant Examiner(s): Tran, Paul B.
• Application Number:
  US08/424,663
• Time in Patent Office: 1,121 Days
• Field of Search: 435/6, 435/91.52, 435/91.53, 935/77, 935/78, 536/24.33, 536/25.32, 436/94
• US Class Current: 435/6.19
• 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

Method and compositions are provided for analyzing nucleic acid sequences based on repeated cycles of duplex extension along a single stranded template. Preferably, such extension starts from a duplex formed between an initializing oligonucleotide and the template. The initializing oligonucleotide is extended in an initial extension cycle by ligating an oligonucleotide probe to its end to form an extended duplex. The extended duplex is then repeatedly extended by subsequent cycles of ligation. During each cycle, the identity of one or more nucleotides in the template is determined by a label on, or associated with, a successfully ligated oligonucleotide probe. Preferably, the oligonucleotide probe has a blocking moiety, e.g. a chain-terminating nucleotide, in a terminal position so that only a single extension of the extended duplex takes place in a single cycle. The duplex is further extended in subsequent cycles by removing the blocking moiety and regenerating an extendable terminus. The invention provides a method of sequencing nucleic acids which obviates electrophoretic separation of similarly sized DNA fragments, and which eliminates the difficulties associated with the detection and analysis of spacially overlapping bands of DNA fragments in a gel or like medium. The invention also obviates the need to generate DNA fragments from long single stranded templates with a DNA polymerase.

Claims

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

(a) providing a probe-target duplex comprising an initializing oligonucleotide probe hybridized to a target polynucleotide, said probe having an extendable probe terminus;

(b) ligating an extension oligonucleotide probe to said extendable probe terminus, to form an extended duplex containing an extended oligonucleotide probe;

(c) identifying, in the extended duplex, at least one nucleotide in the target polynucleotide that is either (1) complementary to the just-ligated extension probe or (2) a nucleotide residue in the target polynucleotide which is immediately downstream of the extended oligonucleotide probe;

(d) generating an extendable probe terminus on the extended probe, if an extendable probe terminus is not already present, such that the terminus generated is different from the terminus to which the last extension probe was ligated; and

(e) repeating steps (b), (c) and (d) until a sequence of nucleotides in the target polynucleotide is determined.

2. The method of claim 1 wherein each extension probe has a chain-terminating moiety at a terminus distal to said initializing oligonucleotide probe.

3. The method of claim 2 wherein said step of identifying includes removing said chain-terminating moiety and extending said extended oligonucleotide probe with a nucleic acid polymerase in the presence of one or more labeled chain-terminating nucleoside triphosphates.

4. The method of claim 3 wherein said extension probe includes a subsequence of four ribonucleotides and wherein said step of regenerating includes cleaving said extended oligonucleotide probe with RNase H.

5. The method of claim 4 wherein said chain-terminating moiety is a 3'"'"' phosphate.

6. The method of claim 2 further including a step of capping an extended oligonucleotide probe whenever no extension probe has ligated to the extendable terminus in the ligation step.

7. The method of claim 2 wherein said step of regenerating includes cleaving a chemically scissile internucleosidic linkage in said extended oligonucleotide probe.

8. The method of claim 7 wherein said chemically scissile internucleosidic linkage is a phosphoramidate.

9. The method of claim 2 wherein said step of regenerating includes enzymatically cleaving an internucleosidic linkage in said extended oligonucleotide probe.

10. The method of claim 9 wherein at least one extension probe includes a subsequence of four ribonucleotides and wherein said step of regenerating includes cleaving said oligonucleotide probe with RNase H.

11. The method of claim 1, wherein step (a) includes providing, in separate aliquots, a plurality of distinct target-primer duplexes, each distinct duplex comprising an initializing oligonucleotide primer hybridized to a target polynucleotide, wherein

the target polynucleotide in each duplex is the same, but the initializing oligonucleotide in each duplex is bound to a different sequence of the target polynucleotide;

and

steps (b) to (e) are carried out independently on each aliquot.

12. The method of claim 11 wherein for each aliquot, said extension oligonucleotide probe has a chain-terminating moiety at a terminus distal to said primer.

13. The method of claim 12 wherein for each aliquot, said step of identifying includes removing said chain-terminating moiety and extending said extended oligonucleotide probe with a nucleic acid polymerase in the presence of one or more labeled chain-terminating nucleoside triphosphates.

14. The method of claim 13 wherein for each aliquot, said extension probe includes a subsequence of four ribonucleotides and wherein said step of regenerating includes cleaving said extended oligonucleotide probe with RNase H.

15. The method of claim 14 wherein said chain-terminating moiety is a 3'"'"' phosphate.

16. The method of claim 11 further including a step of capping an extended oligonucleotide probe whenever no extension probe has ligated to the extendable terminus in the ligation step.

17. The method of claim 11 wherein for each aliquot, said step of regenerating includes cleaving a chemically scissile internucleosidic linkage in said extended oligonucleotide probe.

18. The method of claim 17 wherein said chemically scissile internucleosidic linkage is a phosphoramidate.

19. The method of claim 11 wherein for each aliquot, said step of regenerating includes enzymatically cleaving an internucleosidic linkage in said extended duplex.