Nucleic acid sequencing with solid phase capturable terminators comprising a cleavable linking group

• US 6,046,005 A
• Filed: 09/16/1998
• Issued: 04/04/2000
• Est. Priority Date: 01/15/1997
• Status: Expired due to Fees

• Current Assignee: Incyte Pharmaceuticals, Inc. (Incyte Corporation)
• Original Assignee: Incyte Pharmaceuticals, Inc. (Incyte Corporation)
• Inventors: Konrad, Ken, Ju, Jingyue
• Primary Examiner(s): Horlick, Kenneth R.
• Application Number:
  US09/154,525
• Time in Patent Office: 566 Days
• Field of Search: 435/6, 435/91.1, 435/810, 536/23.1, 536/25.3
• US Class Current: 435/6.12
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2525/186 incorporating a non-extendable or blocking moiety
• CPC Class Codes: C12Q 2525/204 specific length of the oligonucleotides
• CPC Class Codes: C12Q 2535/101 Sanger sequencing method, i.e. oligonucleotide sequencing using primer elongation and dideoxynucleotides as chain terminators
• CPC Class Codes: C12Q 2563/101 radioactivity, e.g. radioactive labels
• CPC Class Codes: C12Q 2565/518 characterised by the immobilisation of the nucleic acid sample or target

Abstract

Methods of enzymatic nucleic acid sequencing are provided in which solid-phase capturable chain terminators are employed. In the subject methods, sequencing fragments are generated, where the fragments comprise capturable chain terminators. The fragments are then captured on a solid phase and separated from the remaining components of the sequencing reaction. The fragments are then released from the solid phase, size separated and detected to yield sequencing data from which the sequence of the nucleic acid is determined.

Claims

1. A method of determining the sequence of a nucleic acid, said method comprising:

(a) preparing a reaction mixture corresponding to each base of said nucleic acid, wherein each of said reaction mixtures comprises a family of enzymatically produced capturable primer extension products produced by combining said nucleic acid, polymerase, deoxynucleotides, oligonucleotide primer and capturable dideoxy chain terminator in a reaction mixture under conditions sufficient to produce said capturable primer extension products, wherein said capturable dideoxy chain terminator comprises a capture moiety bonded to a dideoxynucleotide through a cleavable linking group.

(b) contacting each of said reaction mixtures with a solid phase capable of capturing said primer extension products comprising said capturable chain terminator through interaction of said capture moiety with a moiety present on said solid phase and to produce solid phase captured primer extension products;

(c) separating said solid phase captured primer extension products from said reaction mixture;

(d) releasing said primer extension products from said solid phase;

(e) size separating and detecting said released primer extension products to produce sequencing data; and

(f) determining the sequence of said nucleic acid from said sequencing data.

2. The method according to claim 1, wherein the primer extension products are labeled.

3. The method according to claim 2, wherein said oligonucleotide primer used to prepare said labeled primer extension products is labeled.

4. The method according to claim 2, wherein said released primer extension products from each of said reaction mixtures are size separated in different electrophoretic media and said sequencing data is a separation pattern.

5. The method according to claim 4, wherein said different electrophoretic media are adjacent lanes in a slab gel.

6. The method according to claim 4, wherein said different electrophoretic media are present in different capillaries.

7. The method according to claim 4, wherein the labeled primer extension products from each of said reaction mixtures comprise the same label.

8. The method according to claim 2, wherein said released primer extension products from each of said reaction mixtures are size separated in the same electrophoretic medium.

9. The method according to claim 3, wherein said labeled oligonucleotide primer is fluorescently labeled and said sequencing data is an electropherogram.

10. The method according to claim 2, wherein the labels of the labeled primer extension products in each reaction mixture are excited at substantially the same wavelength and provide distinguishable signals.

11. The method according to claim 1, wherein said cleavable linking group is a photo-cleavable linking group.

12. The method according to claim 1, wherein said cleavable linking group is a chemically cleavable linking group.

13. The method according to claim 1, wherein said releasing step comprises cleaving said cleavable linking group.

14. The method according to claim 1, wherein said primer extension products are unlabeled and said sequence is determined by mass spectrometry.

15. A method of determining the sequence of a DNA, said method comprising:

(a) preparing four different reaction mixtures corresponding to each base of said nucleic acid, wherein each of said reaction mixtures comprises a family of enzymatically produced fluorescently labeled primer extension products comprising a first member of a specific binding pair produced by combining said nucleic acid, polymerase, deoxynucleotides, fluorescently labeled oligonucleotide primer and dideoxynucleotide bonded to said first member of a specific binding pair member through a cleavable linking group;

(b) contacting each of said reaction mixtures with a solid phase comprising a second member of said specific binding pair whereby said first and second members bind and solid phase captured fluorescently labeled primer extension products are produced;

(c) separating said solid phase captured fluorescently labeled primer extension products from said reaction mixture;

(d) cleaving said cleavable linking groups in a manner sufficient to release said captured fluorescently labeled primer extension products from said solid phase;

(e) size separating and detecting said released fluorescently labeled primer extension products to produce an electropherogram; and

(f) determining the sequence of said nucleic acid from said electropherogram.

16. The method according to claim 15, wherein said fluorescently labeled primer extension products from each of said four reaction mixtures are pooled prior to step (e).

17. The method according to claim 15, wherein said four reaction mixtures are pooled prior to step (b).

18. The method according to claim 15, wherein said four reaction mixtures are pooled prior to step (d).

19. The method according to claim 15, wherein said first member of a specific binding pair is biotin.

20. The method according to claim 15, wherein said second member of a specific binding pair is avidin or streptavidin.

21. The method according to claim 15, wherein said cleavable linking group is a chemically cleavable linking group.

22. The method according to claim 15, wherein said cleavable linking group is a photo-cleavable linking group.

23. A kit for use in determining the sequence a nucleic acid, said kit comprising:

at least one of a labeled moeity selected from the group consisting of labeled oligonucleotide primers and labeled dNTPs; and

solid phase capturable chain terminator comprising a capture moiety bonded to a dideoxynucleotide through a cleavable linking group.

24. The kit according to claim 23, wherein said kit comprises at least one of:

(a) four distinguishably labeled oligonucleotide primers; and

(b) four distinguishably labeled dNTPs.

25. The kit according to claim 23, wherein said labeled oligonucleotide primers are fluorescently labeled.

26. The kit according to claim 23, wherein said capture moiety is a member of a specific binding pair.

27. The kit according to claim 26, wherein said specific binding pair consists of biotin and a member selected from the group consisting of avidin and streptavidin.

28. The kit according to claim 23, wherein said cleavable linking group is photocleavable.

29. The kit according to claim 23, wherein said cleavable linking group is chemically cleavable.